Physiologically clotted fibrin – Preparation and characterization for tissue engineering and drug delivery applications

Biologicals 42 (2014) 277e284

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Physiologically clotted fibrin e Preparation and characterization for tissue engineering and drug delivery applications Weslen S. Vedakumari, Thotapalli P. Sastry* Bio-Products Laboratory, Central Leather Research Institute, Adyar, Chennai, Tamilnadu 600 020, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 January 2014 Received in revised form 11 June 2014 Accepted 24 June 2014 Available online 16 July 2014

Fibrin used for biomedical applications is prepared by mixing concentrated solutions of fibrinogen and thrombin in presence of cross-linking agents such as Factor XIII or glutaraldehyde. The main drawbacks associated with this procedure include cost, complexity and time required for fibrin preparation. Hence, present study deals with the characterization of physiologically clotted fibrin (PF) for bone tissue engineering and drug delivery applications. For this the physico-chemical properties of PF were compared with those of the conventionally prepared fibrin (CF). Further MTT and haemolytic assays were performed for both PF and CF to compare their biocompatibility. The amount of alkaline phosphatase produced and calcium secreted by MG-63 cells in the presence of PF and CF were used to relate the osteogenic potency of PF with that of CF. Gallic acid, an anti-cancer drug was loaded within PF and CF and their role in drug delivery was compared. © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Keywords: Physiologically clotted fibrin Bone tissue engineering Drug delivery Alkaline phosphatase Gallic acid Fluorescence microscopy

1. Introduction Fibrin is a fibrous and viscoelastic protein, commonly used in surgical practices for haemostasis and wound healing [1]. It is a biocompatible and biodegradable polymer and hence serves as a perfect matrix for stem cell differentiation and tissue regeneration [2] and [3]. Human umbilical cord mesenchymal stem cells encapsulated within fibrin-alginate microbeads showed excellent proliferation, osteogenic differentiation and bone mineral formation when compared with the non-encapsulated cells [4]. Keratinocytes loaded within fibrin capsules showed five time increase in the production of transforming growth factor b1 and recombinant human platelet derived growth factor-BB [5]. Apart from tissue engineering applications, fibrin has also been utilized in the form of drug delivery vehicles to attain continuous discharge of drugs and proteins over a specific period of time [6] and [7]. When fibrin was used as a carrier of carboplatin, it lingered cytotoxic on retinoblastoma cells proving its possible use in the field of drug delivery [8].

* Corresponding author. Tel.: þ91 9444382361; fax: þ91 44 24912150. E-mail addresses: [email protected], [email protected] (T.P. Sastry).

Fibrin used in the above mentioned applications is prepared using the conventional method of mixing concentrated solutions of fibrinogen with thrombin in the presence of cross-linking agents such as Factor XIII or glutaraldehyde [9] and [10]. The main drawbacks associated with this procedure include cost, complexity and time required for fibrin preparation [11]. In some of the slaughter houses at India, bovine blood is collected to prepare pharmaceutically important products such as haemoglobin and serum [12]. During the process physiologically clotted fibrin (PF) is obtained as a by-product. It has been reported that fibrin obtained naturally (PF) or therapeutically (conventional method), deems to be biocompatible and biodegradable [13]. However the therapeutic applications of PF has not yet been studied extensively; thus the main objective of this study is to investigate the possible use of PF as an alternative for conventionally prepared fibrin (CF) for bone tissue engineering and drug delivery applications. For this purpose, the physical and chemical properties of CF and PF were compared and the similarities between them were assessed. Haemolysis and MTT assay were performed to evaluate the biocompatibility of PF. For bone tissue engineering applications, MG-63 human osteosarcoma cells were treated with PF and the amount of alkaline phosphatase produced and calcium secreted were quantified. Gallic acid was loaded within

http://dx.doi.org/10.1016/j.biologicals.2014.06.004 1045-1056/© 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

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PF and its anti-cancer potency was determined using MCF-7 human breast cancer cells.

2.4. Haemolysis

Fresh bovine blood was collected from municipal slaughter house under sterile condition. PF was prepared by churning the blood with a glass rod. It was washed thoroughly with cold distilled water and treated with 0.5 M sodium acetate and 30% hydrogen peroxide [14]. CF was purchased from Hi-Media Laboratories Pvt. Ltd., Mumbai, India. All the chemicals used in this study were of analytical grade.

Haemolytic assay is deemed to be an easy and dependable method to evaluate the blood compatibility of bio-materials [17]. Human blood samples were collected with the consent from healthy volunteers and centrifuged at 1000  g for 10 m. The pellet obtained was washed thrice with phosphate buffered saline (PBS) to remove the residual blood plasma. A 5% haematocrit was prepared by suspending the purified red blood cells (RBCs) in PBS. 50 ml of the RBC suspension was treated with 1000 ml of different concentrations of PF and CF (25, 50, 75 and 100 mg/ml) and incubated at 37  C for 60 min. RBCs treated with PBS and water were used as negative and positive control [18]. After incubation, all the samples were centrifuged and their supernatants were used for determining the optical density at 540 nm.

2.2. Physicochemical characterization of PF and CF

2.5. Mechanical strength

UVevis spectroscopic measurements were performed using Jasco dual beam UVeviseNIR spectrophotometer (model V-570). SDSePAGE analysis was carried out to compare the electrophoretic mobility of PF and CF. For this study, 5 mg per sample was treated with sample buffer containing bemercaptoethanol, boiled at 100  C for 5 min and then subjected to electrophoresis. Coomassie

The tensile strength presents an indication of mechanical strength which is a very important feature for hydrogels used for tissue engineering applications. Tensile strength of PF and CF was determined using Instron 4501 tensile system. The formula used to calculate the tensile strength of the sample is as follows;

2. Materials and methods 2.1. PF and CF

Tensile strength (N/mm2) ¼ Breaking force (N)/Cross-sectional area of sample (mm2)

brilliant blue Re250 was used to stain the protein bands. JASCO model 815 circular dichroism spectropolarimeter was used to analyse the secondary structure of PF and CF. For circular dichroism, a total volume of 500 ml per sample was prepared in PBS at a concentration of 0.5 mg/ml. To evaluate the functional groups present in PF and CF, FTIR was performed using ABB MB3000 Fourier transform infrared spectrophotometer. The samples were mixed with potassium bromide, made into pellet and then used for the study. To determine the amino acid composition of PF, 50 mg of sample was placed in 15 ml ampoule and treated with 6 N HCl at 110  C for 24 h under vacuum. The hydrolysed sample was neutralized using 1 N NaOH and diluted with 0.2 M citrate buffer (pH 2.2). 20 ml of the sample was then loaded into the SUPELCOSILTM LCeDABS HPLC column (Agilent Technologies, Santa Clara, California, United States) and the amino acids were analysed [15]. 2.3. Cytotoxicity test MTT (3-(4,5-dimethylazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay was performed to evaluate the cytotoxicity of PF and CF using NIH 3T3 cells (mouse embryonic fibroblasts cell line). MTT is a water soluble yellow coloured substrate which gets reduced into an insoluble purple coloured formazan on reduction by dehydrogenases produced in living cells. For this study, 1  104 cells were seeded separately in 96-well plate and incubated overnight under controlled atmosphere. Subsequently the cells were exposed to similar concentrations of PF and CF (25, 50, 75 and 100 mg/ml) for 24 h. Cells without any treatment served as negative control and those treated with Triton X-100 served as positive control. Following incubation, the cells were incubated with 100 mL of MTT solution (0.5 mg/mL) for 3 h at 37  C. Finally 50 ml of DMSO was added to dissolve the formed formazan and the absorbance was measured at 570 nm [16].

2.6. Rate of degradation To assess the biological stability of PF and CF, a known weight of each sample was incubated in PBS at 37  C for 1, 3, 5 and 7 days respectively. After each incubation, both the samples were removed air-dried and weighed. The percentage of weight loss was calculated as follows; Weight loss (%) ¼ Initial weight  Final weight  100

2.7. Bone tissue engineering Various biochemical events such as synthesis of alkaline phosphatase (ALP) and extracellular calcium deposition ensue during new bone formation. Hence these biochemical markers were quantified after treating MG-63 cells with similar concentrations of PF and CF at different time intervals. This was done to compare the osteogenic property of PF with CF. 2.7.1. Cell proliferation assay MG-63 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin with 5% CO2. 1  105 cells were seeded in 96-well plate and incubated overnight. They were treated with 25, 50 and 100 mg of PF and CF and incubated for 1, 3 and 5 days. After each exposure, the cell culture medium was discarded, 100 ml of 1 mg/ml MTT was added and incubated for 4 h [15]. After incubation, 100 ml of DMSO was added and subjected to UV detection at 570 nm. 2.7.2. ALP Activity The amount of alkaline phosphatase secreted by PF and CF treated cells was determined by measuring p-nitrophenol present in the cell culture supernatants. Briefly, cells were treated with various

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concentrations of PF and CF (25, 50 and 100 mg) for 4, 7 and 10 days. After treatment, cells were lysed using 0.1% Triton x-100 and centrifuged at 15,000 rpm for 15 min, 4  C. To the resultant supernatant, 100 ml of 5 mM p-nitrophenylphosphate was added and incubated for 30 min at 37  C. A 100 ml aliquot of 0.2 N NaOH was added to stop the reaction and the absorption was measured at 405 nm [19]. 2.7.3. Calcium assay Alizarin red S (ARS) assay was performed to study the formation of calcium ions by PF and CF treated cells after 14 days of incubation. For this, the cells were fixed in 4% formaldehyde and stained with 2% Alizarin red for 15 min. They were then treated with 10% cetylpyridinium chloride and the optical density was read at 562 nm [20]. 2.7.4. Formation of bone like apatite 0.1 g of PF and CF were separately made into 1.5 mm thick pellets and soaked in 10 ml of SBF (simulated body fluid) for 21 days. The solution was changed once in every two days to avoid pH variation or microbial contamination. After 21 days of incubation, the pellets were removed from SBF, air dried and viewed under scanning electron microscope (SEM). 2.8. Drug delivery 2.8.1. Loading Gallic acid into PF and CF Gallic acid (GA), a phytomedicine was used as a model drug to compare the drug loading and release characteristics of PF. For this, a clear, homogeneous solution of PF was prepared by dissolving each sample (5 mg) in 5 ml of 1 N NaOH. The pH of this solution was reduced to 7, followed by dropwise addition of aqueous solution of GA (2.5 mg). The reaction mixture was stirred overnight, centrifuged and redispersed in deionised water to remove free gallic acid. The resultant GA loaded PF was characterized using fourier transform infrared spectroscopy (ABB MB3000). Similar procedure was carried out to prepare GA loaded CF (GAeCF). This was done to compare the drug delivering properties of PF with CF. 2.8.2. Entrapment efficiency of GA and its release profile Entrapment efficiency of GA was determined by treating 1 mg of each composite (GAePF and GAeCF) in 1 ml of PBS containing 25 mg of pepsin. The mixture was vortexed for 15 min, centrifuged and the supernatant was analysed using UV spectrophotometer. Following formula was used to calculate the percentage of GA entrapped within the composites.

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ethidium bromide (AO/EtBr) and viewed under fluorescence microscope (Olympus-BX-51). 2.9. Statistical analysis SPSS software version 13.0 was used to carry out the statistical analysis. Data were expressed as mean ± standard deviation (n ¼ 3). One way analysis of variance (ANOVA) with Duncan's test for multiple comparison was used to evaluate the parameters. P values <0.05 were considered significant. 3. Results and discussion 3.1. Characterization In the current study, PF e isolated from the blood collected from slaughter house has been investigated for its potential use as an alternative for CF. The physical and chemical properties of PF and CF were compared and their similarities were explored in this study. Fig. 1a shows the UVevis absorption spectra of PF and CF with both the samples exhibiting maximum absorption at 280 nm, a characteristic peak of proteins with phenyl groups. FTIR spectroscopy was used to study the functional groups present in PF and CF. From Fig. 1b the sharp peak at around 1650 cm1 (amide I) is attributed to the stretching vibrations of C]O groups present along the polypeptide backbone of PF and CF [21]. Bands around 1550 cm1 (amide II) and 1250 cm1 (amide III) represent the NeH bending and CeH stretching vibrations of CF and PF respectively [22] and [23]. When SDS-PAGE (Fig. 1c) was performed, three major bandseAa, Bb chains and geg dimers were obtained at similar positions in PF and CF [24]. Based upon the presence of geg dimers, fibrin is generally classified into two different types e cross linked and non-cross linked fibrin [24]. Cross linked fibrin is commonly used for biological applications due to its increased stability and rigidity. Based up this observation, PF falls under cross linked form of fibrin and hence could be widely used for biological applications as CF. Secondary structure was determined using CD spectroscopy in the fareUV region. Two negative bands (Fig. 1d) at 218 nm and 209 nm, characteristic of besheet and aehelix were observed in both the samples proving the similarity between them. The amino acid composition of PF is in good agreement with those reported earlier [25]. PF contains high percentage of glutamic acid and aspartic acid (Table 1) and is supposed to possess negative charge. Thus, the physical and chemical properties of PF observed in this study are comparable with those of CF.

Entrapment efficiency ¼ (Amount of GA within PF/Total amount of feeding GA)  100%

2.8.3. Anti-cancer activity MTT assay was performed to test the anti-cancer activity of GAePF and GAeCF (10, 25 and 50 mg) on MCF cells. 2.8.4. Analysis of cell death using fluorescence microscopy 1  106 cells were seeded on 24 well plate and incubated overnight. The cells were then treated with 250 mg/ml of PF, CF, GAPF and GA-CF and incubated in a CO2 incubator for 24 h. Cells without any treatment served as control. Following incubation, cells were washed with PBS and stained with acridine orange/

3.2. Cytotoxicity test MTT assay is often used as a primary test to estimate in vitro cytotoxicity of a material. It is an efficient method for testing mitochondrial injury and well correlates with cell viability. NIH 3T3 cells were treated with similar concentrations of PF and CF; MTT assay was performed after 24 h of incubation. As shown in Fig. 2a, no sign of toxicity was observed with any of the concentrations of PF and CF used for the study. Also the percentage of viable cells was similar with PF and CF, thus proving their biocompatible nature.

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Fig. 1. (a) UVevis absorption spectra of PF and CF displaying their maximum absorption at 280 nm. (b) FTIR spectra showing amide I, II and III bands of PF and CF respectively. (c) SDS-PAGE analysis of PF and CF demonstrating the presence of Aa, Bb chains and geg dimers respectively. (d) CD spectral results of PF and CF in the fareUV region.

Table 1 Amino acid composition of PF. Amino acid

Number of residues

Aspartic acid Glutamic acid Serine Histidine Glycine Threonine Alanine Arginine Tyrosine Valine Methionine Phenyl alanine Isoleucine Leucine Lysine

412 464 235 309 154 225 187 131 8 37 204 131 185 275 256

Cell membrane is negatively charged due to the presence of sialic acid and phospholipids. So, they have reduced association with negatively charged materials. Based upon the amino acid composition, it is evident that PF is negatively charged due to the presence of high levels of glutamic acid, which may be the possible reason for good cell viability in PF treated cells [26]. 3.3. Haemolysis Haemolysis is a vital parameter to determine the biocompatibility of a biomaterial [27]. It is widely used to study the interaction of a material with red blood cells (RBCs). When a foreign material is exposed to blood circulation, it communicates with RBCs and may cause damage to their cell membranes resulting in the release of haemoglobin. The amount of haemoglobin released indicates the degree of toxicity of the material. In the current study, zero percent haemolysis was observed with PBS and 100% haemolysis was observed with water. As shown in Fig. 2b the percentage of hemolysis for all the concentrations of PF used for the analysis was less than 2% and comparable with those obtained for CF. As per ASTM standard F756, a material with percentage of haemolysis <2% is considered to

Fig. 2. (a) MTT assay illustrating the viability of NIH 3T3 after treating with similar concentrations of PF and CF. (b) Haemolytic assay for similar concentrations of PF and CF, after 1 h of incubation in red blood cells.

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be non-haemolytic. Hence, PF is proved to be hemocompatible and could be extensively used for various applications. 3.4. Mechanical strength Mechanical strength is an important characteristic feature of a biomaterial for tissue engineering applications. Tensile strength serves as an indicator for mechanical strength of a material. In the current study, both PF and CF exhibited similar tensile strength of 0.98 MPa and hence can be used as suitable biomaterials for tissue engineering applications. 3.5. Rate of degradation In vitro degradation of PF and CF were studied by incubating each sample in PBS for 1, 3, 5 and 7 days. Both PF and CF exhibited

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similar rate of degradation which is as follows e On day 1, 11% weight loss was observed in both PF and CF. On day 3, 20% weight loss was observed in both PF and CF. On day 5, 31% weight loss was observed and on day 7, 48% weight loss was observed in both PF and CF respectively. This shows the increased biostability of PF and CF which is an important factor for tissue engineering applications. 3.6. Bone tissue engineering 3.6.1. Cell proliferation assay Fig. 3a and b shows the density of MG-63 cells in the presence of similar concentrations of PF and CF at different time of incubation. In both the cases, about 97e99% of the cells were viable on days 1 and 3 with significant increase on day 5. It has been reported that fibrin is involved in cellular division and tissue formation by activating various growth factors, integrins and extracellular matrix

Fig. 3. (a and b) Proliferation of MG-63 cells treated with similar concentrations of PF and CF. (c and d) Impact of PF and CF on the ALP activity of MG-63 cells at different time interval. (e and f) Alizarin red assay for MG-63 cells after 14 days of incubation with PF and CF. The inset shows the microscopic images of alizarin red stained cells. (g and h) SEM image of the SBF soaked PF and CF. The inset shows the EDX analysis of SBF soaked PF and CF respectively.

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components, which could the potential cause for such increased cellular viability in PF treated cells. 3.6.2. ALP assay ALP acts an essential marker of osteoblastic differentiation as it promotes bone formation by generating inorganic phosphate (Pi), an inducer of hydroxyapatite formation [28]. During the initial stages of osteogenesis, ALP activity is found to be unregulated and serves as an important feature of ossification. Fig. 3c and d shows significant increase in the level of ALP on all days of experimentation in PF and CF treated cells when compared with control. The increased ALP activity may be due to PF, as it has been proved that fibrin acts as an excellent modulator of Pi leading to the bone formation. 3.6.3. Calcium assay Deposition of calcium and phosphate ions may be used as an effective indicator of osteogenesis. It has been proved that Ca2þ and Pi ions serve as nucleating agents for the formation of hydroxyapatite, a main component of bone. ARS binds with calcium to form an orange red coloured alizarin red S-calcium complex and acts an efficient marker for matrix mineralization [29]. When compared with the control cells, increased staining is observed in PF (Fig. 3e) and CF treated cells (Fig. 3f). These results prove that both PF and CF facilitate good mineralization and bone formation in similar manner. 3.6.4. Formation of bone like apatite Incubating a material in SBF for about 21 days is a widely used technique to determine the bone bonding ability of a biomaterial. SBF contains ion concentrations approximately equal to that of human blood plasma and commonly used for studying the bioactivity of artificial materials. Fig. 3g and h shows the SEM images of apatite coatings deposited on the surface of PF and CF after 21 days of incubation. EDX analysis shows the Ca/P ratio as 1.51 in both the

cases, thus demonstrating the formation of Ca rich apatite layers on the surface of SBF soaked PF and CF. In general, the apatite formation induced by SBF immersing can be achieved by carboxyl acid functional group existed on the materials surface. From these results, it is evident that PF exhibits similar osteogenic properties as CF and hence it can serve as an effective alternative of CF for bone tissue engineering applications. 3.7. Drug delivery GA is a polyphenolic compound with promising anti-oxidant, anti-cancer and anti-inflammatory properties [30]. GA is found to inhibit the growth of prostrate, breast and lung cancer cells without damaging normal cells [31] and [32]. GA was used as a model drug to study the use of PF for drug delivering applications. It was also loaded within CF to compare the drug delivery properties of GAePF and GAeCF. Fig. 4a represents the FTIR spectra of GA, PF and GAePF respectively. GA displayed the typical phenolic features with the presence of a benzene ringeOH stretching in the range of about 3201e3549 cm1 and OH in plane bending at 1360 cm1 and an aromatic ring C]C stretching within 1400e1610 cm1 [33]. PF exhibited the characteristic amide I, II and III peaks at 1650 cm1 (C]O stretching), 1550 cm1 (NeH bending) and 1250 cm1 (CeN stretching) respectively [14]. The prominent peak at 1550 cm1 corresponding to the NeH bending group of primary amine in PF has disappeared in GAePF depicting the change of primary amine to secondary amine due to the reaction at NH2 sites on PF. Moreover, appearance of a new band at 1737 cm1 (C]O) in GAePF shows the formation of ester bond between hydroxyl groups of PF and carboxyl groups of GA. PF was found to entrap 69% of GA, with its release lasting up to 72 h. The release properties of GA from GAePF was initially fast up to 5 h followed by slow and steady release up to 72 h of incubation (Fig. 4b). The variation in release profile may be due to the initial

Fig. 4. (a) FTIR spectra of PF, GA and GA-PF respectively. (b) Cumulative release of GA from GA-PF and GA-CF. (c) Anti-cancer activity of GA-PF and GA-CF against MCF-7 cells (d) Fluorescence microscopic analysis of control, GA-PF and GA-CF treated cells stained with AO/EtBr.

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burst release of GA (about 15%) absorbed onto the surface of PF, followed by its constant release. PF immersed in PBS buffer system may degrade slowly resulting in the formation of interconnected pores on the exterior and interior of PF. This porous structure must have helped in the slow and sustained release with 80% in 72 h. Interestingly CF also exhibited similar drug loading and release characteristics, thus proving its similarity with PF.

3.7.1. Anti-cancer activity MTT assay showed dose-dependent toxicity of GAePF and GAeCF on MCF-7 cells (Fig. 4c). Significant reduction in cell viability was observed for up to 49% in GAePF (50 mg) treated cells. Moreover the anti-cancer activity of GAePF was significantly higher when compared with the pure form of GA. This increased antiproliferative activity of GAePF may be due the non-specific interface between GAePF and cell membrane. Nevertheless, empty PF did not exhibit any toxic effect on treated cells. As shown in Fig. 3c, GAeCF reduced the viability of MCF-7 cells in similar percentage as observed with GAePF. These results prove that both PF and CF exhibit similar drug delivering properties and hence PF can be used as an effective vehicle for drug delivery applications.

3.7.2. Analysis of cell death using fluorescence microscopy Fluorescence microscopy was used to study the cell death induced after treating with 25 mg (IC50 value) of GAePF and GAeCF. When cells were treated with AO/EtBr, some appeared green representing normal cells and some appeared red representing necrotic cells. Those cells with mid green to orange colour (in web version) indicated apoptotic bodies formed due to nuclear shrinkage and blebbing (Fig. 4d). It has proved previously that GA can inhibit the growth of HeLa cervical cancer cells via apoptosis and/or necrosis. GA induced apoptosis is related with oxidative stress derived from reactive oxygen species, increased intracellular Ca2þ level and mitochondrial dysfunction [34,35]. From the above results, PF exhibits similar properties as those of CF and hence can be as an efficient alternative of CF in bone tissue engineering and drug delivery applications.

4. Conclusion In the present study, the physico-chemical properties of PF and CF were studied and the similarities between them were identified. MTT and haemolytic assay performed under different exposure conditions demonstrated the non-cytotoxic behaviour of PF and CF. Moreover MG-63 cells treated with PF and CF showed similar proliferation and mineralization. Increased synthesis of alkaline phosphatase and calcium deposits were observed in both PF and CF treated cells, thus proving their osteogenic property. Additionally 69% of GA was loaded within PF, with the sustained GA release for up to 72 h. Dose-dependent toxicity of GAePF and GAeCF was observed on MCF-7 cells. Fluorescent microscopic results showed that both GAePF and GAeCF inhibited the growth of MCF-7 cells through apoptosis and/or necrosis. From these findings, it is apparent PF can be used as efficient alternative for CF in bone tissue engineering and drug delivery applications.

Acknowledgement We thankfully acknowledge the funding support granted by the Department of Science and Technology, India (IF 10511).

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