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Journal of Microencapsulation, February 2006; 23(1): 7990

Characteristics of vitamin C encapsulated tripolyphosphate-chitosan microspheres as affected by chitosan molecular weight


K. G. DESAI1, C. LIU1, & H. J. PARK1,2
1 2

School of Life Sciences and Biotechnology, Korea University, Seoul, South Korea and Clemson University, Clemson, SC, USA

(Received 17 January 2005; in final form 10 May 2005)

Abstract In this paper, the effect of chitosan molecular weight on the characteristics (size, encapsulation efficiency, zeta potential, surface morphology and release rate) of vitamin C encapsulated tripolyphosphate cross-linked chitosan (TPP-chitosan) microspheres. The molecular weight of chitosan had a noticeable influence on the size, encapsulation efficiency, zeta potential, surface morphology and controlled release behaviour of the vitamin C encapsulated TPP-chitosan microspheres. The mean particle size and encapsulation efficiencies of TPP-chitosan microspheres were 3.1, 4.9 and 6.7 mm and 67.25, 60.43 and 52.74% for the microspheres prepared using low, medium and high molecular weight chitosan, respectively. All the TPP-chitosan microspheres (low, medium and high molecular weight) had positive charge on their surface. The zeta potential of the TPP-chitosan microspheres prepared using low, medium and high molecular weight chitosan was 41.25, 40.84 and 39.13 mV, respectively. The particle sizes of TPP-chitosan microspheres increased with increases in chitosan molecular weight. Molecular weight of chitosan did not affect significantly the % yield of TPP-chitosan microspheres prepared by spray-drying. The influence of chitosan molecular weight on the surface morphology of vitamin C encapsulated TPP-chitosan microspheres was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). It was observed that, as the molecular weight of chitosan increases, TPP-chitosan microspheres with uniform spherical shape could be obtained. The physical state of vitamin C (amorphous or crystalline) in TPP-chitosan matrix was studied by X-ray diffraction (XRD) and it was found that vitamin C is dispersed at the molecular level (amorphous) in the TPP-chitosan matrix. Release rate of the vitamin C from TPP-chitosan microspheres was significantly affected by the chitosan molecular weight. The release rate decreased with increase in the chitosan molecular weight. The release of vitamin C from TPP-chitosan microspheres followed Ficks law of diffusion. Keywords: Vitamin C, chitosan, molecular weight, encapsulation, microspheres, tripolyphosphate, controlled release

Correspondence: Hyun Jin Park, School of Life Sciences and Biotechnology, Korea University, 1, 5-Ka, Anam-Dong, Sungbuk-ku, Seoul-136-701, South Korea. Tel: 82-2-3290-3450. Fax: 82-2-953-5892. E-mail: hjpark@korea.ac.uk ISSN 0265-2048 print/ISSN 1464-5246 online 2006 Taylor & Francis DOI: 10.1080/02652040500435360

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Introduction The food industry is taking advantage of the technology of controlled release for food additives including flavouring agents (flavour oils, spices, seasoning), sweeteners, colours, nutrients (vitamins, amino acids, minerals), essential oils, acids, salts, bases, anti-oxidants, anti-microbial agents, preservatives and ingredients with undesirable flavours (Kirby 1991, Augustin et al. 2001, Desai and Park 2005). Controlled release helps to overcome both the ineffective utilization and the loss of food additives during the processing steps novas and Pothakamury 1995). The most commonly used method to achieve (Barbosa-Ca controlled release in the food industry is microencapsulation (Kirby 1991, Augustin et al. 2001). Microencapsulation is defined as the technology of packaging solid, liquid or gaseous materials in miniature sealed capsules that release their contents at controlled rates under the influence of certain stimuli. The advantages of controlled release of food ingredients are: (a) The active ingredients are released at controlled rates over prolonged periods of time; (b) Loss of ingredients, such as vitamins and minerals, during processing and cooking can be avoided or reduced; and (c) Reactive or incompatible components can be separated (Taylor 1983, Shahidi and Han 1993). Vitamin C is an essential nutrient involved in many physiologic functions. Nearly all species of animals synthesize vitamin C and do not require it in their diets, but humans cannot synthesize the vitamin C (Farajzadeh and Nagizadeh 2003). For practical purposes, raw citrus fruits are good daily sources of vitamin C, since appreciable amounts in other foods can be destroyed when cooked, in the presence of air and when in contact with traces of copper (Kirby 1991, Farajzadeh and Nagizadeh 2003). Therefore, vitamin C is widely used in various types of foods as a vitamin supplement and as an anti-oxidant (Jacobs et al. 2001). Vitamin C is distributed throughout the body with high concentrations in specific tissues, such as lungs, white blood cells, pancreas, thyroid, spleen, brain, etc. This is an important anti-oxidant that may reduce the risk of cancer by neutralizing reactive oxygen species or other free radicals that can damage DNA (Jacobs et al. 2001). Since vitamin C is responsible for many biochemical functions in the human body, it is mainly supplied by the diet. In addition, vitamin C is also utilized in food or food additives and has been shown to decrease LDL cholesterol in the plasma of hyper-lipidemia patients (Snyder and Malloy 1998, Jeserich et al. 1999, Mosinger 1999). Hence, microencapsulation and controlled release of vitamin C would broaden its application range in food and pharmaceutical industries. The techniques used for microencapsulation are spray-drying, coating, extrusion, liposome entrapment, coacervation and freeze drying. Of these methods, spray-drying is the most commonly used method in the food industry. This is because the process is economical, flexible in that it offers substantial variation in the encapsulation matrix, adoptable to commonly used processing equipment and produces particles of good quality novas and Pothakamury 1995, Augustin et al. 2001). (Shahidi and Han 1993, Barbosa-Ca The spray-drying method is a one-stage continuous process, easy to scale-up and spraydrying production costs are lower than those associated with most other methods of microencapsulation (Taylor 1983, Desai and Park 2005). The microcapsule sizes prepared by the spray-drying method ranged from microns to several tens of microns and had a relatively narrow distribution. Hence, it is a method of choice for the microencapsulation of food ingredients in the food industry.

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The food additive (active agent) can be encapsulated using carbohydrates, gums, lipids, proteins, polymers such as poly(vinyl) acetate (PVA), a fibre matrix made of polymers and/or novas and Pothakamury 1995). Because few suitable polymers have liposomes (Barbosa-Ca been approved for use in foods, certain food materials can be modified to increase their porosity and to alter other characteristics, thus enabling their use as coating materials in microencapsulation. Chitosan is a hydrophilic, biocompatible and biodegradable polysaccharide of low toxicity which in recent years has been used for development of oral drug delivery systems (Hejazi and Amiji 2003). Since it is a well-known dietary food additive, it was selected as an appropriate encapsulating material for the vitamin C. However, the encapsulating ability of chitosan varies with its molecular weight. Therefore, in continuation of an ongoing programme of research on microencapsulation of vitamin C in a chitosan-based matrix (Desai and Park 2005), this paper describes the effect chitosan molecular weight has on the characteristics (size, encapsulation efficiency, zeta potential, surface morphology and release rate) of thus prepared vitamin C encapsulated TPP-chitosan microspheres.

Materials and methods Materials Vitamin C (99.5% purity) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Chitosan (low, medium and high molecular weight) and tripolyphosphate were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). All other chemicals were of analytical grade and used as received. Ultrapure water (Millipore, USA) water was used throughout the study. Methods Determination of molecular weight of chitosan. The average molecular weight of chitosan was determined by a batch mode method using a multi-angle laser light scattering (MALLS) photometer according to Chen and Tsaih (1998). Every sample was repeated five times in an identical manner. The obtained values are presented in Table I. Determination of degree of deacetylation of chitosan. The % N-deacetylation of chitosan was determined by the 1NMR spectroscopy method (Hirai et al. 1991, Lavertu et al. 2003). The obtained values are presented in Table I.
Table I. Viscosity, molecular weight and degree of deacetylation of chitosan used for the microencapsulation of vitamin C. Viscosity (cps) 20 200 800 Molecular weight 7.270 105 1.336 106 1.743 107 DOD (%) 84.94 82.10 81.29

Chitosan Low Mw Medium Mw High Mw


Brookfield viscosity provided by Aldrich. Degree of N-deacetylation of chitosan.

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Preparation of vitamin C encapsulated TPP-chitosan microspheres. Microencapsulation of vitamin C in a TPP-chitosan matrix was achieved by the spray-drying technique as described previously (Desai and Park 2005). Briefly, the required volume (usually 250 ml) of chitosan (low or medium or high molecular weight) solution (1% w/v) was prepared using aqueous acetic acid solution (0.5% v/v). Vitamin C (2.5 g) was dissolved in 10 ml of ultrapure water (Millipore, USA). The vitamin C solution was then added to the chitosan solution and homogenized at 8000 rpm for 10 min using a Young Ji HMZ 20DN stirrer (Hana Instruments). To the above prepared mixture, 10 ml of 1% w/v aqueous solution of TPP was added dropwise under constant stirring at 8000 rpm for 20 min using a Young Ji HMZ 20DN stirrer. TPP was used as a cross-linking agent. Spray-drying was then performed using a SD-04 spray drier (Lab Plant, UK), with a standard 0.5 mm nozzle. Inlet temperature, liquid flow rate and compressed spray air flow were set at 170 C, 2 ml min1 and 10 l min1, respectively. Encapsulation efficiency. Twenty five milligrams of vitamin C encapsulated TPP-chitosan microspheres were dissolved in 100 ml 0.1 N HCl. The sample was passed through a 0.2 mm filter (Millipore, USA) and then vitamin C content was assayed by measuring the absorbance at 244 nm (lmax of vitamin C in 0.1 N HCL) after suitable dilution using an UV spectrophotometer (Shimadzu 1601PC, Japan). Experiments were performed in triplicate (n 3) and encapsulation efficiencies were calculated as follows. Encapsulation efficiency (%) = Calculated vitamin C concentration 100 Theoretical vitamin C concentration 1

Measurement of particles size. TPP-chitosan microspheres prepared by spray-drying exhibited quick swelling in liquid medium and, hence, sizes could not be determined using a laser diffraction technique in a particle size analyser. Therefore, the particle size was determined by microscopy. Briefly, in each determination, $5 mg of chitosan microspheres were taken on a glass slide and sizes of $100 particles were measured by using biological microscope (Olympus, Japan). Mean size of 100 particles was considered as size of the microspheres from one determination and it was repeated for three times and then mean particle size of the microspheres was calculated from three independent determinations. Zeta potential. TPP-chitosan microspheres concentration of 0.3% w/v was made by dispersing microspheres in KCl solution (pH 7). The zeta potential of TPP-chitosan microspheres was recorded using laser doppler anemometry (Malvern Zetasizer, UK). Zeta potential measurements were done in triplicate. Scanning electron microscopy. The surface morphology of vitamin C encapsulated TPP-chitosan microspheres was examined by means of an Hitachi (Japan) scanning electron microscope. The powders were previously fixed on a brass stub using double-sided adhesive tape and then were made electrically conductive by coating, in a vacuum, with a thin layer of platinum ($35 nm), for 100 s and at 30 W. The pictures were taken at an excitation voltage of 15 kV and a magnification of 8.0, 1.2, 3.0 or 4.5 k. Transmission electron microscopy. The shape of vitamin C encapsulated TPP-chitosan microspheres was examined by TEM. TPP-chitosan microspheres sample was added on a formvar coated grid. The shape of the microspheres was examined using a Philips TECNAI 12 transmission electron microscope (The Netherlands) at an accelerating voltage of 120 kV.

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XRD. X-ray powder diffraction patterns of pure vitamin C and vitamin C encapsulated TPP-chitosan microspheres prepared using different molecular weight chitosan were obtained at room temperature using a Philips X Pert MPD diffractometer (Philips, The Netherlands), with Co as anode material and graphite monochromator, operated at a voltage of 40 kV. The samples were analysed in the 2 angle range of 260 and the process parameters were set as: scan step size of 0.025 (2), scan step time of 1.25 s and time of acquisition of 1 h. In vitro release studies. The in vitro release of vitamin C from TPP-chitosan microspheres was determined using a USP model dissolution apparatus (TW-SM, Wooju Scientific, Co., Korea). In order to suspend the microspheres in the dissolution medium, microspheres equivalent to 25 mg of vitamin C were taken into a cellulose dialysis bag (previously soaked in dissolution medium) containing 3 ml of dissolution medium and tied to the paddle. The in vitro release studies of vitamin C were carried out at a paddle rotation of 100 rpm in 500 ml of phosphate buffer (pH 7.4 and 25 C). In order to facilitate compatible environment for released vitamin C, the temperature of the dissolution medium was maintained at 25 0.1 C until the end of the study. An aliquot of the release medium (5 ml) was withdrawn through a sampling syringe attached with a 0.2 mm filter at pre-determined time intervals (0.5, 1, 2, 3, 4, 5 and 6 h) and an equivalent volume of fresh dissolution medium which was pre-warmed at 25 C was replaced. Collected samples were analysed for vitamin C content by measuring the absorbance at 265 nm using UV spectrophotometer (Shimadzu 1601PC, Japan). In vitro release studies were performed in triplicates (n 3) in an identical manner.

Results and discussion Microencapsulation of vitamin C in low, medium and high molecular weight chitosan microspheres Chitosan, a natural linear biopolyaminosaccharide, is obtained by alkaline deacetylation of chitin, which is the second abundant polysaccharide next to cellulose. Properties such as biodegradability, low toxicity and good biocompatibility make it suitable for use in food and pharmaceutical formulations. Chitosan microspheres are the most widely studied drug delivery systems for the controlled release of drugs viz. antibiotics, anti-hypertensive agents, proteins, peptides and vaccines (Hejazi and Amiji 2003). Due to the easy availability of free amino groups in chitosan, it carries a positive charge and, thus, in turn reacts with many negatively charged surfaces/polymers. Chitosan can form gels by interacting with different types of divalent and polyvalent anions. Using this property, many researchers have prepared cross-linked chitosan microspheres or films to sustain the release of drugs. The word chitosan refers to a large number of polymers, which differs in their degree of N-deacetylation (4098%) and molecular weight (50 0002 000 000 Da). These two characteristics are very important to the physico-chemical properties of the chitosans and, hence, they have a major effect on the biological properties (Sannan et al. 1976). The molecular weight of chitosan has a remarkable influence on the size, encapsulation efficiency, zeta potential, surface morphology and controlled release behaviour of drugloaded microspheres (Hejazi and Amiji 2003). It is, therefore, necessary to establish the effect of chitosan molecular weight on the characteristics of thus prepared vitamin C encapsulated TPP-chitosan microspheres. Hence, this paper mainly focused on the influence of chitosan molecular weight on the properties of vitamin C encapsulated

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TPP-chitosan microspheres. Such information will help a food formulator while choosing the appropriate chitosan for the encapsulation of vitamin C. Effect of chitosan molecular weight on size, % yield, encapsulation efficiency and zeta potential of vitamin C encapsulated TPP-chitosan microspheres Three chitosan materials (low, medium and high molecular weight) were used in the present study. The effect of molecular weight of chitosan on the size, % yield, encapsulation efficiency and zeta potential of TPP-chitosan microspheres is presented in Table II. The results of the present study indicated that the size, encapsulation efficiency and zeta potential were remarkably influenced by the molecular weight of chitosan. For instance, the size of the vitamin C encapsulated TPP-microspheres prepared using low, medium and high molecular weight chitosan was 3.1, 4.9 and 6.7 mm, respectively. As the molecular weight of chitosan increases, size of the TPP-chitosan microspheres increased. It is well-known that the viscosity of the polymer solution increases with increase in its molecular weight. Therefore, under the same preparation conditions, the droplets formed from the higher viscosity chitosan solution will be larger in size and result in larger microspheres. Pavenetto et al. (1993) prepared PLA microspheres by a spray-drying method. At same polymer concentration (1.25%), the particles size was similarly increased with polymer molecular weight. In the preset study, the encapsulation efficiencies of the TPP-chitosan microspheres ranged from 52.7467.25%. Encapsulation efficiency decreased with increase in the molecular weight of chitosan. This is in agreement with the report of Filipovic-Grcic et al. (2003), where the encapsulation efficiency of spray-dried chitosan microspheres decreased for carbamazepine with increases in chitosan molecular weight. The influence of chitosan molecular weight on the surface charge of vitamin C encapsulated TPP-chitosan microspheres was determined by measuring the zeta potential. The zeta potential of the vitamin C encapsulated TPP-chitosan microspheres did not vary significantly with increase in the molecular weight of chitosan. However, zeta potential of vitamin C encapsulated TPP-chitosan microspheres decreased slightly with an increase in the molecular weight of chitosan. Effect of chitosan molecular weight on the surface morphology of vitamin C encapsulated TPP-chitosan microspheres The surface morphology of vitamin C encapsulated TPP-chitosan microspheres prepared using low, medium and high molecular weight chitosan is presented in Figure 1. It can be emphasized that non-cross-linked chitosan microspheres did not exhibit a smooth surface (Figure 1a). Non-cross-linked chitosan microspheres had wrinkles on their surface. Vitamin C encapsulated TPP-chitosan microspheres prepared using low, medium and high molecular weight chitosan exhibited a smooth surface. This can be clearly observed from
Table II. Effect of chitosan molecular weight on particle size, encapsulation efficiency, % yield and zeta potential of vitamin C encapsulated TPP-chitosan microspheres (M S.D., n 3). Molecular weight of chitosan 7.270 105 1.336 106 1.743 107 Mean particle size (mm) 3.1 0.2 4.9 0.4 6.7 0.3 Encapsulation efficiency (%) 67.25 2.6 60.43 1.5 52.74 1.3 Zeta potential (mV) 41.25 3.4 40.84 1.3 39.13 1.5

Yield (%) 59.26 1.4 60.56 1.8 58.46 2.1

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(a)

(b)

(c)

(d)

Figure 1. Scanning electron microscopic pictures of non-cross-linked non-loaded chitosan microspheres (a) and vitamin C encapsulated TPP-chitosan microspheres prepared using low (b), medium (c) and high (d) molecular weight chitosan (volume of 1% w/v TPP solution employed for cross-linking process is 10 ml).

the SEM pictures (Figure 1b, c and d). However, TPP-chitosan microspheres prepared using medium and high molecular weight chitosan depicted more uniform spherical shape (Figure 1c and d) than those prepared using low molecular weight chitosan (Figure 1b). In other words, with increase in the viscosity of the chitosan, TPP-chitosan microspheres with uniform spherical shape could be prepared by a spray-drying method. Some of the porous TPP-chitosan microspheres were formed from low molecular weight chitosan (Figure 1d). Shape of the vitamin C encapsulated TPP-chitosan microspheres was further confirmed by TEM and the pictures are presented in Figure 2. It can also be noticed that uniform spherical shape microspheres could be obtained from even non-cross-linked chitosan (Figure 2a). In the case of TPP-chitosan microspheres, medium and high molecular weight chitosan microspheres (Figure 2c and d) were having a more uniform spherical shape than the low molecular weight chitosan microspheres (Figure 2b). Physical state of vitamin C in TPP-chitosan matrix The physical state of the core material (crystalline or amorphous) in the polymeric matrix can be studied by XRD studies (Filipovic-Grcic et al. 2003). Therefore, in this paper,

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(a)

(b)

(c)

(d)

Figure 2. Transmission electron microscopic images of non-cross-linked non-loaded chitosan microspheres (a) and vitamin C encapsulated TPP-chitosan microspheres prepared using low (b), medium (c) and high (d) molecular weight chitosan (volume of 1% w/v TPP solution employed for cross-linking process is 10 ml).

the physical state of the vitamin C (crystalline or amorphous) in TPP-chitosan matrix was studied by XRD studies. The X-ray diffractograms of pure vitamin C and vitamin C encapsulated TPP-chitosan microspheres prepared using different molecular weight chitosan are presented in Figure 3. From Figure 3(a), it is evident that vitamin C exhibited characteristic crystalline peaks at 2 of 10.3, 14.09, 17.3, 25.24, 40.29, 48.19 and 54.3, indicating the presence of crystalline vitamin C. The characteristic crystalline peaks of vitamin C disappeared after microencapsulation in the TPP-chitosan matrix prepared using low, medium and high molecular weight chitosan (Figure 3b, c and d), but instead only placebo TPP-chitosan microspheres pattern was obtained. This indicates that vitamin C

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2
Figure 3. XRD spectra of pure vitamin C (a) and vitamin C encapsulated TPP-chitosan microspheres prepared using low (b), medium (c) and high (d) molecular weight chitosan (volume of 1% w/v TPP solution employed for cross-linking process is 10 ml).

is dispersed at the molecular level (amorphous form) in the TPP-chitosan matrix and, hence, no crystals were found in the vitamin C encapsulated TPP-chitosan microspheres. Effect of chitosan molecular weight on the release rate of vitamin C from TPP-chitosan microspheres The effect of chitosan molecular weight on the release rate of vitamin C from TPP-chitosan microspheres having the same cross-linking density is depicted in Figure 4. It is evident that the TPP-chitosan microspheres prepared using low molecular weight chitosan (i.e. 7.270 105) exhibited a higher vitamin C release rate than those prepared using medium (1.336 106) and high (1.743 107) molecular weight chitosan. These results are in agreement with the reports of Al-Helw et al. (1998). Although the volume of TPP solution (10 ml) used in all the preparations for cross-linking reaction was the same, the vitamin C release rates were remarkably different. The chain length between cross-links in the higher molecular weight chitosan is expected to be quite long, whereas the lower molecular weight chitosan possesses smaller free chains. Thus, in the microspheres with the longer chain segments can be held responsible for the slower release rate of vitamin C from the higher molecular weight chitosan microspheres. In addition, lower release rates of vitamin C with increase in the molecular weight of chitosan can also be attributed to the increased viscosity of the chitosan. This behaviour was predictable, taking into the account the relationship between the molecular weight and viscosity (Adusumilli and Bolten 1991). By increasing the viscosity of the chitosan, the diffusion of the vitamin C through the gel into

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the release medium was retarded. Chitosan with higher molecular weight has more acetyl groups on molecular chain. The presence of acetyl groups leads to significant decrease in charge density as well as its intra-molecular hydrogen bonds and makes the chitosan molecules exist in an extended form. So, high deacetylated degree molecules tend

100

80

Cumulative release (%)

60

40 Low molecular weight Medium molecular weight High molecular weight

20

0 0 1 2 3 4 5 6

Time (h)
Figure 4. The influence of chitosan molecular weight on the release rate of vitamin C from TPPchitosan microspheres (volume of 1% w/v TPP solution employed for cross-linking process is 10 ml).

120 y = 45.919x - 10.27 100 R2 = 0.9641 y = 44.03x - 13.827 80 R2 = 0.9828 y = 40.991x - 14.513 60 R2 = 0.9848

Cumulative release (%)

40 Low molecular weight 20 Medium molecular weight High molecular weight 0

0.5

1.5

2.5

Time

(h1/2)

Figure 5. Higuchi plot of vitamin C encapsulated TPP-chitosan microspheres prepared using different (low, medium and high) molecular weight chitosan.

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to be a random coil in the solution, which will result in less entanglement and weaken the structure to hold the vitamin C. Therefore, microspheres prepared from higher deacetylated degree chitosan were higher in release percentage than those prepared with lower deacetylated degree chitosan. Mechanism of vitamin C release from TPP-chitosan microspheres In the present study, an effort has been made to understand the mechanism of vitamin C release from chitosan microspheres prepared using different molecular weight chitosan. In order to understand the release mechanism, the release study data obtained were fit to the Higuchi equation (Bravo et al. 2004). Q k H t 1=2 2

where Q is the amount of vitamin C release at time t, kH the Higuchi rate constant. The dissolution data were plotted as the percentage of vitamin C release against the square root of time (see Figure 5). Linearity was observed with the plots since the correlation coefficient (R2) ranged from 0.96410.9848. However, good correlation (R2) 0.9848 was observed with high molecular weight chitosan than those of medium (0.9828) and low (0.9641) molecular weight chitosan. This indicates that the release of vitamin C from TPP-chitosan microspheres followed Ficks law of diffusion.

Conclusions The influence of chitosan molecular weight on the characteristics of vitamin C encapsulated TPP-chitosan microspheres was thoroughly studied. The study indicated that molecular weight of chitosan had a noticeable influence on the size, encapsulation efficiency, zeta potential, surface morphology and release rate of the TPP-chitosan microspheres. The particle size increased with increase in the molecular weight of chitosan. Encapsulation efficiencies and release rate decreased with increase in the molecular weight of the chitosan. The vitamin C could be homogeneously dispersed (amorphous form) in the TPP-chitosan matrix by the spray-drying method. Vitamin C release from TPP-chitosan microspheres followed Ficks law of diffusion.

Acknowledgements This study was supported by a grant of the Korea Health 21 R & D Project, Ministry of Health & Welfare, Republic of Korea (A050376).

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