Application of Chitosan for Preparation of Arbutin Nanoparticles as Skin Whitening

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1 Journal of Metals, Materials and Minerals, Vol.20 No.3 pp , 2010 Application of Chitosan for Preparation of Arbutin Nanoparticles as Skin Whitening Pimporn LEELAPORNPISID*, Phuriwat LEESAWAT, Surapol NATAKARNKITKUL and Porjai RATTANAPANADDA Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiangmai, Thailand Abstract The arbutin loaded chitosan nanoparticles were prepared by ionotropic gelation technique. Variations in chitosan concentration, ph of chitosan solution, volume ratios of chitosan/tripolyphosphate and weight ratios of arbutin/chitosan were systematically examined for their effect on the physicochemical properties of the nanoparticles which were characterized by the zeta sizer and transmission electron microscope (TEM). These nanoparticles presented high arbutin loading efficiency. The entrapment efficiency increased with increasing arbutin/chitosan weight ratios. This study demonstrated that complexation between chitosan and TPP forms stable cationic nanoparticles for subsequent arbutin loading. Key word : Arbutin, Chitosan nanoparticle, Ionotropic gelation Introduction Chitosan, a biodegradable polymer has shown favorable biocompatibility characteristics as well as the ability to increase membrane permibility, both in vitro and in vivo. It is prepared by the partial N-deacetylation of chitin, natural biopolymer derived from crustacean shells such as crabs, shrimps and lobsters. It is the second abundant polysaccharide and a cationic polymer present in nature. Chitosan is soluble at ph less than 6.5 in aqueous acids including formic, lactic, malic, malonic, propionic, pyruvic, succinic, nitric acid. It is insoluble in phosphoric and sulfuric acid. Thus, chitsan has attracted much attention in pharmaceutical and drug delivery. Arbutin is a derivative of hydroquinone that has been found in the leaves of various types of plants, most notably the bearberry plant (Arctostaphylas uva-ursi), cranberries and blueberries. Arbutin is also an inhibitor of melanin formation and is used in some skin-lightening products. Arbutin is readily hydrolyzed by diluted acids to yield D-glucose and hydroquinone. Arbutin is highly hydrophilic and hygroscopic substance. However, the formidable barrier property of stratum corneum and the high hydrophilicity of arbutin (log P value = -1.49) make it difficult to permeate through the skin and reach to its site of action (i.e. melanocytes). Liposome is previously formulated but it have many disadvantage such as low drug entrapment efficiency, poor reproducibility and stability. (1-2) Thus this research aimed to prepare arbutin nanoparticles from chitosan and to study the physicochemical properties of the obtained arbutin nanoparticles for controlling the absorption via the skin and protecting from degradation. The arbutin loaded chitosan nanoparticles were prepared by ionotropic gelation technique. The mechanism of chitosan nanoparticles formation is based on electrostatic interaction between the amine group of chitosan and the negatively charge group of sodium tripolyphosphate. This technique offers a simple and mild preparation method in the aqueous environment. (3-4) Materials and Experimental Procedures Chitosan with 90% degree of deacetylation and the molecular weight (MW) of 30,000-50,000 was obtained from Specialty Natural Products Co.,Ltd. Abutin and sodium tripolyphosphate were bought from Nam Siang Co.,Ltd. and Sigma Chemical Co. (USA), respectively Preparation of Chitosan Nanoparticles Arbutin loaded nanoparticles were prepared using chitosan as coating material and tripolyphosphate (TPP) as cross-linking agent. Firstly, chitosan was *Corresponding author Tel: ; Fax: ; pim_leela@hotmail.com

2 102 LEELAPORNPISID, P. et al. dissolved at differrent concentrations (0.1, 0.2, 0.3, 0.4 and 0.5%w/w) with 1% (v/v) acetic acid and then raised to ph 5-6 with 1 N NaOH. Sodium tripolyphosphate was separately dissolved in de-ionized water at concentration 0.3%w/w. Nanoparticles were formed instantaneously upon the dropwise addition of a different volume of TPP solution to a fixed volume of chitosan solution (chitosan to TPP weight ratios of 3:1, 4:1, 5:1, 6:1 and 7:1) The nanoparticle suspensions were gently stirred for 60 minutes at room temperature before characterization. Then the optimum condition of chitosan nanoparticle preparation was selected for loading arbutin and further investigated for entrapment efficacy, particle size and shape as well as releasing profile and stability. Physicochemical Characterization of Nanoparticles Chitosan nanoparticles, and arbutin-loaded chitosan nanoparticles were characterized by following measurements. Particle size and the zeta potential were determined using Zetasizer Nano-ZS (Malvern Instruments). The analysis was performed at a room temperature using samples diluted with de-ionized water. The morphological examination of nanoparticles was observed by transmission electron microscopy (TEM). The samples weren t stained and placed on copper grids,dried at room temperature for 30 minutes and then examined using a TEM. The chitosan nanoparticles which separated from suspension were also dried into freeze dried powder. Arbutin Entrapment Efficiency in Nanoparticles The arbutin entrapment efficiency was determined both indirect method and direct method by HPLC using BDS HYPERSIL C18 column ( mm, particle size 5 µm). The mobile phase was the mixture of methanol:h 2 O = 10:90(adjusted to ph 4.0 by adding phosphoric acid). The flow rate was 1.0 ml/min at ambient temperature, and the wavelength was set at 222 nm. Each 100 ul of the sample was injected. Resorcinol (5.0 µg/ml) was used as an internal standard. All the measurement were performed in triplicate. (5) Indirect Method The amount of arbutin entrapped into the nanoparticles was calculated by measuring the amount of arbutin in the supernatant after ultracentrifugation at 10,000 rpm, 4 C for 10 minutes. The determination of entrapment efficiency (%EE) was evaluated using the difference between initial amount of arbutin and untrapped amount in the supernatant. (6) Direct Method The freeze dried powder of arbutin nanoparticles (50 mg) were accurately weighed into a 50 ml volumetric flask. The arbutin entrapped into nanoparticles was then extracted with about 50 ml of H 2 O at ph 3 and sonicated for 1 hour. The homogeneous suspension was then diluted with deionized water until final concentration and mixed with resorcinol solution. (7) Arbutin Releasing from the Nanoparticles, in Vitro The in vitro release of entrapped arbutin was evaluated using the Franz Cell apparatus. Firstly, the arbutin-loaded chitosan nanoparticle was separated from the aqueous suspension medium through ultra-centrifugation. Then this was re-dispersed in 0.2 mol/l PBS solution (ph 5.5) and placed on the dialysis membrane(molecular weight cut-off of 1.2 kda) located in Franz cells. Each Franz cell was clamped and the PBS was also pipetted into receiver compartment. The assembled Franz cells were placed on a magnetic stirring block in a water bath. The entire system was kept at 32 C with continuous magnetic stirring. A volume of 300 ul of the receptor solution was withdrawn at appropriate time intervals and replaced with the same volume of fresh PBS. The amount of arbutin in the release medium was determined by HPLC. Thermal Stability of the Arbutin-Loaded Nanoparticles Every weight ratio of arbutin/chitosan nanoparticles was seperately kept at room temperature, 4 C and 45 C for 3 months and compared with the unentrapped arbutin powder. The amount of arbutin in each sample was then analyzed after 1 and 3 month storage at various conditions (45 C, room temp, 4 C). Results and Discussion Factors Influencing the Preparation of Chitosan Nanoparticles Effect of Chitosan Concentration on the Colloidal Properties of Chitosan Nanoparticles Figure 1 shows the influence of chitosan concentration on particle size and zeta potential

3 Application of Chitosan for Preparation of Arbutin Nanoparticles as Skin Whitening 103 values of nanoparticles. Particles size increased with the increasing of chitosan concentration due to the storage of cations inside the particles and the decreasing in particle surface area. But not significantly change in the zeta potential was observed. Effect of ph of Chitosan Solution on the Colloidal Properties of Chitosan Nanoparticles The dividing line between stable and unstable suspensions is generally taken at either +30mV or -30mV. Particles with zeta potentials more positive than +30mV or more negative than - 30mV are normally considered stable. The most important factor that affects zeta potential is ph. (8) The chitosan nanoparticles formed at solution ph 5.5 revealed the highest particle zeta potential where the colloidal system is the most stable and also at the range that arbutin is stable. (4) Chitosan concentration Figure 1. Chitosan concentration affecting on the particle size ( ) and zeta potential ( ) of Effect of Volume Ratios of Chitosan/TPP on the Colloidal Properties of Chitosan Nanoparticles There is no significantly difference of the particle size and zeta potential at all the volume ratio of chitosan/tpp more than 5:1. But at the lower volume ratios of chitosan/tpp, it demonstrated the excess of anions that affected to the decreasing of zeta potential, leading to flocculation that caused larger particle size. The results in Figure 2 shows that the optimum volume ratios of chitosan/tpp is 5: ph of chitosan solution Figure 3. ph of chitosan solution affecting on the particle size ( ) and zeta potential ( ) of Effect of Weight Ratios of Arbutin/Chitosan on the Colloidal Properties of Chitosan Nanoparticles The results in Figure 4 shows that all the weight ratio of core:polymer gave similar particle size and zeta potential. Therefore, the selection of optimal core:polymer for application is considered from the entrapment efficiency. Volume ratios of chitosan/tpp Figure 2. Volume ratios of chitosan/tpp affecting on The particle size ( ) and zeta potential ( ) of Weight ratio of core:polymer Figure 4. Weight ratio of arbutin/chitosan on the particle size ( ) and zeta potential ( ) of

4 104 LEELAPORNPISID, P. et al. Morphology of Arbutin Nanoparticles Figure 5 shows the spherical shape of arbutin loaded chitosan nanoparticles under TEM measurement which were about nm. But exhibited larger size when measured by zeta sizer ( nm) which was in the colloidal suspension stage. 200 nm chitosan concentration at ph 5.5, volume ratio of chitosan/tripolyphosphate as 5/1 and weight ratio of arbutin/chitosan as 10/1. The size and zeta potential of chitosan nanoparticles in colloidal suspension were in the range of nm and mv, respectively. Arbutin Releasing from the Nanoparticles, in Vitro Figure 6 displayed the release profile of arbutin from chitosan nanoparticles. The arbutin nanoparticles showed a slow release profile, suggest that the remaining arbutin entrapped inside the nanoparticles structure are tightly bound and entangled with the chitosan polymer chain. (10) Control Chitosan nanoparticles Figure 5. TEM image of arbutin chitosan nanoparticles prepared at weight ratio of arbutin: polymer =10:1. Determination of Arbutin Entrapment Efficiency in Nanoparticles In general, the indirect method provides the higher entrapment efficiency than direct method. The indirect method was to determine the difference between initial amount of arbutin and unentrapped amount in supernatant of the system, where the direct method was to determine the amount of arbutin that extracted from nanoparticles. However, the direct method was more believable than indirect method eventhough its weakness was always complication and time-consuming. (9) As shown in Table 1, entrapment efficiency of the prepared chitosan nanoparticles were varied between 6.47% and 40.89%, which depended on the ratios of arbutin/chitosan. Figure 6. Release profile of arbutin from nanoparticles. Thermal Stability of the Arbutin-Loaded Nanoparticles Stability of arbutin nanoparticles under various storage conditions (45 C, room temp, 4 C). As shows in Figure 6, the arbutin nanoparticles and arbutin were stable in their solid-state within 3 months in all tested conditions. Table 1. The influence of weight ratios of arbutin/ chitosan on entrapment efficiency in each method. core:polymer direct method indirect method 1:2 1:1 3:1 5:1 10: The optimum condition for arbutin nanoparticles preparation was obtained. This was by using 0.3% Figure 7. Effect of time and temperature on the stability of chitosan nanoparticles.

5 Application of Chitosan for Preparation of Arbutin Nanoparticles as Skin Whitening 105 Conclusions Optimum condition in arbutin loaded chitosan nanoparticles preparation was successfully obtained with small size ( nm), high zeta potential (30.87 mv) and highest loading efficiency (40.89%). Arbutin and arbutin nanoparticles was stable in their solid-state under various stability tested condition. The efficacy of arbutin nanoparticles as skin whitening will be further investigated. Acknowledgments The authors gratefully thank TRF-MASTER RESEARCH GRANTS, Thailand Reserch Fund (TRF) for financial support and Specialty Natural Products Co.,Ltd for supplying the chitosan. References 1. Wen, A.H., Choi, M.K., & Kim, D.D. (2006). Formulation of liposome for topical delivery of arbutin. Arch Pharm Res. 29(12) : Nation Toxicology Program. (2006). Chemical information review document for arbutin [CAS No ] and extracts from arctostaphylos uva-ursi. 7. Huang, S.C., Lin, C.C., Huang, M.C. & Wen, K.C. (2004). Simultaneous determination of magnesium ascorbyl phosphate, ascorbyl glucoside, kojic acid, arbutin and hydroquinone in skin whitening cosmetics by HPLC. J. Food and Drug Anal. 12(1) : Muller, R.H., Jacobs, C. & Kayser, O. (2001). Nanosuspensions as particulate drug formulations in therapy: Rationale for development and what we can expect for the future. Adv. Drug Deliver. Rev. 47(1) : Xu, X., Fu, Y., Hu, H., Duan, Y. & Zhang, Z. (2006). Quantitative determination of insulin entrapment efficiency in triblock copolymeric nanoparticles by high-performance liquid chromatography. J. Pharmaceut. Biomed. 41(1) : Gan, Q. & Wang, T. (2007). Chitosan nanoparticle as protein delivery carrier--systematic examination of fabrication conditions for efficient loading and release. Colloid. Surface. B 59(1) : Tiyaboonchai, W. (2003). Chitosan nanoparticles : A promising system for drug delivery. Naresuan University Journal. 11(3) : Gan, Q., Wang, T., Cochrane, C. & McCarron, P. (2005). Modulation of surface charge, particle size and morphological properties of chitosan-tpp nanoparticles intended for gene delivery. Colloid. Surface. B 44(2-3) : Thongchai, W. (2005). Development of high performance liquid chromatography method for the determination of arbutin on creams and medicinal plant extracts. Master Thesis, Chiang Mai University,Chiangmai. 6. Wu, Y., Yang, W., Wang, C., Hu, J. & Fu, S. (2005). Chitosan nanoparticles as a novel delivery system for ammonium glycyrrhizinate. Int. J. Pharm. 295(1-2) :