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2. Materials and methods
2.1. Materials
The chitosan used in this study to prepare the CS¨CMMTnanohydrogels was supplied by Aldrich¨CSigma and usedwithout purification. The same type of chitosan was usedby Darder et al., who reported that it has an averagemolecular weight of 342,500 g mol1 and a deacetylationdegree (DD) of ca. 75% [14]. Acetic acid and sodium phosphatefor the preparation of buffers were purchased fromAldrich Chemicals. Vitamin B12 (Sigma¨CAldrich Co.) waschosen as a model molecule to characterize the releasebehavior from the nanohydrogel. Na+-montmorillonite,supplied by Nanocor Co., is an Na+ form of layered smectiteclay with a cationic exchange capacity (CEC) of120 meq. (100 g)1. The MMT platelet shows a surfacedimension of about 200¨C500 nm in length and several tensof nanometers in width.2.2. Preparation of CS¨CMMT nanohydrogelsTo prepare the CS¨CMMT nanohydrogels, the preparationprocedure is separated into two stages. The first stageis to prepare a suspension containing MMT and CS with aweight ratio of 1:2 (where the CS solution was prepared bydissolving predetermined amounts of CS in 1 wt.% aceticacid solution and stirring for about 4 h until the CS wascompletely dissolved). The CS¨CMMT suspensions wereobtained by adding CS to an aqueous solution containing2 wt.% MMT (i.e. 0.5 g of Na+-MMT dispersed in 25 mlof double-distilled water), followed by stirring at 50 Cfor 24 h. To enhance the formation of exfoliation of theMMT in the final nanohydrogel, the suspension with aCS to MMT ratio of 2:1 was then subjected to ball-millingfor 24 h, after which the as-prepared final CS¨CMMT suspensionwas used to form nanohydrogel.In the second stage of the CS¨CMMT nanohydrogelpreparation, 2 wt.% CS solution was obtained by dissolvingCS in 1 wt.% acetic acid solution. A small amount ofthe ball-milled CS¨CMMT suspension was then added tothe prepared CS solution to form a CS-rich suspension,with the MMT content controlled in the range of 1, 2, 3and 4 wt.%, relative to the total weight of CS in the suspensions,under continuous stirring at 60 C for 1 h. This finalsuspension was then cast onto Petri dishes and dried at30 C for 24 h, to form final dried nanohydrogels. Thedried nanohydrogels were then rinsed with an aqueoussolution of 1 M NaOH to remove any residual acetic acid,followed by washing with distilled water and drying for 1week at 40 C in vacuum until use. The compositions ofthe nanohydrogels are expressed using the value of n todefine the content of MMT in CS¨CMMTn, wheren = C mmt, the content of the MMT incorporated in thenanohydrogels, which ranged from 1% to 4%.2¡£ ²ÄÁϺͷ½·¨
2. 1 ²ÄÁÏ
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