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Calcium ion modulates protein release from chitosan-hyaluronic acid poly electrolyte gel.

INTRODUCTION

Polymeric architectures responsive to local stimuli have enabled extensive applications in areas such as drug delivery, scaffolds for tissue engineering, antibacterial surfaces, and as highly selective sensors. In spite of the availability of wide range of synthetic materials, attention has been drawn to biopolymers to mold components to suit the required applications. Again emphasis has been given to generate appropriate materials by assembling through physical interaction such as H-bonding and electrostatic interaction considering the fact that toxic liability may arise from moieties used for stabilizing the architecture through covalent linking.

Scaffold design and fabrication are major areas of biomaterial research during the past two decades [1], Their function is to provide mechanical support, promote cell-biomaterial interaction, cell adhesion, extra cellular matrix (ECM) deposition etc. [2]. Scaffolds have also been referred as regeneration templates as they induce formation of tissues and organs that do not revive spontaneously [3], Biopolymers such as CH, HA, gelatin etc. are used to develop scaffolds for skin and cartilage regeneration owing to their multiple bioactivities that are beneficial for embedded cells since they contain cell-specific domains like RGD (Arg-Gly-Asp) [4-8], Detailed reviews on biopolymer hydrogels as tissue engineering scaffolds are given by Van et al. [9] and Dhandayuthapani et al. [3].

Chitosan [poly (1, 4-jS-D-glucopyrosamine)], 70-80% N-deacetylated derivative of chitin is the most abundant polysaccharide (PS) in nature. The exoskeleton of marine arthropods like shrimp, lobster, and crabs are the main industrial sources of CH [10], Since CH is biodegradable, biocompatible, and nontoxic it has received considerable attention in various fields of pharmaceutical technology [11, 12], Excellent properties of CH, which make it a promising biomaterial for tissue engineering are highlighted in a review by Muzzarelli et al. [13]. In recent years enhanced attention has been given to CH based materials and their applications in the field of orthopedic tissue engineering. Interesting characteristics that render CH suitable for this purpose are minimal foreign body reaction, its intrinsic antibacterial nature and ability to be molded in various geometries [14, 15],

HA is a naturally occurring linear polysaccharide with a high molecular weight. It has a repeating disaccharide structure, consisting of 2-acetamide-2-deoxy-[beta]-D-glucose and [beta]-D-glucuronic acid residues, linked by alternating (1-3) and (1-4) glycoside bonding. HA is a component of extracellular matrix of all higher animals, which influences several cellular functions, such as migration, adhesion, and proliferation. It occurs naturally in the synovial fluid that surrounds the joints [16]. It has been recently reported that HA increases osteoblastic bone formation in vitro. Recent biomedical applications of HA include as a component in implant materials, ophthalmic surgery, arthritis treatment, scaffolds for wound healing, tissue engineering, and drug delivery applications [17, 18].

The aforementioned favorable properties along with the poly cationic and poly anionic characteristics of CH and HA respectively make their combination (PEC) a very versatile and functional scaffold for tissue engineering. Recently, it is reported that CH/HA matrices have a pore network configuration and exhibit enhanced ECM cartilage formation, thus proved to have potential use in cartilage repair [19-21]. The mechanical, thermo-physical, and biological assays of these scaffolds are studied in detail by Nazhat and Showan [22]. Tan et al. reported on the development of a composite hydrogel derived from water-soluble CH and oxidized HA without the addition of a chemical crosslinking agent [6]. On the other hand, Xu et al. used films cast by mixing CH and HA solutions together in different ratios for wound dressing applications [23]. In various reports Choi et al. and Kim et al. followed the same method for the preparation of PEC of CH and HA and studied the response of these films in various external pH conditions [24-26].

Approaches based on specific microenvironment created by disease/infection as molecular cue to activate drug release have attracted much interest in designing "drug on demand" delivery systems. The ageing populations and the increasing occurrence of osteoporosis indicate that the repair of bone defects and fractures will be a major challenge. Biocompatible materials, which could respond to the local concentration of [Ca.sup.2+] seems to have potential in repairing bone defects by rapidly releasing drugs/ growth factors to the site. In this study, FITC conjugated to BSA has been incorporated into the CH-HA for release studies. Fluorescent BSA conjugates have been used for quantitative studies of electroporation [27], the rate of receptor-mediated endocytosis and exocytosis [28],

Our aim in this study is to check the fate of CH-HA complex in the presence of [Ca.sup.2+] and also to look into role of ionic environment on the release of the incorporated drug. Napier and Haider [29], with the help of [C.sup.13] NMR thoroughly investigated conformational status of HA in the presence of varied quantities of [Ca.sup.2+]. Their observation suggest that flexibility of HA chains increased subsequent to the addition of [Ca.sup.2+]. They also observed that changes are minimal when [Na.sup.+] ions are added reflecting that [Ca.sup.2+] has a significant role in influencing the structural features of HA. Gesssler et al. have studied the effect of [Na.sup.+] and [Ca.sup.2+] ions on the thermodynamics of HA solutions [30], Their study indicated that [Ca.sup.2+] form a tight sheath around the polymer backbone while [Na.sup.+] ions are distributed in a diffuse cloud. The sheath, to a considerable extent can influence the interaction between the polymer chains and the surrounding hydration layer. Kim et al. have reported that the equilibrium swelling ratio of CH-HA decreased with increase in the NaCl concentrations, which they attributed to the association state of the ionic groups within the polymer and the affinity of the complex for water [31].

On implantation, these scaffolds come in contact with [Ca.sup.2+] especially when they are used in applications related to cartilage and bone surgery. The behavior of CH-HA in the presence of [Ca.sup.2+], which can significantly affect their morphology has not been studied seriously so far. Along with [Ca.sup.2+], we also studied the effect of [Na.sup.+] ions considering the exposure of biopolymers to this prominent monovalent cation at physiological conditions.

EXPERIMENTAL

Materials

CH (75% deactivated, molecular weight 195 kDa), HA, FITC, and BSA were purchased from sigma Aldrich Bangalore, India. All other reagents used were of analytical grade from SD Fine Chemicals, Mumbai, India.

Preparation of Chitosan-Hyaluronic Acid Poly Electrolyte Complex (CH-HA)

CH (2.5%) in 2% acetic acid was prepared by dissolving 2.5 g of CH in 100 mL, 2 % (v/v) aqueous acetic acid by continuous stirring at room temperature using a mechanical stirrer (IKA RW 20 digital, India) at 700 rpm for 8 h. HA (1 g) was dissolved in 100 mL distilled water by magnetic stirring to form 1% solution. PEC was prepared by physical mixing of CH and HA in 1:5 ratio at room temperature for 15 mts.

CH-HA formed was deep freezed at -80[degrees]C overnight followed by lyophilization at -84[degrees]C, 0.31 mbar for two days using a Lyophilizer (Martin Christ, Alpha 1-4, Germany). Before placing the sample, the lyophilizer was set in the freezing mode till temperature comes below -40[degrees]C and the vacuum pump was kept for 20 mts warming up.

For crosslinking, 2 g of the lyophilized CH-HA was immersed in 4 ml of EDC/NHS solution (20 rnM EDC and 8 mM NHS in 90% ethanol v/v), overnight. The cross-linked matrix was dried at room temperature.

Conjugation of FITC to BSA

Two hundred fifty microliter FITC (1 mg/ml) was added to 5 ml of BSA (2 mg/ml) and kept overnight. The resulting solution was then dialyzed against DMSO followed by distilled water.

Preparation of PEC Containing FITC Conjugated BSA

FA (1.5 ml) (BSA conjugated FITC) was added to 20 ml, 1% HA solution, and 4 ml of 2.5% CH solution. PEC containing FA was dried at 37[degrees]C in a vacuum oven (Rays Scientific Instruments, India). The quantity of loaded FA was estimated to be 316 pg/g weight of the PEC.

Characterization

Fourier Transform Infrared Spectroscopy. The FTIR spectra were recorded in the mid IR range 600-4000 [cm.sup.-1] using Nicolet 5700 FTIR Spectrometer (Madison, WI) in conjunction with a Diamond-ATR accessory. Before analysis the diamond ATR crystal was cleaned with isopropanol. Background spectrum was measured, which will automatically be subtracted from the sample spectrum by the spectrum analysis program. After placing the sample on the crystal, the pressure arm was positioned over the sample area and locked into a precise position above the diamond crystal so that on applying force the sample was fully in contact with the crystal. DTGS-KBr and KBr are the detector and beam splitter, respectively. Samples were in the form of film and spectra were recorded at room temperature at a resolution of 4 [cm.sup.-1], wave number accuracy 0.1 [cm.sup.-1] and signal to noise ratio 50,000:1.

Thermal Analysis. Thermogravimetric analysis (TGA) to determine the thermal stability and compositional analysis of PEC containing FA (CH-HA-FA) was done on a SDT Q600, simultaneous DTA-TGA system (TA Instruments, USA). Periodic calibration of temperature and mass signals of the SDT Q600 is done using standard reference materials. The test method is based on ASTM E 1131-08 [32], Before starting the experiment the mass of the sample and reference pans are tared using the software. Five milligram of the freeze dried sample was taken in a platinum sample cup and heated under nitrogen atmosphere at a heating rate of 10[degrees]C [min.sup.-1] from room temperature to 800[degrees]C. Calcined alumina is used as the reference material.

Surface Morphology. The surface morphology of the freeze dried sample was studied by Environmental Scanning Electron Microscope (ESEM, Model FEI Quanta 200, Netherlands). The samples were directly placed on the stub, voltage of 12.5 kV was applied and images were recorded at 1000X magnification, using LFD detector in low vacuum mode. Both dry and wet samples were analyzed using the same method. The surface morphology of FA loaded PEC kept in De.W and [Ca.sup.2+] was analyzed by Scanning electron microscope (S2400 Hitachi, Japan). Samples were gold coated in an ion sputter (Hitachi E101) prior to SEM observation. Voltage of 15 kV was applied and images were recorded at 1000X magnification.

[FIGURE 1 OMITTED]

Swelling Study. The swellability of the samples in De.W was compared with that of aqueous solutions of [Na.sup.+] and [Ca.sup.2+] (1M each). The swelling % of the matrix in different concentrations of [Ca.sup.2+] (0.02, 0.5, and 1.0M) was also studied. Preweighed pieces of the lyophilized sample were immersed in the respective solutions for different time intervals, (1, 24, and 48 h) and the percentage swelling was calculated using the following equation:

% Swelling = [([W.sub.f] - [W.sub.i]) / [W.sub.i]] x 100

where [W.sub.i] is the initial weight of the dry gel and [W.sub.f] is the final weight of the gel after dipping in the respective solutions for different time intervals.

Drug Release Study Using FITC Conjugated BSA. Release study was conducted in De.W and 1M aqueous solutions of each of [Na.sup.+] and [Ca.sup.2+]. The drug release property was also studied in varying concentrations of [Ca.sup.2+] (0.02 and 0.5M) to get information on the effect of concentrations of the ions on the release pattern and the total amount released.

Weighed pieces (~0.05 g) of gel were immersed in 2 ml of aqueous solutions of [Ca.sup.2+] and [Na.sup.+]. After definite time intervals i.e., 0.25, 0.5, 1, 2, 3, 4, 5, 24, and 48 h, the 2 ml solution added was completely withdrawn and replaced with fresh solution. Release of FA was estimated spectrophotometrically using a Varian Cary 100 Bio UV-Visible spectrophotometer (Melbourne, Australia) based on ASTM procedure [33], A calibration curve using the absorbance of different concentration of FA at a wavelength of 493 nm was constructed and the amount of FA released was calculated from the curve. The experiments were carried out at 37[degrees]C.

[FIGURE 2 OMITTED]

Kinetics of Swelling and Release studies

Korsmeyer et al. [34] derived a relationship to explain the drug release from a polymer system. To study the release kinetics, data obtained from in vitro release studies were plotted as 'log cumulative percentage drug release' against 'log time'. To find out the mechanism of drug release, the first 60% drug release data were to be fitted to the Korsmeyer-Peppas model exponential equation, Q = k[t.sup.n] where Q is the percentage of drug release at time 't', 'k' is the release rate constant, and V is the release exponent. The same method was adopted for the kinetics of swelling study. A value of n [less than or equal to] 0.5 is classified as Fickian diffusion [35],

Statistical Analysis of the Results. The data from each experiment were the average from triplicate samples. 'One way anova' was used for statistical analysis and the statistical differences were established as p < 0.05.

RESULTS AND DISCUSSION

A fibrous solid mass of PEC of polycationic CH and polyanionic HA was formed instantaneously on mixing an aqueous acetic acid solution of CH (2.5% w/v) and an aqueous solution of HA (1% w/v). The PEC of HA and CH was prepared by varying the ratios of CH and HA. It was observed that 1:5 (CH: HA) is the appropriate one to get a completely reacted electrostatically crosslinked PEC for the projected application. Moreover, the ratio of the constituents in a matrix definitely matters depending on the application of the scaffold. HA has been used to deliver drugs, like dexamethasone to treat rheumatoid arthritis [36, 37] and has clinically been used for joint and cartilage diseases such as osteoarthritis [38].

[FIGURE 3 OMITTED]

The ratio of CH:HA was fixed as 1:5 considering the beneficial effect of HA as highlighted in the aforementioned applications.

The non toxic nature of CH and HA is already reported in Ref. [39]. Chemical crosslinker was not used during the preparation step. Both the air dried and freeze dried PEC was insoluble in De.W at physiological pH for several weeks. The PEC is formed between the strong electrostatically crosslinked interaction of positively charged CH and the negatively charged HA. This may be the reason for its high stability and insolubility in De.W. In the presence of salts like [Ca.sup.2+] the interacting charges will be screened and their interaction is reduced leading to the collapse of the formed PEC [40], The unusual stability exhibited by these PEC scaffolds in De.W prompted us to check their behavior in response to [Ca.sup.2+] ions. Such interactions are possible when PEC scaffolds are employed in cartilage or bone tissue regeneration. We extended the study to the freeze dried PEC of same composition to view the fate of such a porous scaffold, which is always preferred for various tissue engineering applications. As mentioned in the experimental part, FA was incorporated in the matrix to study and compare the release property of the same in water and ionic solutions. Figure 1 shows the photographic image of the scaffolds prepared with and without FA under a UV lamp. The matrix without FA (Fig. 1A) appears as light green while FA incorporated CH-HA (Fig. IB) showed distinctively intense green color. The light green color of the CH-HA is due to the auto fluorescent nature of the scaffold, while the intense green color of CH-HA-FA (which is highly visible to the naked eye) because of the fluorescence of FA when viewed at 365 nm under the UV lamp.

[FIGURE 4 OMITTED]

Figure 2 depicts the FTIR spectra of CH, HA, and CH-HA. The characteristic peaks at 3309, 1651, and 1548 cm" due to OH/-N[H.sub.2] stretching, amide 1 (--C--O-- stretching) and amide II (--N[H.sub.2] bending), respectively can be observed for CH in the FTIR spectra [41, 42], In the case of HA the peak at 1658 [cm.sup.-1] is due to --C=0-- stretching of the carboxylic acid groups.

[FIGURE 5 OMITTED]

Peak at 1404 [cm.sup.-1] in CH-HA has already been reported for CH-HA (--C--H bending) by Peniche et al. [43], It can be seen that when CH-HA is formed from CH and HA, the corresponding peaks due to --OH stretching (3309 [cm.sup.-1] and 3297 [cm.sup.-1] for CH and HA, respectively) and --C=0 stretching (1652 and 1659 [cm.sup.-1] for CH and HA, respectively) are shifted to lower frequency region, viz. 3272 and 1634 [cm.sup.-1], respectively due to hydrogen bonding and electrostatic crosslinking among the functional groups.

The FTIR spectra of air dried and freeze dried CH-HA samples are almost similar and the peak at around 1400 [cm.sup.-1] due to (--C--H bending) is present in air dried sample also (Fig. 3). FTIR spectra of CH-HA-FA before and after release are shown in Fig. 4. The CH-HA-FA shows broad and fused bands in the --C=0- (~1600 [cm.sup.-1]) and --C--0--C--(~1020 [cm.sup.-1]) region due to the interaction between the PEC and FA, on the other hand these peaks become sharp and resolved for the FTIR spectra of CH-HA-FA after release, which also resembles the FTIR spectra of the air dried/freeze dried CH-HA. Moreover, the peak at ~1429 [cm.sup.-1] is prominent for the CH-HA-FA after release, which is ascribed to be the specific peak for CH-HA [43],

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

The thermal stability of materials used for biomedical applications are relevant only upto physiological temperature, 37[degrees]C. However, in order to study the thermal behavior and to have a compositional analysis of the samples, thermal analysis was conducted upto 800[degrees]C. Percentage weight loss against temperature curves of CH-HA and CH-HA-FA are given in Fig. 5. It can be observed that the thermograms of both CH-HA and CH-HA-FA are almost same upto 500[degrees]C. For both the samples, around 10% weight loss is observed below 100[degrees]C due to the loss of trapped moisture. The initial decomposition starts at 176[degrees]C for both samples. Fifty percent weight loss for CH-HA and CH-HA-FA were found to be around 301 and 304[degrees]C, respectively. From this we can assume that the initial thermal stability is same for both CH-HA and CH-HA-FA. However, at ~795[degrees]C the residue for CH-HA is 12% and that for CH-HA-FA is 18% confirming the presence of FA in the CH-HA matrix.

A porous matrix was formed by freeze drying the CH-HA gel at -59[degrees]C. The ESEM image in Fig. 6A at 1000X magnification shows a fibrous structure for the scaffold. Figure 6B is the wet matrix showing open irregular pores and an interconnected frame work, which enables the smooth infiltration of cells and nutrients when it is used for tissue engineering purposes.

[FIGURE 8 OMITTED]

The % swelling of the CH-HA scaffolds in De.W, [Ca.sup.2+] and [Na.sup.+] solutions are given in Fig. 7. The scaffold was found to be highly stable in aqueous solutions at physiological pH. Tan et al. have shown that hydrogel prepared by mixing CH and HA solutions (1:9) became water soluble after 2 h incubation due to poor crosslinking [6], In the present case, the CH-HA showed 235% swelling within 1 h on immersion in water at pH 7.4 and no further substantial increase (only upto 240%) was observed even after 48 h. At the same time the PEC scaffold kept in 1.0M [Ca.sup.2+] solution exhibited high swelling ~600% within 1 h and further increased to 1120% at the end of 24 h, which remained the same after 48 h. The matrix in 1M [Na.sup.+] solution showed swelling of 120% within 1 h and was slightly increased to 130% after 2 h and remained the same at the end of 48 h. Thus the % swelling in [Na.sup.+] solution was less when compared to that in De.W and [Ca.sup.2+] solution. This may be due to the lesser extent of interference of [Na.sup.+] ions with the matrix.

[FIGURE 9 OMITTED]

To confirm the effect of salt on the stability of the scaffold, the swelling study was repeated for the crosslinked CH-HA matrix also. Figure 8A and B show the percentage swelling of the bare and the crosslinked CH-HA matrix in De.W and [Ca.sup.2+] solution for varied time intervals. As mentioned earlier the uncrosslinked matrix exhibited swelling ~240% in De.W and ~1120% in presence of [Ca.sup.2+] after 24 h. The crosslinked matrix kept in De.W showed comparatively less swelling (230% in 1 h) as expected and the swelling percentage did not undergo a significant change with time (290% after 24 h). However, the effect of [Ca.sup.2+] induced dissociation of the PEC is observed for the crosslinked matrix also as evident from the increased swelling of the matrix kept in [Ca.sup.2+] solution. The extent of swelling was found to be 1586% after 48 h. The swelling study strongly point out the remarkable influence of [Ca.sup.2+] on the morphology of PEC formed between CH and HA. On mixing CH with HA a completely reacted, electrostatically crosslinked PEC is obtained. On adding an external cross linker, viz EDC/NHS, additional effective crosslinking between --N[H.sub.2] and --COOH groups may also be formed depending upon the availability of free functional groups. Such a matrix may have more loosened structure. In the presence of [Ca.sup.2+] such a structure is destabilized more and thereby could imbibe more water. This may be the reason for more swelling behavior shown by externally crosslinked CH-HA in [Ca.sup.2+] environment. The significant swelling induced by [Ca.sup.2+] strongly indicates that the structural integrity of the gel is severely affected even if it is in the crosslinked form. This aspect is to be taken into consideration while using such biopolymer scaffolds for tissue engineering purposes, especially in environment having a high concentration of [Ca.sup.2+].

[FIGURE 10 OMITTED]

[FIGURE 11 OMITTED]

The swelling % of the matrix in different concentrations of [Ca.sup.2+] (0.02, 0.5, and 1.0M) was also studied and it was found that swelling % increases with increase in [Ca.sup.2+] concentration (Fig. 9). The role of ions such as [Ca.sup.2+] in modulating the conformation of HA has been actively pursued due to the imminent encounter of HA with [Ca.sup.2+] in the bio environment. Our observation of the swelling behavior of the CH-HA complex in media containing Ca and Na ions is in line with the earlier findings [29, 30],

The release property exhibited by the CH-HA-FA scaffolds is conducive to their swelling nature. From the graph shown in Fig. 10 it is clear that the release of FA from CH-HA matrix is significantly higher in the presence of [Ca.sup.2+]. [Ca.sup.2+] may bind with the COO groups of the HA present in the CH-HA and this may lead to the dissociation of the ionic bonding between the CH and HA, thus facilitating the trapped drug to release into the medium. Nearly, 30% of the drug is released within 1 h and almost 90% is released after a period of 24 h. Interestingly, in the other two media (De.W and aqueous Na+ solution), even after 50 h, the quantity of drug released is remarkably lower (<20%) as compared to the quantity released in the presence of [Ca.sup.2+]. The drug release at different concentrations of calcium ions ranging from 0.02 to 1.0M is also depicted in Fig. 10. Significant difference was observed for the amount of drug released on varying the concentration of [Ca.sup.2+] ions. Enhanced release was observed for increased concentrations of [Ca.sup.2+] apparently suggesting that drug release can be modulated in response to concentration of [Ca.sup.2+].

[FIGURE 12 OMITTED]

From the SEM images of FA loaded PEC kept in De.W (Fig. 11A) and [Ca.sup.2+] (Fig. 1 IB), it can be observed that the PEC kept in [Ca.sup.2+] is having a collapsed structure losing its network morphology due to the strong screening of their charges by the [Ca.sup.2+]. Both these observations, the increased swelling with increase in [Ca.sup.2+] concentration and the collapsed morphology of the matrix in presence of [Ca.sup.2+], explain the increased release of trapped FA from the PEC in [Ca.sup.2+] rich environment.

From the photographic image in Fig. 12, the green fluorescence of the scaffold after 48 h of release in De.W (Fig. 12B) is comparable with the image shown in Fig. IB (FA incorporated CH-HA) indicating the presence of the remaining FA within the matrix, whereas scaffold after the release in calcium solution shows no fluorescence at all, Fig. 12A (image is similar to the photograph of the gel without FA shown in Fig. 1A) thus confirming its rapid release in presence of [Ca.sup.2+].

The kinetic release exponent, V values generated for drug release and swelling, as per the Korsmeyer-Peppas exponential equation are summarized in Table 1. It can be seen that the V values obtained for the kinetics of both drug release and swelling are less than 0.5 and hence can be classified as Fickian diffusion.

Calcium, the most abundant element in the human body is present in the form of calcium phosphate salts (1 kg, 99% of it in the skeleton). The extracellular fluid (ECF) contains ~22.5 and 500 mM of calcium is exchanged between bone and the ECF over a period of 24 h [44]. Ross et al. reported that damaged bone tissue exposes mineral crystals and therefore calcium binding sites, otherwise surrounded by collagen and coated with noncollgeneous proteins. Carboxylate, phosphonate, and bisphosphonate functionalized gold nanoparticles were chosen for targeting microdamage via chelation of these ligands with calcium ions on these mineral surfaces, which are exposed in damaged and disrupted tissue [45, 46], In the light of these observations it is convincing that the CH-HA matrix can be used for drug delivery at the damaged bone tissue sites where the tissue exposes calcium ions which in turn induces the [Ca.sup.2+] responsive drug delivery.

CONCLUSION

PEC loaded FA was synthesized and characterized by FTIR, TGA, and ESEM. Swelling and release study were carried out in De.W and aqueous solutions of [Ca.sup.2+] and [Na.sup.+]. This study apparently show that our matrix CH-HA swells significantly in presence of [Ca.sup.2+]. Rapid release of FA from the complex in presence of [Ca.sup.2+] was also observed. This matrix seems to have potential to target [Ca.sup.2+] rich sites like bone crack and subsequently empty drug or growth factors to facilitate rapid bone repairing. Strategies aimed at targeting damaged bones could open up new paradigms for the precise treatment of fractured bones.

ACKNOWLEDGMENTS

The authors also thank Mr. Nishad for the ESEM and SEM analysis. The authors thank DBT, New Delhi, for providing funding.
ABBREVIATIONS

CH         Chitosan
HA         Hyaluronic acid
PEC        Polyelectrolyte complex
CH-HA      PEC of CH and HA
BSA        Bovine serum albumin
FA BSA     conjugated FITC
CH-HA-FA   CH-HA containing FA
De. W      Deionized water


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A. Shanti Krishna, C. Radhakumary, K. Sreenivasan

Laboratory for Polymer Analysis, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Thiruvananthapuram, Kerala 695012, India

Correspondence to: K. Sreenivasan; e-mail: sreeni@sctimst.ac.in or C. Radhakumary; e-mail: radha.changerath@gmail.com

Contract grant sponsor: DBT, New Delhi.

DOI 10.1002/pen.24050
TABLE 1. Kinetic analysis for swelling and release experiments.

CH-HA-FA            'n' Value for Drug      V Value for
                     Release Kinetics    Swelling Kinetics

De.W                       0.12                0.02
[Na.sup.+]                 0.09                0.01
0.02M [Ca.sup.2+]          0.13                0.07
0.5M [Ca.sup.2+]           0.17                0.11
1.0M [Ca.sup.2+]           0.17                0.13
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Author:Krishna, A. Shanti; Radhakumary, C.; Sreenivasan, K.
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