Thermal and UV radiation effects on dynamic viscosity

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Nov 16, 2016 - were protected from the light since the UV sensitive riboflavin content acts as an onset .... UV/visible absorbance and fluorescence measurements .... tin based on spectroscopic and electrophoretic analysis, J. Food Pharm.
Journal of Molecular Liquids 225 (2017) 147–150

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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq

Thermal and UV radiation effects on dynamic viscosity of gelatin-based riboflavin solutions Barış Demirbay a, Can Akaoğlu a, İlke Ulusaraç b, F. Gülay Acar b,⁎ a b

Physics Engineering Program, Institute of Science and Technology, İstanbul Technical University, 34469 Maslak, İstanbul, Turkey Physics Engineering Department, Faculty of Science and Letters, İstanbul Technical University, 34469 Maslak, İstanbul, Turkey

a r t i c l e

i n f o

Article history: Received 15 July 2016 Accepted 2 November 2016 Available online 16 November 2016 Keywords: Biocompatible materials UV sensitivity Newtonian fluids Arrhenius model Shear thickening Dilatant fluids

a b s t r a c t Two samples of gelatin based solutions which consist of 2% of biocompatible gelatin content and 0.5% of UV sensitive riboflavin sample were constituted at the room temperature. During the preparation process, the solutions were protected from the light since the UV sensitive riboflavin content acts as an onset initiator agent of photocrosslinking reaction in case of light radiation. To clarify the effect of UV radiation on viscosity of specimens at different temperature range, one of the prepared specimens were radiated to UV for a while at the wavelength value where the biologically suitable one of the peaks is determined in UV/visible absorbance. Afterwards, the viscosity measurements of UV radiated and un-radiated gelatin based riboflavin solutions were performed with the speed value where the most reliable torque value is shown, at different temperature values. Experimentally, it was understood that there was no change in fluid types as a result of UV radiation, however; viscosity values of both solutions diminished at the elevated temperature levels. The viscosity of pure gelatin content was lower than gelatin-RF content and addition of RF increased the viscosity. All samples were mathematically modeled by Boltzmann equation as each specimen obeyed Arrhenius model. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The use of biocompatible materials, particularly biopolymers which can be obtained from natural resources, are the new trend for diverse applications including the fields of drug delivery systems, tissue engineering and medicine since these materials can be integrated to biological systems of living beings [1–3]. Biopolymers are as long as being biocompatible which means biodegradable, adaptive and no toxic influence on living cells and medium, these materials can be implemented in many ways in the form of films, liquids or gels. At this point, gelatin which can extensively be used either in gel phase or film form, can be given as one of the beneficial biopolymers. Gelatin is a natural biopolymer that can easily dissolve in water, and it can be obtained from skin, bones and collagen of animals via the hydrolysis or the reaction of thermal denaturation [4–5]. Gelatin can be extracted from different animals like fish, porcine and so forth. From food industry to encapsulation of the pharmaceuticals, the usage area of gelatin extends [6–8]. Besides of industrial applications of gelatin, the medical implementations have been taken a significant place as it brings about tremendous benefits to mankind. Basically, gelatin sponges act as tampauning influence when it is used whereon wounds [1]. As protein-like gelatin is a natural chemical cross-linker, the cross-linked bonds of gelatin molecules can trap the target pharmaceuticals in drug delivery systems [9]. In tissue ⁎ Corresponding author. E-mail address: [email protected] (F. Gülay Acar).

http://dx.doi.org/10.1016/j.molliq.2016.11.053 0167-7322/© 2016 Elsevier B.V. All rights reserved.

engineering, gelatin is a beneficial resource to be used as scaffolds since it possesses a great regenerative impact on tendons, bones and scaffolds [10–11]. Particularly, in the division of the ophthalmology, gelatin molecules act as cell carrier in endothelial cells and the tissue of retina can be stabilized and protected as well [12]. Apart from chemical cross-linking (CXL), photo-CXL in the presence of other UV sensitive biomaterials can be an effective solution to heal and to fix up some ocular disorders related to sight problems such as the disease called Keratoconus which is defined as a cone growth to outer layer of cornea. Riboflavin (vitamin B2) is a UV sensitive biocompatible material which has been widely used to deactivate the progression of the corneal disorder in the treatment process of Keratoconus under UV light as the flavin agents inside the whole macromolecule give rise to realize the reaction of photo-CXL [13–16]. As a crosslinking agent, the use of RF varies from medical treatments to bio-inks as it can strengthen the mechanical properties of the solution form in case of UV radiation [17]. Thus, the ability of crosslinking of biomaterials are quite significant as that of property brings about benefits to health science and medicine. It is no doubt that the adaptation and biocompatibility of materials in the living systems like cells and tissues depends upon the environment of interior liquids and blood. For example, in drug delivery systems, for the transportation of drugs to the target regions in physiological systems of human body, viscosity of environment takes a significant place to assure the homeostasis. Because the biomaterials must be adapted and obeyed to flow type and the viscosity of the medium. Depending on the fluid type of the biological environment for UV sensitive biomaterials, the

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viscosity of the fluid biomaterials which are aimed to be used for any purpose, can be controlled and modified via either the degree of photo-CXL in the wide range of applied UV or the concentration factor of used substances. For drug delivery systems, the mixture containing biocompatible gelatin with riboflavin can be adapted to biological systems and viscosity of interior body, can be controlled by UV radiation at internal temperature levels of human body. Therefore, in our present research, we focused on how UV effects the fluid type and viscosity of gelatin based RF solutions at changing temperature values. The viscosity and fluid types of gelatin based RF solutions were investigated before and after UV radiation at different temperature levels ranged from 18 °C to 45 °C. The fluid types were classified depending upon their mathematical models and types of the shear stress and strain curves. The change in viscosity based on temperature and time were observed at corresponding temperature values respectively. 2. Materials and methods 2.1. Reagents The gelatin (bloom) powder (purchased from Doğa drug industry) and (−)-riboflavin (purchased from Sigma-Aldrich) were used as chemicals in biocompatible solutions. 2.2. Experimental procedure At the first stage, the fluid types of samples were determined at the room temperature about 21 ± 2 °C and the appropriate speed values were obtained based on experimental results. Afterwards, the viscosity measurements were done between 18 °C and 45 °C at these speed values. 2.2.1. Mathematical abbreviations of biocompatible solutions Before experimental process, all the prepared solutions were encoded to make them more understandable in the evaluation step of the experimental charts. The codes of the solutions were given in Table 1. 2.2.2. Preparation of biocompatible solutions Gelatin (bloom) powder was dissolved in distilled water (dH2O) with the proportion of 2% by using magnetic stirrer equipment at 80 °C with the speed of 1200 rpm about 30 min to completely mix the pure gelatin content. Then, riboflavin powder was dissolved in distilled water considering the ratio of 0.5% at 30 °C with the speed of 1200 rpm for 40 min. 2% of gelatin content was used in several biomedical purposes in the literature [18]. It was also found in our previous study that 2% of gelatin content was found to be Newtonian fluid among different amount of gelatin concentrations in the range between 1% and 5% [19]. Besides of this, the usage of Riboflavin even with low concentrations such as 0.5% is well enough to start interactions between the molecules by using UV light and it can be used as a reinforcing agent in order to strengthen mechanical properties of materials [20]. Therefore, these concentrations were used as reference. During the preparation and dissolution of pure RF solution which is miscible with gelatin solution, the glass beaker of the specimen was coated by an aluminum foil to protect the sample from light exposure. 18 ml of two samples of gelatin based

Table 1 Mathematical codes of the solutions. Code of specimen

Gelatin content (%)

Riboflavin content (%)

UV radiation state

2G 2G0.5RF-a 2G0.5RF-b

2 2 2

– 0.5 0.5

– Radiated Un-radiated

RF solutions consist of 80% of pure gelatin solution and 20% of RF solution. 2.2.3. UV/visible absorbance and fluorescence measurements The peaks of absorbance values of one of the prepared samples containing 2G0.5RF were found at the room temperature through using UV/ visible absorbance spectrophotometer (Shimadzu UV-150-02). One of the gelatin based RF solutions were radiated to 6 mW/cm2 UV during 8 min to excite each flavin molecules within prepared gelatinous solutions at 372 nm which corresponds to the determined second absorbance peak. Because in biomedical implementations, it was determined that the radiation at 278 nm which corresponds to the first absorbance peak, leads to distort the structure of DNA while the wavelength of 444 nm which corresponds to third peak causes chemical burns. Corresponding mathematical calculations were given in results and discussion. In order to investigate how UV radiated sample emits back the excitation energy at 372 nm, the UV/visible fluorescence measurements were done at the room temperature by using fluorescence spectrophotometer (Perkin Elmer LS 50). By this way, radiation which is biologically harmless in visible region were observed. 2.2.4. Viscosity measurements The viscosity measurements of UV radiated, un-radiated gelatin based RF and pure gelatin solutions were performed by using rotational low viscometer (Fungi-Lab premium series) and temperature control unit which is laboratory heat bath at temperature range from 18 to 45 °C separately. In the measurements, 18 ml of samples were added to the jacket of viscometer due to its capacity and LCP spindle was used for our low viscous bio-solutions. 3. Results and discussion 3.1. Absorbance and fluorescence measurements In absorbance measurements, the Fig. 1 indicates that the prominent peak value of the solution containing 2% of gelatin and 0.5% of RF, was found at 372 nm respectively. The studies in the literature indicate that the peaks of pure RF content are originally shown at 278 nm, 371 nm and 444 nm [21–22]. The pure gelatin content within gelatin based RF solution caused 1 nm shift only for the second absorbance peak. It was understood that the addition of 2% of gelatin content to pure RF solutions did not alter the original UV range of the material. One sample of 2G0.5RF solutions were radiated to UV at 372 nm for 8 min to initiate the photo-CXL reaction. In fluorescence measurements, one sample of 2G0.5RF was excited by 372 nm where the peak came out in absorbance measurements, to observe in which wavelength the larger energy emits from the material. Based on panel b, the highest energy emission observed at 528 nm a range of green color wavelength in visible region with corresponding intensity value about 300 a.u. 3.2. Viscosity measurements At the beginning of the experiment, the fluctuation which is shown in Fig. 2, took a place for all the solutions since the angular momentum did not conserve. After a retardation time which is about 25 s, the spindle of viscometer resists to flow with regular shear stress values. That's why, after a while the viscosity of 2G and 2G0.5RF solutions did not change with time. In that case, flow types of the solutions were classified as Newtonian fluids and time independent non-Newtonian fluids because corresponding viscosity did not change with the time. Even if the solutions of 2G0.5RF-a and 2G0.5RF-b were observed as dilatant (shear thickening) fluids under constant shear rate, they can not be considered as thixotropic or rheopectic fluids and it means that the fluid behavior of these fluids was independent of time. In order to understand the effect of low and high values of temperatures on

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Fig. 3. Viscosity change vs. 1/Temperature of 2G and 2G0.5RF before and after UV.

Fig. 1. a Absorbance measurement results of 2G0.5RF solutions b Fluorescence measurement results of 2G0.5RF solutions.

viscosity behavior, the measurements were taken at various temperatures separately. The viscosity change by temperatures of 2G, 2G0.5RF-a and 2G0.5RF-b solutions were found and the results were given in Fig. 3. The viscosity measurements were performed at the

Fig. 2. Viscosity vs. time graph of pure gelatin and gelatin based RF solutions at the room temperature before and after UV.

reference speed value of 120 rpm for 2G solutions and 150 rpm for 2G0.5RF solutions since the rotational viscometer gives the most reliable results in the torque range between 40 and 60. As it is seen in the given Fig. 2, the viscosity of 2G value is higher than 2G0.5RF solutions at the room temperature. The addition of RF to pure gelatin content lowered the viscosity value. However, the application of UV radiation almost did not alter the viscosity of 2G0.5RF solutions as the photo-CXL was ineffective. As it is shown in Fig. 3 that the rapid decrease in viscosity for each specimen at the temperature values below 23 °C. Almost the viscosity values were similar to each other for all prepared solutions up to 23 °C. Above this temperature, the viscosity kept slightly decreasing for each specimen. It is clearly seen that there is no specific difference on viscosity behavior of the solutions containing RF before and after UV radiation process. It means that the cross-linked bonds between gelatin and RF molecules did not affect the viscosity of these samples up to 23 °C. However, pure gelatin content had lower viscosity values when it is compared to 2G0.5RF solutions in the range between 23 and 45 °C. It is because 2G0.5RF solutions have higher molecular weight so that the viscosity increased as expected. Increase in temperature stimulate the system of molecular interactions and molecules gain dynamism depending upon its chemical structure and medium. Since the gelatin molecule consists of aliphatic groups, long chain segments, primer amine and NH groups, the mobility of molecule is high and the backbone of whole molecule is flexible. In addition, due to gelatin structure includes its own water compounds per molecule, it ensures more likely to be viscous liquid behavior. Therefore, less viscous flow of pure gelatin content that is 2G was observed. It is possible to be said that cross-linking may occur between hydroxide groups of the chemical structures of gelatin and RF molecules. As it might be expected that the connections of aliphatic groups on the backbone of the gelatin structure, it is possible to restrict movement of whole 2G0.5RF molecule in case of crosslinking where the hydroxide groups break out. Moreover, the UV radiation did not change both fluid type of specimen and viscosity of prepared 2G0.5RF solutions. In our previous study, we found that radiation time was no effect on flow behavior of samples [19]. In response to experimental results, mathematical models of fit lines of points which are shown in charts of viscosity change vs. 1/temperature, reliably obeyed the Arrhenius model. Another name for a powerlaw fluid like studied gelatin based riboflavin solutions with exponential dependence of viscosity on temperature is a first-order fluid. Its formula is given by ΔH

η ¼ η0 þ Ae RT

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Table 2 Mathematical models of samples. Name of specimen

η0 (cP)

A (cP)

ΔH (J/mol)

Adj · R2

2G 2G0.5RF-a 2G0.5RF-b

1.38 1.66 1.70

7.33 1.12 3.40

136.30 ±1.96 163.03 ±1.85 227.92 ±2.91

0.94 0.98 0.96

where η is viscosity constant, η0 is viscosity constant at the reference temperature, A is curve constant, ΔH is activation energy, R is universal gas constant and T is temperature. All corresponding equation constants were given in Table 2 even including approximate errors. Because of the positive activation energy or enthalpy change which is difference between the enthalpy of the products and the reactants, the reaction is endothermic, that is heat is absorbed by the system due to the products of the reaction having a greater enthalpy than the reactants. As heat was added to the investigated solutions, their volumes increase due to their molecules gain speed and spread out. As a result, the density, buoyancy and surface tension was decreased. The movement of their molecules disrupts the imbalanced forces on the surface of the solution and weakens their sheet-like barrier of tightly bound molecules, thereby lowering the surface tension. The penetration becomes easier due to low surface tension. Viscosity will also be affected, as particles accelerate by slipping up and moving apart on each other in random direction. Therefore, it gets easier for them to move past each other and viscosity decreases. Since the temperature of the solutions increased, the molecules make these samples gain more energy and vibrated faster. In case of increment in temperature, the bonds among the molecules get weaker and free spaces tend to increase so that the resistance of the samples diminishes. It was understood that addition of RF into gelatin solution caused increment in activation energy, in other words activation enthalpy of viscous flow which is required to stimulate the molecules per mole, because the molecular weight and molarity of the solutions increased [23]. However, UV radiation lowered activation energy of 2G0.5RF solution due to weakening of bonds between gelatin and RF molecules. Therefore, 2G0.5RF-b have greater activation energy among all the samples. 4. Conclusion To sum up, in this research, viscosity behaviors of the substances which are given by 2G, 2G0.5RF-a and 2G0.5RF-b were investigated at different temperature values. It was experimentally understood that the fluid types of each specimen were classified as Newtonian and time independent Dilatant fluids. Besides, UV radiation time did not influence on viscosity behavior of biocompatible samples containing gelatin and RF. In entire range of the temperature, corresponding viscosity values of the pure gelatin content were almost low when it is compared to 2G0.5RF samples. Addition of RF to gelatinous solutions gave rise to enhance the viscosity of solutions and flow resistance as molecular weight of the mixture were increased. All prepared solutions were mathematically modeled by Boltzmann equation since the fit curves of all samples obeyed Arrhenius model and activation energy of the solution of 2G0.5RF-b was found higher than each other. Since the viscosity level of these solutions can not be changed in the presence of UV radiation at 372 nm, fluidity of solutions did not influence. However, by

taking concentration of used material and medium into consideration, these solutions can be used as potential solutions in the fields of drug delivery systems to mark cancer cells and hydrogels to heal wounds.

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