Preparation, characterization, and in vitro release study of curcumin-loaded cataractous eye protein isolate films
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Curcumin, a naturally occurring polyphenol despite having therapeutic properties, has limited application due to its poor solubility in aqueous medium. The aim of this study is to explore a new protein source developed as a protein film, for the delivery of curcumin. The cataractous eye protein isolate (CEPI) is a mixture of proteins obtained after phacoemulsification surgery of the eye lens. The films have been prepared from the discarded CEPI and investigated for potential applications as a delivery system for curcumin. The films were prepared using glycerol as the plasticizer and glutaraldehyde as the cross-linker. The mechanical properties of the films were monitored by using nanoindentation techniques (NINT). The curcumin-loaded films were characterized by Fourier transform spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), and thermogravimetric analysis (TGA) which established the incorporation of curcumin in the protein matrix. The loading efficiency of the films was found to be dependent on the degree of cross-linking of the films. In vitro release studies showed an initial burst release followed by a sustained release. The release rate is higher at pH 4.5 compared with pH 7.4. The released aliquots of curcumin-loaded films also exhibit antibacterial effects against Staphylococcus aureus. Our findings will be generally beneficial for the further reshaping of protein films as potential delivery carriers.
KeywordsCataractous eye protein Cross-linked film Curcumin Drug delivery system
The biomaterials used for the purpose of transfer are mainly comprised of proteins, polysaccharides , carbohydrates , biocomposites [26, 27, 28, 29, 30, 31, 32, 33, 34], etc. The main advantage of the protein films as drug delivery vehicles over traditional dosage systems is their biodegradability and biocompatibility. Most efforts are aimed at preparing films, which show a sustained release of the drug over a long period of time . Apart from their potency as delivery vehicles, protein films also exhibit good water and gas barrier properties. In order to achieve desired physicochemical properties of films, several protein sources have been employed for film formation [36, 37, 38, 39].
In this respect, the cataractous eye protein isolate (CEPI) is a novel source of protein that has not been explored for possible applications in this area. The concentration of the soluble crystallin proteins in the eye lenses is significantly high and accounts for over 90% of the total protein content in the lens [40, 41]. In the course of time, the crystallin proteins are modified and/or aggregate within the eye lens causing blurred vision and cataract [42, 43, 44]. Post cataract removal surgery, the concentrated and aggregated protein mass in the cataractous lens is discarded. According to the World Health Organization (WHO), the number of cataract surgeries performed in 2010 was estimated to be around 20 million that is expected to reach ~ 32 million by 2020 [45, 46, 47]. These reports indicate that there is unlikely to be a dearth of the discarded cataractous eye emulsion that is constituted primarily of aggregated crystallin proteins, the natural component of the human eye lens. The main reason for selecting this protein source is due to its purity of protein content and because it is a highly economical protein source that has not been used earlier for film preparation. A previous study demonstrates that our prepared films are fully biodegradable by the trypsin enzyme with a similar range of Young’s modulus as that of the collagen, zein, and soy protein films. Cataract surgery involves the removal of the cataractous lens and the insertion of an intraocular lens. The lens proteins present in the aspirated emulsion isolated from the aggregated mass after surgery is referred to the cataractous eye protein isolate (CEPI). This study also explores the efficacy of this new source of protein films as delivery systems. Our research group has previously worked on this new source of protein for nanoparticle formation . In general, proteins require small molecules to improve their functionality to make the films . Glycerol (GL) is a small polyol compound, which is majorly used as a plasticizer to reduce the inter-molecular attraction within the polypeptide chains . Chemical cross-linking is also used to improve the mechanical properties of the protein film for which different cross-linkers like glutaraldehyde , formaldehyde , glyoxal , and genipin [54, 55] are available to improve the mechanical properties of the protein films. Earlier, these CEPI films have been used as a delivery carrier for ampicillin sodium, a member of the β-lactam group of antibiotics. Studies demonstrated that these films exhibited a slow and sustained release of drug from the matrix over a long period of time. The main motive behind the study is to explore the effectiveness of this protein film for the delivery carrier of hydrophobic and hydrophilic compounds. The findings of this study will allow us to explore the plausible modifications and improvements of these CEPI films for future applications.
The CEPI protein films were loaded with curcumin and the curcumin-loaded films characterized by Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), and thermogravimetric analysis (TGA). The mechanical property was evaluated by nanoindentation (NINT). The loading efficiency of curcumin was determined with varying the amounts of the cross-linker glutaraldehyde. In vitro release studies were performed at two different pH values. We have also checked the antibacterial property of the released aliquots collected at different time intervals against Staphylococcus aureus, which is mainly present in skin infections.
2 Materials and methods
Glutaraldehyde (grade I, 70% solution) and cellulose membrane (MWCO 12.6 kDa) were purchased from Sigma Chemical Co. (St. Louis, USA). Curcumin was purchased from Central Drug House Pvt. Ltd. (India). The bacterial culture media soybean-casein digest medium and agar were purchased from Himedia (India). The discarded cataractous eye protein obtained from phacoemulsification surgery was collected from Sathi Eye Clinic, Kharagpur, India (approved by the ethical committee of IIT Kharagpur letter No IIT/SRIC/DR/2018). All other reagents were of analytical grade from SRL, India, and used as received.
2.2 Isolation of cataractous eye protein isolate
The protein was isolated from the discarded cataractous protein obtained through phacoemulsification surgery on patients aged above 50 years according to the previously reported method with slight modifications . The discarded protein emulsion samples were stored in a refrigerator at 4 °C in sterile bottles. Prior to experiments, the samples were centrifuged at 7900 rpm at 4 °C for 40 min. The supernatant was collected leaving the insoluble mass at the bottom of the centrifuge bottle undisturbed. The supernatant was concentrated by lyophilization using liquid nitrogen at ~ − 45 °C at 25 Pa pressure. The concentrated soluble protein solution obtained was dialyzed using cellulose membranes (MWCO 12.6 kDa) for 48 h in 10 mM phosphate buffer (pH 7.4) followed by double distilled water for the next 48 h. At this stage, the concentration of the purified protein solution as determined by the Bradford assay from this isolate is found to be ~ 8.0 mg/ml which is quite significant. This amount of protein is sufficient for the protein film preparation as we have observed from this study. It is possible to scale up this process of film preparation given the abundance of this pure form of discarded protein content. The dialyzed protein solution was further lyophilized again under the previous conditions and the dry mass that was obtained comprised the cataractous eye protein isolate (CEPI). The powder was stored at 0 °C in sterile vials until further use. The homogeneity of the CEPI has been checked by SDS-PAGE before any experiments and it was found to comprise a range of crystallin proteins, with molecular weights varying from 10 to 50 kDa obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. S1). It has been observed that the purified protein mixture always shows the same set of bands in SDS-PAGE with the above range of molecular weights for each cataractous emulsion sample.
2.3 Preparation of CEPI-based films
The dry protein powder was dissolved in 10 mM potassium monobasic phosphate and sodium hydroxide buffer of pH 8 to make 5% (w/v) protein concentration in the solvent. The protein solutions were allowed to stir for homogeneous mixing. In the alkaline pH range (8–12), the protein structure dissociates and unfolds [57, 58, 59]. These changes enrich the formation of S-S bonds between polypeptide chains in close proximity. To these solutions, glycerol (GL) (50% w/w of CEPI) was added as a plasticizer and stirred for 30 min for the interaction with the peptide chains. Glutaraldehyde (GD) as a cross-linker was added to the plasticized film solutions with 20–40% weight percentage of the amount of protein. These film-forming solutions were heated at 50 °C under constant stirring conditions and then allowed to cool to room temperature. Finally, the solutions were cast in polypropylene petri dishes and dried for 24 h at room temperature. The films were peeled off the casting plates and stored in desiccators maintained with fused CaCl2 as the desiccant till further experiments were performed.
2.4 Preparation of curcumin-loaded CEPI films
The films were washed several times and soaked overnight in water in order to wash off any unreacted glutaraldehyde or impurities. The washed dry films were then dipped in 1 mg/ml curcumin solution (1: 1 methanol: water) for 24 h. The films were then removed and dried at room temperature. Three samples of each type of film (curcumin-loaded GD 20, GD 30, and GD 40 where numerical denotes the wt.% glutaraldehyde with respect to protein weight) were prepared for the analysis.
In a nanoindentation test, the displacement of the indenter is recorded as a function of the applied force in the course of a complete cycle of loading and unloading. The tests were performed at room temperature using a Hysitron TI 950 TriboIndenter (Bruker USA), fitted with a Berkovich indenter with the machine. Each film sample (1 cm × 1 cm) was subjected to displacement-controlled indentation cycles. The indentation was performed with a penetration depth at 400 nm and the cycle of 10-s loading and 10-s unloading time frame with 10-, 20-, and 40-s holding time (creep time). Grid of (5 × 2) indents with 50 μM separation was employed for each measurement. The unloading section of nanoindentation curve has been analyzed by the TriboIndenter machine software that follows the Oliver and Pharr method . The machine-generated hardness and modulus values are reported as mean ± S.D. for at least three measurements for each sample.
2.6 Fourier transform infrared spectroscopy
Attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy was used to understand the features of cross-linking and drug loading on the CEPI films. A Shimadzu IRTracer-100 FTIR spectrophotometer equipped with diamond ATR accessory was employed for analysis. The scanning was performed between 4000 cm−1 and 400 cm−1 ± 0.01 cm−1 wavenumber accuracy. The number of acquisitions and the resolution of each measurement were kept at 32 scans and 4 cm−1. The air was taken as background and correction was done accordingly.
2.7 Field emission scanning electron microscopy
Field emission scanning electron microscopy (FESEM) was used to understand the morphology of the films. The films were cut into pieces of 1 cm × 1 cm dimension and were mounted on aluminum stubs with both-sided adhesive tape. The films were coated with gold. The morphology of the films was observed using field emission scanning electron microscope model no. NOVA NANOSEM 450. The microscope was operated at an accelerating voltage of 5 kV. The samples were examined under low vacuum.
2.8 Thermogravimetric analysis
The thermal degradation of the films was analyzed in a thermogravimetric analyzer TG 209 F3 Tarsus (Netzsch) instrument with a resolution of 0.1 μg. The experiments were performed in the temperature range of 25 to 500 °C at a heating rate of 10 °C min−1. The nitrogen flow was maintained at 50 cm3 min−1. The analyses were performed in triplicate.
2.9 Loading efficiency
2.10 In vitro release
The in vitro release property of the curcumin-loaded films was studied using a dialysis method  at pH 7.4 and 4.5. The films were placed in a measuring cylinder containing 5 ml of respective buffers. Aliquots were drawn at measured intervals and fresh buffer solution was added to keep the volume of the solution unchanged. The absorbance of the aliquots was measured at 427 nm by UV spectrophotometer (Shimadzu 1800) . The concentration of the released curcumin from the loaded protein films at pH 7.4 and 4.5 was calculated from the calibration plot. The calibration plot was constructed using standard curcumin solutions with concentrations in the range 1–18 μM.
2.11 Antibacterial activity
The antibacterial activity of released aliquots of curcumin-loaded films in different pH was performed by the disc diffusion technique. The activity was tested against gram-positive bacteria Staphylococcus aureus (MTCC96). The culture medium was grown overnight by inoculation of bacterial stain into a soybean-casein digest broth at 37 °C. Around 20 ml of soybean-casein digest agar was poured on sterile glass petri dishes and bacteria cells were inoculated on the agar using a spreader. Paper discs (6 mm) were placed on solidified agar plates and 20 μl of each aliquot applied on these discs. The plates were then allowed to incubate at 37 °C overnight and photographed. The antibacterial potency was determined by measuring the diameter (in mm) of the inhibition zone.
2.12 Statistical analysis
All experiments were performed at least thrice and the values reported are the means ± standard deviation (SD).
3.1 Characterization of curcumin-loaded films
Reduced modulus and hardness values of the films with 20% (w/w of CEPI) glutaraldehyde (GD) for different creep times
GD 20% (w/w of CEPI)
Creep time (s)
Reduced modulus (GPa)
0.3679 ± 0.02
0.0163 ± 0.0001
0.4868 ± 0.12
0.0136 ± 0.004
0.5101 ± 0.17
0.0167 ± 0.008
3.3 Fourier transform infrared spectroscopy
3.4 Field emission scanning electron microscopy
3.5 Thermogravimetric analysis
3.6 Loading efficiency
3.7 In vitro release
3.8 Antibacterial activity
Curcumin is one of the most popular natural polyphenols widely used for a variety of purposes. However, the inherent solubility problem of curcumin in aqueous medium restricts its application, particularly in the therapeutic field. In order to address this problem, we have attempted to prepare film formulations prepared from a biodegradable and low-cost protein source, the cataractous eye protein isolate (CEPI). We have prepared curcumin-loaded films with various amounts of glutaraldehyde as a cross-linker. We have found that the curcumin-loaded films are yellow in color. A similar color of curcumin-loaded gelatin films has been reported previously . The Nanoindentation study reveals that the films are elastic in nature and have sufficient modulus and hardness to remain intact and malleable. The film stability mainly depends on various interactions like van der Waals, electrostatic, and hydrogen-bonded and covalent interactions like the disulfide bridges [81, 82, 83, 84]. Apart from the abovementioned interactions, in the presence of glutaraldehyde, new covalent bonds are formed in the film structure. In this cross-linked network, the mobility of polypeptide chains is restricted resulting in the formation of mechanically strong protein film that can be used for further applications. A previous study demonstrates that our prepared films are fully biodegradable by the trypsin enzyme and that they have a similar range of Young’s modulus with that of the collagen, zein, and soy protein films. In addition, the films were also found to be biodegradable and were completely disintegrated by the trypsin enzyme (Fig. S4). The cataractous eye protein isolate preparation results in the content of crystallin proteins which are globular in nature with a high extent of β-sheet. This protein source contains a wide variety of the amino acids that impart higher water solubility and biodegradability compared with the other proteins used such as collagen . Collagen is a fibrous protein which is water-insoluble and has a major content of primarily proline, hydroxyproline, and glycine making it resistant against trypsin [86, 87]. The higher solubility of the cataractous eye protein isolate film over collagen and several other proteins renders it beneficial for specific applications related to biodegradation. This implies that the study gives an insight to the wide range of possibilities that the film may be utilized for which is the subject of a future study. The FTIR study is indicative of the loading of the curcumin in the film matrix. The peak values of the protein film and curcumin appear together in the curcumin-loaded protein film, which indicates that both retain their chemical identity on loading. The values are shifted slightly due to the various non-bonded interactions that occur between curcumin and the protein film, which are most likely comprised of hydrogen-bonded interactions. Liu et al. and Reddy et al. have also reported similar observations in the case of soy protein and carrageenan where they have mentioned that primarily hydrogen bond interactions occur within the matrix [19, 72]. Further, the loading of the curcumin is also confirmed by SEM analysis. The curcumin-loaded film shows a crystalline type of morphology that has previously been reported [20, 73]. The thermal stability of the films is significantly improved by the incorporation of curcumin as revealed from the thermogravimetric analysis. Literature reports suggest that the improvement occurs due to the less polar nature of curcumin. In some cases, it has been reported that the thermal stability remained unchanged  or decreased  upon curcumin loading. It has been suggested that the lower thermal stability is likely due to either a reflection of the small amount of curcumin present or due to the weakening of the interaction within the polymer matrix. Interestingly, the improvement in thermal stability in this case is attributed to the even dispersion of curcumin in the film and the relatively strong interaction between these two components . The loading efficiency of curcumin from the films strongly depends on the amount of cross-linker. Generally, with an increase in the amount of cross-linker, there is an increment of tortuosity, i.e., more turns and tangles that develop in the polymer matrix which in turn reduces the void spaces to accommodate the drug molecules. Curcumin encapsulation is found to be reduced with an increase in the degree of cross-linking. Similar trends in the change of curcumin loading efficiency with the degree of cross-linking for soy protein and elastin protein films have been reported by Reddy et al.  and Martinez et al. . The in vitro release profiles of curcumin show an initial burst release up to 5 h followed by a sustained release up to the time of observation. The burst release occurs due to the loosely bound curcumin on the film surface while the entrapped curcumin is released slowly over time. Previously, Govindaraj et al.  reported ~ 12% curcumin release from polyacrylonitrile films after 24 h. On the other hand, Manna et al.  observed 50% curcumin release from curcumin-loaded carboxymethylated guar gum grafted gelatin film in PBS after 24 h of incubation. We observe ~ 51% curcumin release from the CEPI film at physiological pH after 24 h. Although the solid curcumin-loaded film does not possess antibacterial effects [18, 79], released aliquots demonstrate moderate antibacterial effect against Staphylococcus aureus. Curcumin has been reported to bind with the peptidoglycan part of the bacterial cell wall and cause leakage to the cell membrane that eventually leads to cell death [92, 93]. A similar zone of inhibition has been reported earlier for curcumin-loaded films . Low amounts of curcumin used for the formation could be the reason that it does not show a large area of inhibition compared with the previously reported literature . The CEPI protein film alone does not possess any antibacterial activity against Staphylococcus aureus. This type of material could thus prove useful as wound-healing material as it may provide a mild but quick antibacterial effect at the target sites. All of the above findings suggest that our biodegradable films have good mechanical properties and moderate antibacterial properties that make them good candidates as potential carriers for possible therapeutic applications.
Curcumin-loaded CEPI films have been prepared by a simple casting and adsorption method. This discarded protein source is a highly economical protein source that has not been used earlier for film preparation. It has been observed that the Young’s modulus of the films prepared from this new source of protein is comparable with protein films from other sources. The cross-linked films are mechanically strong with a reduced Young’s modulus of 0.5 GPa and hardness of 0.016 GPa. The Hydrogen-bonded interactions play a major role between curcumin and the protein as evidenced from the FTIR study. The thermogravimetric study suggests an improved thermal stability of the curcumin-loaded films due to the evenly dispersed curcumin within the film network. The SEM images depict the crystal-like morphology of curcumin on the film surface. The loading efficiency of curcumin is found to be dependent on the degree of cross-linking and is highest for 20% (w/w of CEPI) for the glutaraldehyde cross-linked film. In vitro release studies revealed a slow and sustained release of curcumin from the films. An extent of release of ~ 85% and ~ 51% of curcumin is observed pH 4.5 and 7.4 respectively after 24 h from the film matrix. Aliquots extracted show a fair extent of antibacterial activity against Staphylococcus aureus. The potential use of the films that have been shown to be biodegradable will help unveil newer applications. Further applications in the pharmaceutical area appear to be a possibility for the curcumin-loaded CEPI films that could be extended to other naturally occurring compounds with therapeutic potential.
The authors are grateful to Prof. Madhab Chandra Das (Department of Chemistry, IIT Kharagpur) and his group for providing TGA facilities. The authors would like to acknowledge the Central Research Facility, IIT Kharagpur for providing experimental facilities.
SDG is grateful to SSLS (Signals and Systems for Life Science) project grant Ministry of Human Resource Development, Government of India (F.NO. 4-23/2014-TS.I, Dt 14-02-2014). SP and PG are thankful to IIT Kharagpur and CSIR respectively for their fellowship. The authors acknowledge the International year of chemistry (IYC) grant from the Department of Science and Technology for the FESEM facility in the Department of Chemistry, IIT Kharagpur.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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