Doi: http://dx.doi.org/10.15446/rev.colomb.quim.v45n3.58760

Anti-pepsin activity of silicon dioxide nanoparticles

Actividad antipepsina de nanopartículas de dióxido de silicio

Atividade antipepsina de nanopartículas de dióxido de silício

Hussein Kadhem Al-Hakeim^1*, Khlowd Mohammed Jasem², Shatha Rouf Moustafa³

¹ Department of Chemistry, Faculty of Science, Kufa University, Kufa, Iraq.
² Department of Chemistry, College of Science, Kerbala University, Kerbala, Iraq.
³ Clinical Analysis Department, College of Pharmacy, Hawler Medical University, Havalan City, Erbil, Iraq.
Corresponding author: headm2010@yahoo.com

Recibido: 30 de Junio de 2016. Aceptado: 9 de Septiembre de 2016.

Abstract

SiO₂NPs as an inhibitor of pepsin enzyme for treatment of gastro-esophageal reflux disease (GERD) were investigated. Silicon dioxide nanoparticles (pepsin coated SiO₂NPs) are among the safest nanoparticles that can be used inside the human body. The activity of pepsin before and after the addition of certain amounts of the NPs to the reaction mixture was measured spectrophotometrically. Furthermore, these experiments were repeated at different temperatures, different weights of NPs, and different ionic strengths. The kinetic parameters (K_m & V_max) of the pepsin-catalyzed reactions were calculated from the Lineweaver-Burk plots. The results showed that there is a significant reduction of pepsin activity by SiO₂NPs (V_max of free pepsin = 4.82 U and V_max of the immobilized pepsin = 2.90 U). The results also indicated that the presence of ionic strength causes remarkable reduction of pepsin activity. It can be concluded the best condition for inhibition of pepsin activity is by using a combinationof SiO₂NPs and high concentration NaCl at 37 °C.

Keywords: GERD, SiO₂nanoparticles, pepsin, enzyme inhibition.

Resumen

Se usaron nanopartículas de dióxido de silicio como inhibidores de la pepsina para el tratamiento del reflujo gastroesofágico (GERD). Estas nanopartículas (SiO₂NPs recubiertas de pepsina) son unas de las más seguras y pueden usarse en el cuerpo humano. Se midió a través de espectrofotometría la actividad de la pepsina antes y después de añadir cierta cantidad de NPs a la mezcla reactante. Adicionalmente, se repitieron estas pruebas a diferentes temperaturas, variando el peso de las NPs y la fuerza iónica. Se calcularon los parámetros cinéticos (K_m y V_max) de las reacciones catalizadas con pepsina a través de las gráficas de Lineweaver-Burk. Los resultados mostraron que, usando SiO₂NPs (V_max de pepsina libre = 4.82 U y V_max de pepsina inmovilizada = 2.90 U) y a través de la presencia de fuerza iónica, la actividad enzimática se reduce significativamente. Se concluye que la mejor condición para inhibir la actividad enzimática es usando una combinación de SiO₂NPs y una alta concentración de NaCl a 37 °C.

Palabras clave: GERD, nanopartículas de SiO₂, pepsina, inhibición enzimática.

Resumo

Palavras-Chave: GERD, nanopartículas de SiO₂, pepsina, inibição enzimática.

Introduction

Gastroesophageal reflux disease (GERD) evolves when reflux of stomach contents causes complications into the esophagus (1). GERD is a popular disease with a prevalence of 10% - 20% in the western countries (2) and some researchers reported up to 35.9% (3), but its risk factors and causes are not clearly known (4). The disease causes various symptoms, among which are heartburn and regurgitation, which affect up to 30% of the population and continue to increase (5-7). Pepsin, which is the most important substance in the gastric contents for the necrosis of the mucosal tissues, plays the main role in the formation of GERD and associated diseases (8).

Although proton-pump inhibitors are known to alleviate symptoms in most patients, a significant portion of patients continue to present GERD (9). There is a wide range of specific inhibitors that can bind to the active site and effectively remove the activity of pepsin, one of the best known ones is pepstatin, a specific pepsin inhibitor, which at acidic pH, tightly binds to the catalytic site of both pepsin and its precursor pepsinogen (10). The best way to determine K_m (the concentration of substrate at V_max/2) and V_max (Maximum velocity of an enzyme-catalyzed reaction at definite conditions) values of the basic Michaelis-Menten equation is by taking the reciprocal of both sides of equation to give the double reciprocal equation or Lineweaver-Burk's equation.

Where V₀ represents the initial velocity or the activity of the enzyme-catalyzed reaction. A plot of 1/V₀ versus 1/[S] yields a straight line with an intercept of 1/V_max and a slope of K_m/V_max (11).

Use of NPs to inhibit the activity of papsin in vitro as a model for the treatment of GERD was performed and published previously (12). Silicon dioxide nanoparticles (SiO₂NPs), also known as silica NPs or nanosilica, are the basis for a great deal of biomedical research due to their stability, low toxicity, and ability to be functionalized with a range of molecules and polymers (13). SiO₂NPs have received an intensive attention by scientific community due to its broad applications in biomedical and biotechnological fields such as drug delivery, gene therapy and molecular imaging, cancer therapy, and enzyme immobilization (14). In addition, it is widely used in cosmetics, food, varnishes, papermaking, and drugs (15). Fruijtier-Polloth et al. (16) evaluated the toxic effects and safety of SiO₂NPs and concluded that they are as safe as conventional SiO₂. Amorphous SiO₂NPs are widely used in food products, for example, as thickeners, anticaking agents, carriers of fragrances and flavors, and additives (17). The aim of the present study is to optimize the process of pepsin inhibition by SiO₂NPs as a possible new treatment for GERD.

Materials and methods

Reagents

Characterization of NPs

The SiO₂NPs used in the present study were visualized using SEM and TEM techniques to further confirmation for their shape and size. The TEM studies were performed by using a JEM-2010 instrument working with an acceleration voltage of 200 kV. SEM images were arried out by using a Hitachi S-4800 SEM at 20 kV.

Estimation of pepsin activity

The enzyme activity was determined by a kinetic method (18). The principle depends on the fact that pepsin cleaves peptides from hemoglobin which are soluble in trichloroacetic acid (TCA). The tyrosine and tryptophan content of these TCA-soluble peptides is determined by the measurement of the extinction at 280 nm. Briefly, pepsin is dissolved in 0.01 N HCl to obtain a concentration of 0.5 mg/ mL. Just prior to assay there is another dilution in 0.01 N HCl to a concentration of 5-20μg/mL.

The steps of the method were as follows: 1 mL of hemoglobin substrate was pipetted into test tubes containing 0.2 mL of the diluted pepsin at 37 °C. After 10 min, the reaction was stopped by adding 2 mL of 5% TCA. The tubes containing the reaction mixture were removed from water bath after 5 min and clarified (filtrates should be clear).

E280 nm was read of filtrate and subtract E280 nm of the appropriate blank using spectrophotometer (Model 721-Taiwan) set at 280 nm and 37 °C. The method of estimation of pepsin activity was repeated at different temperatures (22, 27, 32 and 42 °C). A unit of pepsin enzyme was defined as an amount of enzyme which renders TCA soluble 0.001 E280 nm per minute at 37 °C, using a denatured hemoglobin substrate.

Inhibition of pepsin activity by SiO₂NPs

To study the inhibition of pepsin by SiO₂NPs, a weight of 19.8 mg of SiO₂NPs was weighed by Sartorius balance (Model A200) dissolved in 10 mL of 0.01N HCl, and agitated by ultrasonic water bath (IsoLab, Germany) at 37 °C for 20 min. Five mg of pepsin were added to the SiO₂NPs-containing tubes and incubated at 37 °C for 30 min. These amounts of pepsin and NPs were calculated to produce pepsin monolayer on the SiO₂NPs. SiO₂NPs and pepsin surface areas were calculated in order to estimate theoretically the enough number of pepsin to cover the surface of one spherical SiO₂NP in one layer manner.

The calculations describe the pepsin and SiO₂NPs properties (radius, density, mass of one NP, and the volume of one NP) to calculate the surface areas of pepsin and SiO₂NPs which were 1.50 x 10^-13 cm² and 4.94 x 10^-11 cm², respectively. The number of pepsin molecules that can cover one SiO₂NP in a monolayer manner was obtained from the division of one SiO₂NPs surface area on one pepsin surface area ≈ 329 molecules of pepsin per SiO₂NP. This means that a 5 mg of pepsin were required to cover 19.8 mg of SiO₂NPs to obtain a monolayer of the adsorbed pepsin.

Effect of temperature on the inhibition of pepsin activity by SiO₂NPs

To study the temperature effect in the presence of SiO₂NPs, 19.8 mg of SiO₂NPs was dissolved in 10 mL of 0.01 N HCl, and agitated by ultrasonic water bath at different temperatures (22, 27, 32, 37 and 42 °C) for 20 min. Five milligrams of pepsin were added to each tube and incubated at 42 °C for 30 min. The method of estimation of pepsin activity was repeated and the pepsin activity was estimated after adding the SiO₂NPs

Effect of a combination of ionic strength and SiO₂NPs on the inhibition of pepsin activity

To study the temperature effect in the presence of SiO₂NPs7 F9H8 mg of SiO₂NPs was dissolved in F° mL of °H°F N HCl7 and agitated by ultrasonic water bath at different temperatures qkk7 kB7 Ek7 EB and Mk °C8 for k° minH Five milligrams of pepsin were added to each tube and incubated at Mk °C for E° minH The method of estimation of pepsin activity was repeated and the pepsin activity was estimated after adding the SiO₂NPsH.

Effect of a combination of ionic strength and SiO₂NPs on the inhibition of pepsin activity

To study the effect of the ionic strength in the presence of SiO₂NPs, 15 mg of SiOkNPs and FHE mg of NaCl were dissolved in 10 mL of 0.01 N HCl, and agitated by ultrasonic water bath at 37 °C for 20 min. Five mg of pepsin were added to the tubes and incubated at 37°C for 30 min. The method of estimation of pepsin activity was repeated and the pepsin activity was calculated. The protocol of the research is presented in Figure 1.

Results and discussion

The images of SiO₂NPs taken by SEM and TEM are presented in Figure 2. It is clear that the shape of SiO₂NPs is spherical with a particle size around 39 nm.

Effect of SiO₂NPs weight on the pepsin catalyzed reaction

The activity of the enzyme was measured by using seven different concentrations of hemoglobin (3.01, 6.21, 9.31, 12.41, 15.52, 18.62, and 21.72*^10-5M) and five different weights of added SiO₂NPs (19.8, 39.6, 59.4, 79.2 and 99 mg) to the 10 mL of the 0.01 N HCl to prepare SiO₂NPs solution. These experiments were used to examine the effect of SiO₂NPs weights on the activity of pepsin catalyzed reaction. The Lineweaver-Burk plots of the five experiments, were plotted in Figure 3.

The results in Figure 3 revealed that the presence of SiO₂NPs causes reduction in the V_max of pepsin catalyzed reaction. The values of V_max and K_m are shown in Table 1.

The data in Table 1 demonstrate the effect of weight of SiO₂NPs on pepsin activity. These weights were selected from the calculations of the amount of SiO₂NPs needed to be coated with a monolayer of pepsin molecules. The first weight (19.8 mg) of SiO₂NPs in the reaction mixture represents a NPs coated with a monolayer of pepsin molecules. The results showed that V_max of free pepsin (4.82 U) decreases to 2.90 U when the enzyme was immobilized on the surface of SiO₂NPs. The activity continued to decrease as the weight of the added NPs increases until becoming 1.75 U when the weight added is 99 mg. It is clear that the SiO₂NPs has remarkable inhibitory effect on pepsin activity. Most of the reduction in the pepsin enzyme activity is due to the change in the secondary structures of the whole enzyme and particularly in the active site structure. These changes are caused by the adsorption forces between the surface of the NPs and various chemical groups of the pepsin molecules. The attractive forces are strong enough to hold molecules on the NPs surface and modify the H-bonding that constitute the secondary structure of the pepsin molecules. Therefore, the change in the secondary structure is the most probable cause for the decrease in the activity of the immobilized forces.

Studies indicate that the activity of lysozyme adsorbed onto SiO₂NPs is lower than that of the free protein, and the fraction of activity lost correlates well with the decrease in α-helix content (19). Binding of proteins on planar surfaces often induces significant changes in the secondary structure (20). However, a study of a variety of nanoparticle surfaces and proteins indicates that perturbation of protein structure still occurs to varying extents. The proteins show a rapid conformational change at both secondary and tertiary structure levels (20, 21). Numerous studies have found that activity reduction is related to the loss of α-helical content when proteins are adsorbed onto NPs regardless of an increase in the β-sheet (20). Binding of proteins to planar surfaces often induces significant changes in secondary structure; the high curvature of NPs can help proteins to retain their original structure (22). Several in vivo and in vitro studies of the toxicity of SiO₂NPs have been performed and found that they are safe and can be employed in food production (23). These findings and the findings of the present research encourages the use of SiO₂NPs as an inhibitor of pepsin for the treatment of GERD in vivo.

Effect of temperature on the interaction of SiO₂NPs with pepsin

The activity of the immobilized enzyme was measured at different temperatures (22, 27, 32, 37, and 42 °C) by using 19.8 mg of SiO₂NPs and the same concentrations of hemoglobin. These experiments were used to examine the effect of temperature on the activity of pepsin catalyzed reaction in the presence of SiO₂NPs. The Lineweaver-Burk lines of the immobilized pepsin activity at five different temperatures are presented in Figure 4.

The values of V_max and K_m of the pepsin catalyzed reaction at different temperatures in the presence of SiO₂NPs are listed in Table 2. The results revealed that the V_max at 37 °C is 2.91 U and then is reduced as temperature decreases. Whereas the K_m at 37 °C equal to 11.72 x 10^-5 M then increases as temperature decreases. It means that the change in temperature may cause a change in the form of active site and thus change V_max.

The results in Table 2 revealed that K_m and V_max change when the temperature changes. V_max increases as the temperature increases until reaching the optimum temperature and then tends to decrease when the temperature reaches 45 °C, while K_m decreases as temperature increases. However, at 42 °C, the K_m increases again indicating that there are conformational changes in the immobilized pepsin or changes in the interaction between the pepsin molecules and the SiO₂NPs.

In a previous study, it is found that the interaction between SiO₂NPs and three different enzymes are dependent on the functional groups on the surface of NPs (24). These findings indicated the presence of weak electrostatic interactions between the protein molecules and the surface of NPs. When nanomaterials are in contact with a biological environment, the proteins can immediately bind to the surface of the NPs, which creates protein coronas (25).

The bio-distribution of the nanomaterial is affected by this protein coating, which aids in understanding the mechanisms of protein coronas formations on nanomaterial surfaces including the effect of the nanomaterial surface properties (26). Weak protein-NPs interactions were studied previously in a low binding regime as a model for the soft protein corona around NPs in complex biological fluids. Noncovalent and reversible interactions between protein and SiO₂NPs showed significant alteration in conformation and enzymatic activity in a NP-size dependent manner. These facts indicated the presence of very weak interactions between protein and SiO₂NPs (27). Changes of environment temperature can alter the intramolecular attractive forces (hydrogen bonding, dipole-dipole interaction, hydrophobic interaction etc.) of the protein (e.g. enzyme). This can alter the active site of the enzyme rendering it inactive (28).

Effect of SiO₂NPs and ionic strength

The pepsin activity was measured at various conditions, i.e., free pepsin, in the presence of 130 mg of NaCl, immobilized pepsin on SiO₂NPs, and immobilized pepsin in the presence of 130 mg NaCl. All experiments were carried out at 37 °C. These experiments were carried out to explore the effect of the combination of ionic strength and SiO₂NPs on the activity of pepsin. The Lineweaver-Burk plots of the four experiments were plotted in Figure 5.

In Table 3, the comparison of free pepsin activity, immobilized pepsin on SiO₂NPs, and immobilized pepsin in the presence of 130 mg NaCl is shown. The results showed the V_max of free enzyme equal to 4.82 U and then became 2.50 U when 19.8 gm of SiO₂NPs were added. Then V_max decreased and became 0.92 U when 130 mg of NaCl were added to the enzyme reaction mixture, and then it became equal to 0.85 U when NaCl and SiO₂NPs were added together to the reaction mixture. It can be easily noticed that the effect of the combination of the ionic strength and SiO₂NPs causes high reduction of pepsin activity.

Most of the reduction in the enzyme activity is due to the changes in the secondary structures of the whole enzyme and particularly in the three dimensional structure of the active site, as previously mentioned in the Effect of SiO₂NPs weight on the pepsin catalyzed reaction section. Furthermore, a study by Wu et al. (20) showed that both β-lactoglobulin and lysozyme unfolded to a greater extent at lower surface concentration on SiO₂NPs. The proteolytic activity of pepsin is affected by the conditions of the dissolution medium. There is a significant reduction in the activity of pepsin after adding different concentrations of surfactants salts (29). Salts can form weak bonds with the charged functional groups on the protein surface.

Depending on the nature of ions, the balance among the forces are changed. However, higher concentration of salt can lead to salting out effect or decreased solubility of protein (50). There have been many interesting studies done about the inhibition of various enzyme activities upon adsorption on the surface of different nanoparticles (31, 32). Using advanced techniques such as circular dichroism and fluoroscopy, it is found that the most affective factors responsible for the reduction in the enzyme activity, after adsorption on the nanoparticle .surface, is the change in the secondary and tertiary structures of the enzyme especially around 18 the active site (32, 33). The same explanation can be generalized for the pepsin-SiO₂NPs system and it can be concluded that the reduction in the pepsin activity is due to the perturbation in the secondary and tertiary protein structures.

Conclusions

The results of the present study revealed that the SiO₂NPs has an ability to inhibit the pepsin activity. The results also indicates that the presence of high ionic strength causes remarkable reduction of pepsin activity. The increase in the amount of SiO₂NPs in the reaction medium leads to more reduction in the pepsin activity. Furthermore, the optimum temperature for the NPs to inhibit the reaction is 37 °C. Therefore, the best conditions of inhibition of pepsin enzyme is by using higher amounts of SiO₂NPs in the presence of NaCl at 37 °C.

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