Anti-pepsin activity of silicon dioxide nanoparticles

Resumo SiJR6Ps as an inhibitor of pepsin enzyme for treatment of gastrozesophageal reflux disease :àñRé0 were investigatedSilicon dioxide nanoparticles :pepsin coated SiJR6Ps0 are among the safest nanoparticles that can be used inside the human bodyThe activity of pepsin before and after the addition of certain amounts of the 6Ps to the reaction mixture was measured spectrophotometricallyãurthermoreN these experiments were repeated at different temperaturesN different weights of 6PsN and different ionic strengthsThe kinetic parameters :õm d Vmax0 of the pepsinz catalyzed reactions were calculated from the 5ineweaverzjurk plotsThe results showed that there is a significant reduction of pepsin activity by SiJR6Ps :Vmax of free pepsin 9 T-LR U and Vmax of the immobilized pepsin 9 R-B( U0The results also indicated that the presence of ionic strength causes remarkable reduction of pepsin activityôt can be concluded the best condition for inhibition of pepsin activity is by using a combinationof SiJR6Ps and high concentration 6aál at )V ° áSe usaron nanopartículas de dióxido de silicio como inhibidores de la pepsina para el tratamiento del reflujo gastroesofágico :àñRé0ñstas nanopartículas :SiJR6Ps recubiertas de pepsina0 son unas de las más seguras y pueden usarse en el cuerpo humanoSe midió a través de espectrofotometría la actividad de la pepsina antes y después de añadir cierta cantidad de 6Ps a la mezcla reactanteódicionalmenteN se repitieron estas pruebas a diferentes temperaturasN variando el peso de las 6Ps y la fuerza iónicaSe calcularon los parámetros cinéticos :õm y Vmax0 de las reacciones catalizadas con pepsina a través de las gráficas de 5ineweaverzjurk5os resultados mostraron queN usando SiJR6Ps :Vmax de pepsina libre 9 T-LR U y Vmax de pepsina inmovilizada 9 R-B( U0 y a través de la presencia de fuerza iónicaN la actividad enzimática se reduce significativamenteSe concluye que la mejor condición para inhibir la actividad enzimática es usando una combinación de SiJR6Ps y una alta concentración de 6aál a )V °áãoram usadas nanopartículas de dióxido de silício como inibidores da pepsina para o tratamento do refluxo gastroesofágico :àñRé0ñstas nanopartículas :SiJR6Ps cobertas de pepsina0 são uma das mais seguras e podem usarzse no corpo humanoãoi medida a atividade da pepsina mediante espectrofotometria antes e depois de agregar certa quantidade de 6Ps à mistura de reaçãoódicionalmenteN repetiramzse estas provas a diferentes temperaturasN variando o peso das 6Ps e a força iônicaãoram calculados os parâmetros cinéticos :õm e Vmax0 das reações catalisadas com pepsina a través das gráficas de 5ineweaverzjurkJs resultados mostraram queN usando SiJR6Ps :Vmax de pepsina livre 9 T-LR U e Vmax de pepsina imobilizada 9 R-B( U0 e a través da presença de força iônicaN a atividade enzimática se reduze significativamenteãoi concluído que a melhor condição para inibir a atividade enzimática é usando uma combinação de SiJR6Ps e uma alta concentração de 6aál a )V °á

SiJ R 6Ps as an inhibitor of pepsin enzyme for treatment of gastrozesophageal reflux disease :àñRé0 were investigated-Silicon dioxide nanoparticles :pepsin coated SiJ R 6Ps0 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 6Ps to the reaction mixture was measured spectrophotometrically-ãurthermoreN these experiments were repeated at different temperaturesN different weights of 6PsN and different ionic strengths-The kinetic parameters :õ m d V max 0 of the pepsinz catalyzed reactions were calculated from the 5ineweaverzjurk plots-The results showed that there is a significant reduction of pepsin activity by SiJ R 6Ps :V max of free pepsin 9 T-LR U and V max of the immobilized pepsin 9 R-B( U0-The results also indicated that the presence of ionic strength causes remarkable reduction of pepsin activity-ôt can be concluded the best condition for inhibition of pepsin activity is by using a combinationof SiJ R 6Ps and high concentration 6aál at )V °á-Se usaron nanopartículas de dióxido de silicio como inhibidores de la pepsina para el tratamiento del reflujo gastroesofágico :àñRé0-ñstas nanopartículas :SiJ R 6Ps recubiertas de pepsina0 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 6Ps a la mezcla reactante-ódicionalmenteN se repitieron estas pruebas a diferentes temperaturasN variando el peso de las 6Ps y la fuerza iónica-Se calcularon los parámetros cinéticos :õ m y V max 0 de las reacciones catalizadas con pepsina a través de las gráficas de 5ineweaverzjurk-5os resultados mostraron queN usando SiJ R 6Ps :V max de pepsina libre 9 T-LR U y V max de pepsina inmovilizada 9 R-B( U0 y a través de la presencia de fuerza iónicaN la actividad enzimática se reduce significativamente-Se concluye que la mejor condición para inhibir la actividad enzimática es usando una combinación de SiJ R 6Ps y una alta concentración de 6aál a )V °á-ãoram usadas nanopartículas de dióxido de silício como inibidores da pepsina para o tratamento do refluxo gastroesofágico :àñRé0-ñstas nanopartículas :SiJ R 6Ps cobertas de pepsina0 são uma das mais seguras e podem usarzse no corpo humanoãoi medida a atividade da pepsina mediante espectrofotometria antes e depois de agregar certa quantidade de 6Ps à mistura de reação-ódicionalmenteN repetiramzse estas provas a diferentes temperaturasN variando o peso das 6Ps e a força iônica-ãoram calculados os parâmetros cinéticos :õ m e V max 0 das reações catalisadas com pepsina a través das gráficas de 5ineweaverzjurk-Js resultados mostraram queN usando SiJ R 6Ps :V max de pepsina livre 9 T-LR U e V max de pepsina imobilizada 9 R-B( U0 e a través da presença de força iônicaN a atividade enzimática se reduze significativamente-ãoi concluído que a melhor condição para inibir a atividade enzimática é usando uma combinação de SiJ R 6Ps e uma alta concentração de 6aál a )V °á-

Reagents
Pepsin g[R UNANbUNzSx MWt V U8xATL 'x GGNTm purityx was supplied from F'Hx [nglandN Spherical Silicon dioxide nanoparticles gSiO b NPsSx particle diameter V UGN8AA nmx GGNTm purityx was supplied from Nanjing nanotechnologyx RhinaN Lyophilized Hemoglobin ghuman red blood cellsSx G8m purityx was supplied from Lee Fiosolutionsx Missourix US°N Trichloroacetic acid gTR°Sx GIm purity from °lpha Rhemikax IndiaN Hydrochloric acid gHRlx °nalytical Gradex UTNAmS was supplied from Rentral 'rug Housex New 'elhix IndiaN The SiO b NPs used in the present study were visualized using S[M and T[M techniques to further confirmation for their shape and sizeN The T[M studies were performed by using a J[MObLzL instrument working with an acceleration voltage of bLL kVN S[M images were arried out by using a Hitachi SOAILL S[M at bL kVN The enzyme activity was determined by a kinetic method g18SN The principle depends on the fact that pepsin cleaves peptides from hemoglobin which are soluble in trichloroacetic acid gTR°SN The tyrosine and tryptophan content of these TR°Osoluble peptides is determined by the measurement of the extinction at bIL nmN Frieflyx pepsin is dissolved in LNLz N HRl to obtain a concentration of LNT mgj mLN Just prior to assay there is another dilution in LNLz N HRl to a concentration of TObLμgjmLN The steps of the method were as followsv z mL of hemoglobin substrate was pipetted into test tubes containing LNb mL of the diluted pepsin at Uk °RN °fter zL minx the reaction was stopped by adding b mL of Tm TR°N The tubes containing the reaction mixture were removed from water bath after T min and clarified gfiltrates should be clearSN [bIL nm was read of filtrate and subtract [bIL nm of the appropriate blank using spectrophotometer gModel kbzOTaiwanS set at bIL nm and Uk °RN The method of estimation of pepsin activity was repeated at different temperatures gbbx bkx Ub and Ab °RSN °unit of pepsin enzyme was defined as an amount of enzyme which renders TR°soluble LNLLz [bIL nm per minute at Uk °Rx using a denatured hemoglobin substrateN

Inhibition of pepsin activity by SiO 2 NPs
To study the inhibition of pepsin by SiO b NPsx a weight of zGNI mg of SiO b NPs was weighed by Sartorius balance gModel °bLLS dissolved in zL mL of LNLzN HRlx and agitated by ultrasonic water bath gIsoLabx GermanyS at Uk °R for bL minN ]ive mg of pepsin were added to the SiO b NPsOcontaining tubes and incubated at Uk °R for UL minN These amounts of pepsin and NPs were calculated to produce pepsin monolayer on the SiO b NPsN SiO b NPs and pepsin surface areas were calculated in order to estimate theoretically the enough number of pepsin to cover the surface of one spherical SiO b NP in one layer mannerN

Introduction
GastroOesophageal reflux disease gG[R'S evolves when reflux of stomach contents causes complications into the esophagus g1SN G[R' is a popular disease with a prevalence of zLm O bLm in the western countries g2S and some researchers reported up to UTNGm g3Sx but its risk factors and causes are not clearly known g4SN The disease causes various symptomsx among which are heartburn and regurgitationx which affect up to ULm of the population and continue to increase g5-7SN Pepsinx which is the most important substance in the gastric contents for the necrosis of the mucosal tissuesx plays the main role in the formation of G[R' and associated diseases g8SN °lthough protonOpump inhibitors are known to alleviate symptoms in most patientsx a significant portion of patients continue to present G[R' g9SN There is a wide range of specific inhibitors that can bind to the active site and effectively remove the activity of pepsinx one of the best known ones is pepstatinx a specific pepsin inhibitorx which at acidic pHx tightly binds to the catalytic site of both pepsin and its precursor pepsinogen g10SN The best way to determine K m gthe concentration of substrate at V max jbS and V max gMaximum velocity of an enzymeOcatalyzed reaction at definite conditionsS values of the basic MichaelisOMenten equation is by taking the reciprocal of both sides of equation to give the double reciprocal equation or LineweaverOFurkHs equationN Where V L represents the initial velocity or the activity of the enzymeOcatalyzed reactionN °plot of zjV L versus zj[S] yields a straight line with an intercept of zjV max and a slope of K m jV max g11SN Use of NPs to inhibit the activity of papsin in vitro as a model for the treatment of G[R' was performed and published previously g12SN Silicon dioxide nanoparticles gSiO b NPsSx also known as silica NPs or nanosilicax are the basis for a great deal of biomedical research due to their stabilityx low toxicityx and ability to be functionalized with a range of molecules and polymers g13SN SiO b NPs have received an intensive attention by scientific community due to its broad applications in biomedical and biotechnological fields such as drug deliveryx gene therapy and molecular imagingx cancer therapyx and enzyme immobilization g14SN In additionx it is widely used in cosmeticsx foodx varnishesx papermakingx and drugs g15SN ]ruijtierOPolloth et al.
The calculations describe the pepsin and SiO k NPs properties qradius7 density7 mass of one NP7 and the volume of one NP8 to calculate the surface areas of pepsin and SiO k NPs which were FHI°x F°L FE cm k and MH9M x F°L FF cm k 7 respectivelyH The number of pepsin molecules that can cover one SiO k NP in a monolayer manner was obtained from the division of one SiO k NPs surface area on one pepsin surface area ≈ Ek9 molecules of pepsin per SiO k NPH This means that a I mg of pepsin were required to cover F9H8 mg of SiO k NPs to obtain a monolayer of the adsorbed pepsinH The method of pepsin activity estimation was repeated and the activity of the immobilized pepsin was estimatedH To study the effect of weight on the pepsin activity the same method was repeated using different weights of SiO k NPs qE9H* mg7 I9HM mg7 B9Hk mg and 99H°m   Effect of temperature on the interaction of SiO 2 NPs with pepsin

Results and discussion
The activity of the immobilized enzyme was measured at different temperatures 7009 0G9 I09 IG9 and M0 °C4 by using URLE mg of SiO 0 NPs and the same concentrations of hemoglobinL These experiments were used to examine the effect of temperature on the activity of pepsin catalyzed reaction in the presence of SiO 0 NPsL The Lineweaver8Burk lines of the immobilized pepsin activity at five different temperatures are presented in Figure ML Table 1.Effect of weight of SiO 2 NPs at 37 °C on the activity of pepsin coated SiO 2 NPs.
The data in Table U demonstrate the effect of weight of SiO 0 NPs on pepsin activityL These weights were selected from the calculations of the amount of SiO 0 NPs needed to be coated with a monolayer of pepsin moleculesL The first weight 7URLE mg4 of SiO 0 NPs in the reaction mixture represents a NPs coated with a monolayer of pepsin moleculesL The results showed that V max of free pepsin 7MLE0 U4 decreases to 0LRk U when the enzyme was immobilized on the surface of SiO 0 NPsL The activity continued to decrease as the weight of the added NPs increases until becoming ULGH U when the weight added is RR mgL It is clear that the SiO 0 NPs has remarkable inhibitory effect on pepsin activityL 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 structureL These changes are caused by the adsorption forces between the surface of the NPs and various chemical groups of the pepsin moleculesL The attractive forces are strong enough to hold molecules on the NPs surface and modify the H8bonding that constitute the secondary structure of the pepsin moleculesL Therefore9 the change in the secondary structure is the most probable cause for the decrease in the activity of the immobilized forcesL Studies indicate that the activity of lysozyme adsorbed onto SiO 0 NPs is lower than that of the free protein9 and the fraction of activity lost correlates well with the decrease in α8helix content 7194L Binding of proteins on planar surfaces often induces significant changes in the secondary structure 7204L However9 a study of a variety of nanoparticle surfaces and proteins indicates that perturbation of protein structure still occurs to varying extentsL The proteins show a rapid conformational change at both secondary and tertiary structure levels 720, 214L Numerous studies have found that activity reduction is related to the loss of α8helical content when proteins are adsorbed onto NPs regardless of an increase in the β8 sheet 7204L Binding of proteins to planar surfaces often induces significant changes in secondary structureW the high curvature of NPs can help proteins to retain their original structure 7224L Several in vivo and in vitro studies of the toxicity of SiO 0 NPs have been performed and found that they are safe and can be employed in food production 7234L These findings and the findings of the present research encourages the use of SiO 0 NPs as an inhibitor of pepsin for the treatment of GERD in vivoL The values of V max and K m of the pepsin catalyzed reaction at different temperatures in the presence of SiO 0 NPs are listed in Table 0L The results revealed that the V max at IG °C is 0LRU U and then is reduced as temperature decreasesL Whereas the K m at IG °C equal to UULG0  The results in Table 5 revealed that K m and V max change when the temperature changes7 V max increases as the temperature increases until reaching the optimum temperature and then tends to decrease when the temperature reaches 85 °C0 while K m decreases as temperature increases7 However0 at 85 °C0 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 SiO 5 NPs7 In a previous study0 it is found that the interaction between SiO 5 NPs and three different enzymes are dependent on the functional groups on the surface of NPs W24-7 These findings indicated the presence of weak electrostatic interactions between the protein molecules and the surface of NPs7 When nanomaterials are in contact with a biological environment0 the proteins can immediately bind to the surface of the NPs0 which creates protein coronas W25-7 The bioAdistribution of the nanomaterial is affected by this protein coating0 which aids in understanding the mechanisms of protein coronas formations on nanomaterial surfaces including the effect of the nanomaterial surface properties W26-7 Weak proteinANPs interactions were studied previously in a low binding regime as a model for the soft protein corona around NPs in complex biological fluids7 Noncovalent and reversible interactions between protein and SiO 5 NPs showed significant alteration in conformation and enzymatic activity in a NPAsize dependent manner7 These facts indicated the presence of very weak interactions between protein and SiO 5 NPs W27-7 Changes of environment temperature can alter the intramolecular attractive forces Whydrogen bonding0 dipoleAdipole interaction0 hydrophobic interaction etc7-of the protein We7g7 enzyme-7 This can alter the active site of the enzyme rendering it inactive W28-7 9

Effect of SiO 2 NPs and ionic strength
The pepsin activity was measured at various conditions0 i7e70 free pepsin0 in the presence of FqB mg of NaCl0 immobilized pepsin on SiO 5 NPs0 and immobilized pepsin in the presence of FqB mg NaCl7 All experiments were carried out at qM °C7 These experiments were carried out to explore the effect of the combination of ionic strength and SiO 5 NPs on the activity of pepsin7 The LineweaverABurk plots of the four experiments were plotted in Figure U7 Rev. Colomb.Quim.20160 45 Wq-0 UAFF7 In Table q0 the comparison of free pepsin activity0 immobilized pepsin on SiO 5 NPs0 and immobilized pepsin in the presence of FqB mg NaCl is shown7 The results showed the V max of free enzyme equal to 87E5 U and then became 57UB U when FD7E gm of SiO 5 NPs were added7 Then V max decreased and became B7D5 U when FqB mg of NaCl were added to the enzyme reaction mixture0 and then it became equal to B7EU U when NaCl and SiO 5 NPs were added together to the reaction mixture7 It can be easily noticed that the effect of the combination of the ionic strength and SiO 5 NPs causes high reduction of pepsin activity7 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 site0 as previously mentioned in the Effect of SiO 5 NPs weight on the pepsin catalyzed reaction section7 Furthermore0 a study by Wu et al7 W20-showed that both βAlactoglobulin and lysozyme unfolded to a greater extent at lower surface concentration on SiO 5 NPs7 The proteolytic activity of pepsin is affected by the conditions of the dissolution medium7 There is a significant reduction in the activity of pepsin after adding different concentrations of surfactants salts W29-7 Salts can form weak bonds with the charged functional groups on the protein surface7 Depending on the nature of ions0 the balance among the forces are changed7 However0 higher concentration of salt can lead to salting out effect or decreased solubility of protein W30-7 There have been many interesting studies done about the inhibition of various enzyme activities upon adsorption on the surface of different nanoparticles W31, 32-7 Using advanced techniques such as circular dichroism and fluoroscopy0 it is found that the most affective factors responsible for the reduction in the enzyme activity0 after adsorption on the nanoparticle 7surface0 is the change in the secondary and tertiary structures of the enzyme especially around FE the active site W32, 33-7 The same explanation can be generalized for the pepsinASiO 5 NPs system and it can be concluded that the reduction in the pepsin activity is due to the perturbation in the secondary and tertiary protein structures7   The results of the present study revealed that the SiO 5 NPs has an ability to inhibit the pepsin activity7 The results also indicates that the presence of high ionic strength causes remarkable reduction of pepsin activity7 The increase in the amount of SiO 5 NPs in the reaction medium leads to more reduction in the pepsin activity7 Furthermore0 the optimum temperature for the NPs to inhibit the reaction is qM °C7 Therefore0 the best conditions of inhibition of pepsin enzyme is by using higher amounts of SiO 5 NPs in the presence of NaCl at qM °C7 : àñRéN SiJ R nanoparticlesN pepsinN enzyme inhibition-Palabras clave:àñRéN nanopartículas de SiJ R N pepsinaN inhibición enzimática-Palavras-Chave: àñRéN nanopartículas de SiJRN pepsinaN inibição enzimática-Recibido= )( de âunio de R(G&-óceptado= B de Septiembre de R(G&-Resumen Characterization of NPs Rev. Colomb.Quim.2016x 45 gUSx TOzzN g16S evaluated the toxic effects and safety of SiO b NPs and concluded that they are as safe as conventional SiO b N °morphous SiO b NPs are widely used in food productsx for examplex as thickenersx anticaking agentsx carriers of fragrances and flavorsx and additives g17SN The aim of the present study is to optimize the process of pepsin inhibition by SiO b NPs as a possible new treatment for G

g8H 7
Effect of temperature on the inhibition of pepsin activity by SiO 2 NPsTo study the temperature effect in the presence of SiO k NPs7 F9H8 mg of SiO k NPs 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 SiO k NPsHEffect of a combination of ionic strength and SiO 2 NPs on the inhibition of pepsin activityTo study the effect of the ionic strength in the presence of SiO k NPs7 FI mg of SiO k NPs and FHE mg of NaCl were dissolved in F°mL of °H°F N HCl7 and agitated by ultrasonic water bath at EB °C for k°m inH Five mg of pepsin were added to the tubes and incubated at EB °C for E°minH The method of estimation of pepsin activity was repeated and the pepsin activity was calculatedH The protocol of the research is presented in Figure FH The images of SiO k NPs taken by SEM and TEM are presented in Figure kH It is clear that the shape of SiO k NPs is spherical with a particle size around E9 nmH Rev. Colomb.Quim.20167 45 qE87 ILFFH Effect of SiO 2 NPs weight on the pepsin catalyzed reaction The activity of the enzyme was measured by using seven different concentrations of hemoglobin qEH°F7 *HkF7 9HEF7 FkHMF7 FIHIk7 F8H*k7 and kFHBkz F°LI M8 and five different weights of added SiO k NPs qF9H87 E9H*7 I9HM7 B9Hk and 99 mg8 to the F°mL of the °H°F N HCl to prepare SiO k NPs solutionH These experiments were used to examine the effect of SiO k NPs weights on the activity of pepsin catalyzed reactionH The LineweaverLBurk plots of the five experiments7 were plotted in Figure EH

Figure 1 .
Figure 1.Workflow of the protocol of the research.
x Uk 8H M then increases as temperature decreasesL It means that the change in temperature may cause a change in the form of active site and thus change V max L

Table 2 .
Maximum velocities and Michaelis constants of the SiO 2 NPs-immobilized pepsin at different temperatures.
Lineweaver-Burke lines of pepsin catalyzed reaction at 37 °C after adding 130 mg NaCl°19.8 mg SiO 2 NPs and in the presence of both c130 mg NaCl and 19.8 mg SiO 2 NPsy.