Antimicrobial and biocompatible fluorescent hydroxyapatite-chitosan nanocomposite films for biomedical applications

Colloids and Surfaces B: Biointerfaces 171 (2018) 300–307

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Antimicrobial and biocompatible fluorescent hydroxyapatite-chitosan nanocomposite films for biomedical applications

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Somtirtha Banerjeea, Biswajoy Bagchia, ,2, Suman Bhandaryb, Arpan Koola,c, Nur Amin Hoquea,1, ⁎ Prosenjit Biswasa,1, Kunal Pald, Pradip Thakure, Kaustuv Dasa, Parimal Karmakard, Sukhen Dasa, a

Physics Department, Jadavpur University, Kolkata 700032, India Division of Molecular Medicine, Bose Institute, Kolkata 700054, India c Department of Physics, Techno India University, Sector V, Salt Lake, Kolkata 700092, India d Life Science &Biotechnology Department, Jadavpur University, Kolkata 700032, India e Department of Physics, Netaji Nagar College for Women, Kolkata 700092, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Nanocomposites Biocompatibility Chitosan Cytotoxicity Contact inhibition

Development of fluorescent erbium doped hydroxyapatite (eHAp)-chitosan nanocomposite film is reported. Nanocrystalline eHAp has been synthesized by hydrothermal assisted precipitation method using erbium (III) ions as dopant. Physico-chemical characterization by UV/Visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), photoluminiscence spectroscopy (PL) and Field emission scanning electron microscopy(FESEM) confirmed incorporation and uniform distribution of eHAp in the chitosan films. Strong antimicrobial activity was observed using eHAp incorporated chitosan films against E. coli and S. aureus by contact inhibition on agar plates. On the other hand, excellent biocompatibility was observed with human lung fibroblast cells (WI-38) which showed strong attachment and proliferation on the chitosan films with minimal cytotoxicity. Moreover, the doped films showed good biodegradation and mineralization behavior after 2 weeks in simulated body fluid. Thus the doped fluorescent chitosan films with multifunctional attributes can be a strong candidate for diverse applications like in antimicrobial treatments, wound healing, tissue scaffolds and bioimaging.

1. Introduction Chitosan based films and nanocomposites have receiv-ed great attention in recent times for its desirable antimicrobial, biodegradable and biocompatible attributes combined with natural abundance and economical processing. Chitosan is derived from the deacetylation of chitin (which is naturally found in the exoskeleton of crustaceans) and is made up of glucosamine and N-acetyl glucosamine units linked by β (1–4) glycosidic linkages. Due to its microbicidal and nontoxic nature, chitosan has found diverse range of usage such as in food packaging and preservation which includes coatings on bread, fruit, vegetable, eggs, and various meat products and in healthcare and biomedical field such as in drug delivery, wound dressing material, skin grafts and tissue regeneration scaffold [1–5]. The positively charged amino group is instrumental in interacting with biological membranes and makes it a potent antimicrobial moiety. However, antimicrobial property of

chitosan is also depended on several factors such as degree of acetylation, pH, type of microorganism, cell age, presence or absence of metal cations, pKa and molecular weight [2,6]. For example, Kong et al investigated physico-chemical factors that influence the antimicrobial property of chitosan [7]. In another study, Escárcega-Galaz et al reported on the antimicrobial activity of chitosan based films against Salmonella typhimuriumand Staphylococcus aureus [8]. While Rhazi et al illustrated the effect of different chitosan sources and deacetylation process on the physiochemical characteristics of chitosan [9]. Li et al explained the antimicrobial mechanism of chitosan based on molecular weight with low molecular weight chitosan showing high antimicrobial activity due to higher percentage of amino group protonation and cation formation [10]. Jayakumar et al demonstrated that Chitosan based antimicrobial films can be effectively used to treat nosocomial infections arising from Streptococcus, Staphylococcus and Pseudomonas species during surgery and is also an excellent wound or burn dressing

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Corresponding authors. E-mail addresses: [email protected] (B. Bagchi), [email protected] (S. Das). 1 Equal contribution. 2 Present Address: Department of Medical Physics and Biomedical Engineering, University College London, W1W 7TS, United Kingdom. https://doi.org/10.1016/j.colsurfb.2018.07.028 Received 6 April 2018; Received in revised form 7 July 2018; Accepted 12 July 2018 0927-7765/ © 2018 Elsevier B.V. All rights reserved.

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respectively.

material on its own due to its adhesive nature,biocompatibilityand permeability to oxygen [11]. Apart from antimicrobial applications, chitosan based nanocomposites have been widely used as functional biomaterials ranging from drug delivery to tissue regeneration systems that interact with mammalian cells to influence their growth in a positive way. Generally, these nanocomposites consist of various bioactive molecules like drugs, enzymes, genes, nanoparticles embedded in the chitosan matrix [1,3–5]. One of the most commonly used biomaterial is the hydroxyapatite incorporated chitosan for bone tissue engineering applications. Due to closer resemblance to the chemical and mineral components of natural bone and excellent osteoconductive behavior, synthetic hydroxyapatite is widely used for hard tissue repairing, bioceramic coating, and dental applications [12,13]. Thus combining chitosan and HAp into a hybrid composite will showexcellent potential as bioactive material with enhanced mechanical, chemical and biological properties [14,15]. Recently, luminescent biomaterials based on hydroxyapatite have garnered wide interest and are widely used as fluorescent probe to study cellular membranes, imaging tissues and intracellular structures and drug delivery applications. For example, fluorescent rare earth doped HAps were investigated by several research groups to be excellent agents for bioimaging applications [16–21]. Similarly, Li et al developed fluorescein isothiocyanate functionalized HAp nanoparticles for cell labelling, animal imaging, and drug delivery applications [22]. Thus combining a fluorescent bioactive material such as hydroxyapatite with chitosan will be an ideal choice toward developing an advanced biomaterial suitable for not only therapeutic applications but also imaging cell-material interaction. In the present work, we used erbium ion doped hydroxyapatite (eHAp) to develop luminescent chitosan-hydroxyapatite nanocomposite films which can be used both as an antimicrobial dressing material and a fluorescent cell regeneration scaffold or implant material. The developed chitosan films are flexible, easy to process and detailed characterization by various analytical techniques and biochemical assays indicated excellent antimicrobial, bioactive and fluorescent properties under in vitro conditions.

2.3. Materials and methods CH films were characterized by a Bruker AXS (Model D8, WI, USA) model X-ray Diffractometer (XRD) using CuKα radiation (1.5409 Å) and scan range (2θ) from 0 to 70° (at 40 kV voltage). Absorption spectra of the films were measured by UV/Visible spectrophotometer (UV 1800, Shimadzu) in the 200–800 nm wavelength range. The interaction between chitosan and eHAp was studied by fourier transform infrared spectrometry (FT-IR, FTIR-8400S model Shimadzu, Tokyo)with thescanning range set from 400 to 4000 cm−1 under Happ–Genzel configuration and a resolution of 4 cm−1. The photoluminescence spectra of 1 mg/mL eHAp solution in absolute ethanol medium was investigated using Cary Eclipse fluorescence spectrophotometer, Agilent Technologies and microscopic images of eHAp powder and eHAp doped CH films were taken by Zeiss Axiocam MRc fluorescence microscope. Microstructure of the CH films was observed under field emission scanning electron microscope (FESEM, model FEI Quanta 50 (USA). A portion of the films were cut and gold coated via plasma spraying at 0.1 mbar pressure. The emission current and operating voltage were kept at 170 μA and 20KV, respectively. Antimicrobial activity of CH films was studied by agar-diffusion method [9] and plate count method [23]. For agar-diffusion method, 20 ml nutrient broth (0.5% peptone, 0.1% beef extract, 0.2% yeast extract, 0.5% NaCl, (Himedia Pvt. Ltd., India)) containing 20%agar was taken into sterile petri dishes and allowed to solidify. Next, 100 μL of overnight grown microbial culture (cell density- 107 CFU/mL) was dispersed over the medium by sterile swab. The films were then placed carefully on the solid media to ensure direct contact with the film surface. The plates were then incubated at 37 °C for 24 h. Direct contact between film and medium surface is a crucial factor to assess the bactericidal effect of chitosan membranes [9,24]. For plate counting method, microbial culture with same cell density was incubated for 24 h in nutrient broth (composition as described above but without agar) containing three different CH films. After incubation, an appropriate inoculum was plated on nutrient agar plates. Mortality rate was calculated by counting the number of colonies on control and treated plate after 24 [23]. The morphological characteristics of the microbial cells after interaction with the CH films were observed by FESEM. This procedure was done on the suspensions of treated cultures after 12 h of incubation. Briefly, mid exponential phase bacterial culture(final cell density ∼ 107 CFU/mL) were incubated with 1CH, 1.5CH and 2.5CH films at 37 °Cfor 24 h. Control (without the films) was prepared under same conditions. After treatment, the cells were collected by centrifugation at 5000 rpm for 10 min followed by repeated washings with phosphate buffer saline (PBS) and fixation with 2% formaldehyde (37–41% w/v, Merck, India) (in PBS). Samples were then dehydrated by serial dilutions of ethanol and 5 μL was drop casted on a clean glass cover slip. After drying, the samples were sputter coated with gold and observed under electron microscope [23,25]. Qualitative determination of hydrophillicity of CH film was performed by placing a single drop of distilled water on the film and taking a cross-sectional image using DSLR camera (Nikon D3300). Cytotoxic effect of CH filmson human lung fibroblast (WI-38) cells was determined by Methyl tetrazolium based (MTT, Biologos Inc., USA) cell viability assay.Briefly, sterile chitosan films were incubated with human lung fibroblast (WI-38) cells (cell density 5 × 103per well in 96 well plate) in Dulbecco Modified Eagle Medium (DMEM) medium, (Biologos Inc. USA) for 24 h. Thereafter, the cell suspension was treated with 5 μL of 15 mg/mL MTT solution followed by 4 h incubation at 37 °C. The MTT solution was then removed and 100 μL of DMSO was added to dissolve the formazan crystals and generate homogeneous purple solution. Cytotoxicity was determined by measuring absorbance at 570 nm using a microplate reader. By comparing this absorbance

2. Experimental 2.1. Synthesisof erbium doped hydroxyapatite(eHAp) powder Initially, in 40 mL of 0.03(M) calcium chloride (Merck, India) solution, 0.662 g erbium (III) chloride hexahydrate (ErCl3.6H2O, SigmaAldrich) was dissolved by stirring at room temperature. Next, 30 mL of diammonium hydrogen phosphate ((NH4)2HPO4, Merck, India) was prepared and pH of the solution was adjusted to 10.5 by dropwise addition of 1 (M) sodium hydroxide (NaOH, Ranbaxy laboratories limited, India). The first solution was then added drop wise to the second solution by stirring and after 30 min, the total solution mixture was transferred into a teflon lined hydrothermal autoclave for 6 h at 140 °C. The resultant hydrothermally treated precipitate was washed several times by centrifugation to remove excess NaOH. The pale yellow precipitate was then dried at 60 °C to obtain erbium dopedhydroxyapatite powder. The mole ratio of Ca/P and Ca/erbium was 1.66 and 1.5 respectively [17]. 2.2. Synthesis of eHAp doped chitosan film Three different concentrations (1, 1.5 and 2.5% w/v) of chitosan (Mw: 100,000-300,000, DA- 85%, Acros-Organics, USA) films were prepared each containing 25% of eHAp by weight (Fig. 1). Briefly, calculated amount of chitosan and eHAp powders were dissolved in 1% acetic acid solution and stirred overnight at 60 °C. The solutions were then poured into plastic petri dishes and dried at 60 °C. The resultant films were collected and stored for further investigations [15,16]. Henceforth, the films will be designated as 1CH, 1.5CH and 2.5CH 301

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Fig. 1. Schematic representation for synthesis of eHAp doped chitosan (CH) films.

doping with hydroxyapatite does not seem to affect theabsorption spectra of chitosan films [28].

with that of control cell recorded at the same wavelength, the relative cell viability (%) was calculated. The experiment was repeated for 3 times with the average data plotted [23]. Cell attachment of human lung fibroblast (WI-38) on chitosan films were also observed by FESEM. The analysis was carried out on the film surface after 24 h of incubation with WI-38 cells.CH films were fixed with glutaraldehyde for 15 min followed by repeated washings with PBS and serially diluted of ethanol. Finally, the samples were sputter coated with gold and observed under electron microscope [23]. Cellular attachment and viability on 2.5CH film were also observed by fluorescence microscope (model IX53, Olympus, USA)after 24 h incubation and staining with 0.2 mg/mL 4, 6-diamino 2-phenylindole (DAPI) (vectashield) [26]. Bioactivity ofCH film was determined by incubating the film immersed in stimulated body fluid (SBF) for 2 weeks at 37 °C [18,27]. Microstructure of the surface was analyzed by FESEM and degradation of the film was estimated by measuring the weight of film before and after incubation (Fig. 1).

XRD pattern of 1%, 1.5% and 2.5% pure chitosan films exhibited characteristic reflections around 2θ values of 12°, 19° and 26° (Fig. 2b) [28]. The intensity of these peaks increased with concentration of chitosan. In case of doped films (1CH, 1.5CH and 2.5CH), the XRD pattern is dominated by characteristic peaks of eHAp along with some minor peaks of chitosan (Fig. 2c). XRD of pure eHAp (Fig. 2d) shows characteristic reflections of hydroxyapatite along with a minor β-tricalcium phosphate phase (β-TCP) [27,29,30]. The broad peaks suggested nanostructured and amorphous nature of hydroxyapatite phase. Although HAp concentration was same in all the cases, an increase in the peak intensity is observed with chitosan concentration. This might be due to more HAp accumulating on the surface of the film as the viscosity of chitosan increases with concentration.

2.4. Statistical analysis

3.3. Photoluminescence spectroscopy and fluoroscence microscopy analysis

All data are represented as the means + standard deviation n = 3. P < 0.05 was considered to indicate significance. Mean, N and SD or SEM for each column were calculated from raw data. One-way analysis of variance (ANOVA) was used to differentiate among mean followed by Tukey’s test for multiple comparisons, using the software Graph Pad Prism (Graph Pad Software, Inc., La Jolla, CA, USA).

The photoluminescent (PL) spectra of eHAp showed four emission bands after excitation at 500 nm wavelength (Fig. 2f). Green emissions around 526 nm and 569 nm are observed due to 2H11/2-4I15/2and 4S3/ 2-4I5/2 transitions respectively while red emission is observed around 650–730 nm due to n-L shell electronic transitions [18]. Fluorescence microscopic images of chitosan and CH films were observed using blue light excitation (Fig. 2g). Microscopic images clearly indicate uniform distribution of fluorescent HAp particles in the CH films with more agglomeration at higher chitosan concentrations.

3.2. XRD analysis

3. Results and discussions 3.1. UV-Visible spectroscopy

3.4. FTIR analysis The UV/Visible absorption spectra of CH films have been shown in Fig. 2a. All the films show characteristic chitosan peak around 200 nm which is primarily due to absorption by glucosamine moiety. An additional absorption peak was also observed at around 300 nm which also increased in intensity with chitosan concentration. It is well known that exposure to ultraviolet radiation causes photo oxidation of chitosan which leads to formation of new chromophores. In the present case, the absorption around 300 nm is ascribed to the formation of carbonyl and amino groups after ultraviolet exposure during measurement. However,

FTIR spectra of CH films reveal characteristic bands corresponding to both chitosan and hydroxyapatite phases having some overlapping peak positions (Fig. 2d) [31]. The peak intensities became somewhat diffused for 2.5CH which may be due to the thickness of the film. Chitosan fingerprint bands were observed at 1030 cm−1 (C–N stretching vibrations), 1330 cm−1 (−CH2 bending vibration), 1415 cm−1 (−CH3 symmetrical bending), 1587 cm-1 (NeH bending mode vibration) and 1641 cm−1 (C]O stretching vibration for the 302

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Fig. 2. (a) UV–vis spectra of 1CH, 1.5CH and 2.5CH films (b) XRD pattern of 1%, 1.5%, and 2.5% chitosan films without eHAp, (c) XRD pattern of 1CH, 1.5CH and 2.5CH films, (‘*’ indicates the presence of eHAp and ’ indicates the presence of chitosan), (d) XRD pattern of eHAp after calcinations, (e) FTIR spectra of 1CH, 1.5CH and 2.5CH films, (f) Photoluminescence spectra of 2.5CH, (g) Fluorescence images of pure chitosan film and 1CH, 1.5CH and 2.5CH films and (h) Qualitative contact angle image of water on 2.5CH after 0 and 10 min.

acetamido group). A broad band around 3400-3500 cm−1 was also observed which corresponds to NeH vibrations from chitosan and OH vibrations from chitosan, HAp and absorbed water [16,28]. Band vibrations for eHAp were observed around 567 cm−1, 630 cm−1, 1025 cm−1and 1136 cm−1 which were ascribed to the different stretching and bending vibrations for PO43− and HPO42− groups. All band vibrations showed some shifting toward lower wavelength for chitosan which indicates interaction with the hydroxyapatite phase [16,29,30].

the drying stage the increased viscosity leads to accumulation of more HAp particles on the surface consequently resulting in a porous microstructure. 3.7. Antimicrobial assay Fig. 4 shows the antimicrobial activityof CH films against E. coli and S. aureus. Cell mortality calculated from plate count after immersing the films in bacterial broth for 24 h shows a maximum value of around 80% and 60% for S. aureus and E. coli respectively (Fig. 4a) using 2.5CH. The FESEM micrographs of the treated microbial cells shows extensive membrane damage, cytoplasmic leakage and cell rupture leading to agglomerated cell debris for all the CH films compared to normal healthy cells (Fig. 4b, top row- E. coli, bottom row- S. aureus). However, the membrane damage was observed to be comparatively low for E. coli. It can be clearly seen that the antimicrobial activity is not very remarkable when the films were used in broth. However, this changes when the same films were used directly on agar plates containing microbial culture. Fig. 4c, shows the plate images with clear zones of inhibition depending on the dimension of the films. Therefore, it is evident that the CH films are significantly more effective through contact inhibition and only moderately active in broth.This observation is contrast to the work by Foster and Butt, who reported that chitosan films do not exhibit antimicrobial property [34]. Fig. 5 shows the probable mode of action of antimicrobial activity of eHAp doped chitosan film. The microbicidal attribute of chitosan is widely accepted and is generally realized by membrane disruption, loss of cellular proteins and change in membrane permeability. Antimicrobial activity of chitosan is depended on a large number of parameters including degree of deacetylation, molecular weight, pH, temperature, viscosity and ionic strength of medium, solvent, type of microorganism and growth stage [1,2,5]. In general, Gram-positive bacteria are more susceptible to chitosan activity due to the electrostatic interaction of negatively charged teichoic acid on their cell wall and positively charged amino groups on chitosan [5,24,35]. This explains the lower mortality rate observed for Gram-negative E. coli in broth when treated with chitosan films. However, chitosan has also been reported to impart bacterial

3.5. Hydrophilicity of CH films Surface wettability of a biomaterial is an important factor for optimal interaction with cells and tissues. Fig. 2h shows the water droplet image on 2.5CH eHAp film taken between 0 and 10 min. The contact angle is observed to decrease to around 60° after 10 min which confirmed the hydrophilic nature of the film. The other composites i.e., 1CH and 1.5CH also showed similar wettability. Inclusion of hydrophilic hydroxyapatite has been reported to confer hydrophilic attributes to polymer biomaterials for cell scaffold and tissue regeneration applications [26,31]. In the present case, addition of 25% (by weight) of eHAp and porosity of the film act together for optimum hydrophilicity. 3.6. FESEM analysis Morphology of pure chitosan, eHAp and CH films were studied by scanning electron microscopy (Fig. 3). Fig. 3a shows that microstructure of pure chitosan film has smooth texture with no apparent porosity [32]. SEM image of eHAp nanopowder (Fig. 3b) shows rod like nanocrystalline particles with length and width in the range of 100–150 nmand 40–50 nm respectively. Morphology of 1CH, 1.5CH and 2.5CH (Fig. 3c–e) shows presence of eHAp particles in the films [16,18,33]. It can be observed that 1CH has relatively smooth surface with HAp particles embedded mainly in the matrix. In contrast, 1.5 and 2.5CH films exhibited a rough surface with porous microstructure due to presence of high percentage of eHAp particles on the surface. This may be due to the fact that at higher concentration of chitosan, during 303

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Fig. 3. FESEM micrograph showing (a) non-porous even chitosan film, (b) hydrothermally synthesized eHAp particles (c) 1CH, (d) 1.5CH and (e) 2.5CH.

Fig. 4. (a) Cell mortality curve for E. coli and S. aureusas calculated from plate count technique after 24 h incubation, (b) FESEM micrographs of E.coli and S.aureus showing morphological characteristics after the treatment with or without 1CH, 1.5CH, and 2.5CH- top row from left to right, E. coli control and increasing concentration of chitosan. Bottom row indicates similar for S. aureus and (c) Contact inhibition zone for E. coli and S. aureus after treatment with1CH, 1.5CH and 2.5CH films for 24 h. 304

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Fig. 5. Probable mode of action of eHAp doped chitosan film.

determined by MTT assay using human lung fibroblast (WI38) (Fig. 6a). It can be observed that all the CH films shows excellent biocompatibility with > 80% cell viability after 24 h of incubation. However, with increase in chitosan concentration a slight decrease in cell viability (∼5%) was observed compared to pure chitosan film. Morphology of WI38 on CH films was carried out by FESEM and shows healthy adherent cells with extending filopodia typical of fibroblasts on the surface of the films (Fig. 6b–d for 1CH, 1.5CH and 2.5CH respectively). The best morphology was observed in case of 2.5CH (Fig. 6d) where multiple healthy growing cells are observed interconnected through a fibrillar matrix formation on the film surface. It is well known that presence of hydroxyapatite inclusions in chitosan improves the bioactivity of the composite by promoting attachments sites and biochemical interactions requisite for cell and tissue growth. Fibroblasts can be most effectively made to attach, proliferate and differentiate on hydroxyapatite-chitosan based composites [11,16,17,27,38]. Fluorescence imaging was performed to further understand the interaction of WI38 cells with the CH matrix (6e-j). 2.5CH was selected as it showed maximum antimicrobial activity. Fig. 6e–g shows the DAPI stained WI38 cells under blue filter, green filter and merged condition respectively.The images were taken after treatment with extract of

growth inhibition by acting as membrane perturbing agent, damaging DNA and proteins after internalization and disrupting nutrient assimilation by forming impermeable polyelectrolyte complex on Gram-negative bacteria [2,5,24,35–37]. In the present case, the doped chitosan films strongly and equally inhibits E. coli and S. aureus when placed on agar plates which indicates direct interaction with the membrane. However, there was no significant change in antimicrobial activity due to the presence of hydroxyapatite in the chitosan matrix.

3.8. Biocompatibility assay Biomedical application of antimicrobial materials dictates that it should elicit microbicidal effect while remaining minimally cytotoxic and in some cases even providing support and stimulate proliferation of new cells and tissues around the affected region leading to complete healing [5]. Chitosan and hydroxyapatite based nanocomposite materials are excellent in terms of physico-chemical properties where the biocompatibility and antimicrobial activity of the components can be exploited for diverse application like treating bacterial and fungal infection, wound healing, cell proliferation and tissue regeneration [16,17]. Keeping this in mind, cytocompatibility of the CH films was 305

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Fig. 6. (a) MTT assay on human lung fibroblast (WI-38) cell line after treatment with pure chitosan and 1CH, 1.5CH and 2.5CH films. Values are means ± SEM of five separate experiments done in triplicate (one way ANOVA). FESEM micrographs showing WI-38 cells on (b) 1CH, (c) 1.5CH and (d) 2.5CH films. Fluorescence microscopic images of DAPI stained WI-38 cells after treatment with extract of 2.5CH film for 24 h under (e) blue filter, (f) green filter and (g) merged condition. Similarly, h–j represents cells on the surface of 2.5CH after 24 h incubation. DAPI stained cells appear blue while eHAp incorporated cells appear green (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

accumulation of eHAp particles in the cell which shows greater tolerance limit for erbium doped hydroxyapatite (Fig. 6a). Thus the nanocomposite films shows excellent biocompatibility and biodegradability along with fluorescent properties that can be used to monitor implant bioactivity by fluorescence technique.

2.5CH for 24 h. Green cells indicates successful internalization of fluorescent eHAp particles (Fig. 6f and j). This also shows the biodegradable nature of the nanocomposite films whereby due to degradation of chitosan, HAp particles are released and consequently taken up by cells [18,19,39,33]. Fig. 6h–j shows fluorescence images of cells on the surface of 2.5CH film. Living cells can be observed attached to the surface of the fluorescent 2.5CH film (Fig. 6i and j background) and also emitting blue and green fluorescence due to uptake of DAPI and eHAp particles simultaneously (Fig. 6j). Presence of nano eHAp aids in cellular attachment by enhancing surface roughness and interacting with the cells directly. Cellular uptake of fluorescent hydroxyapatite has been observed in a wide variety of cell lineages including macrophages, monocytes, epithelial cells, human hepatoma and fibroblasts and forms the basis for monitoring cellular events in situ.However, the cytotoxicity associated with such rare earth doped fluorescent particles presents an issue in terms of practical application [19,23,40,41]. In the present case, the cytotoxicity profile shows negligible cell death even after

3.9. Simulated body fluid test In vitro bioactivity of 2.5CH was observed after immersing the film in SBF for 2 weeks. Fig. 7a shows the FESEM micrographs of 2.5CH surface before immersion in SBF. After treatment, the surface clearly indicates degradation of chitosan as seen from the blurred regions on the surface. Overall, a weight loss of 20% occurred after 2 weeks which indicates dissolution of components. However, deposition of nanocrystalline apatite layer is also observed in the center of the image (Fig. 7b). Mineralization by apatite formation in SBF occurs by a dynamic solution and precipitation mechanism and is essential for 306

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Fig. 7. FESEM micrograph 2.5CH film surface before and after immersion in simulated body fluid (SBF) for 14 days. A- before immersion in SBF, B- after immersion in SBF.

osteoconductivity which helps in tissue regeneration including bone formation [18,27]· Thus the CH films forms an ideal matrix where biodegradation and mineralization are complemented to ensure optimum cellular attachment and proliferation. 4. Conclusion Antimicrobially active fluorescent chitosan films have been developed. 2.5% chitosan film showed the strongest contact inhibitions against E. coli and S. aureus after 24 h of incubation. Antimicrobial effect of the film is found to be mediated by the chitosan component and is more active against Gram negative Staphylococcus species. Biocompatibility studies on human lung fibroblast (WI-38) showed that all the films are cell friendly with ∼80% viability. Good cellular attachment is observed on the films with fluorescent HAp particles being internalized with minimum cytotoxity. The films also exhibited good mineralization behavior in simulated body fluid. The fluorescent films are therefore a promising candidate for therapeutic and bioimaging applications under both in vitro and in vivo conditions. Acknowledgements We are grateful to the Council of Scientific and Industrial Research

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