Journal of Science: Advanced Materials and Devices (2017) 45e50 Contents lists available at ScienceDirect Journal of Science: Advanced Materials and Devices journal homepage: www.elsevier.com/locate/jsamd Original Article pH-responsive drug release from dependal-M loaded polyacrylamide hydrogels Raman Dwivedi a, Alok Kumar Singh b, Anju Dhillon a, * a b Maharaja Surajmal Institute of Technology, C-4, Janakpuri, Affiliated to Guru Gobind Singh Inderprastha University, New Delhi, India HMR Institute of Technology and Management, Hamidpur, Affiliated to Guru Gobind Singh Inderprastha University, New Delhi, India a r t i c l e i n f o a b s t r a c t Article history: Received 20 September 2016 Received in revised form 12 January 2017 Accepted February 2017 Available online 20 February 2017 A study of pH responsive drug release from dependal-M drug loaded polyacrylamide (PAM) hydrogel matrix is reported PAM hydrogel with different crosslinker concentrations has been taken for the different drug loading capacities The associative interaction of drug in the polymer network complicates the release pattern of drug, and the release kinetics show a dependence on the cross linker and its ratio The drug release kinetics in hydrogel with higher cross linker (H1) and less crosslinked hydrogel (H2) are followed by the Higuchi's model and the KorsmeyerePeppas model, respectively Drug release mechanism is based on diffusion Initial burst of drug release was observed at pH 5.8 The calculated diffusion coefficient (D) is 2.57 for H1 and 1.799 for H2 © 2017 The Authors Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Keywords: pH Drug release Kinetics Hydrogel Diffusion Introduction Numerous devices have come up for drug delivering application, in convoy to everlastingly advancing development in the field of biomedical applications However, logical system is the one wherein the system itself is capable of sensing the varying exterior surrounding conditions to deliver the necessary amount of drug at the desired site [1] Polymeric hydrogels are the best fit in the drug delivery systems (DDS) as they present pulsated release of the desired drug to the affected site in response to the changing temperature, electric field strength and pH These drug carrier polymeric hydrogels are basically hydrophilic polymer structures wherein, the three dimensionally (3-D) cross linked polymer chain networks are reliable of swelling to the highest possible degree in liquid media [2] These water swollen polymer network matrices can be made available in variety of forms such as nanoparticles, micro particles and films for use in various medicinal applications such as tissue engineering, as three dimensional scaffolds in drug delivery system Hydrogel based DDS have gained a lot of curiosity among the researchers, as the loaded drug in the porous structure of hydrogel can be released at a controllable rate depending on the changing * Corresponding author E-mail addresses: anju.dhillon@msit.in, anju.dhillon@gmail.com (A Dhillon) Peer review under responsibility of Vietnam National University, Hanoi structure and physical property of hydrogel in different conditions of pH [3] and temperature [4] pH responsive 3-D polymer network can be most effective as DDS in humans and mammals, as there occurs pH variations at many particular body sites and thus this criterion can be used to deliver the drug at a particular preconceived rate on a particular body site There occurs a prompt change in intraluminal (among tubes of stomach) pH from highly acidic in the stomach to about pH in the duodenum, again pH gradually increases from 6.0 in the small intestine to about pH 7.4 in the terminal ileum [5] The physiological situation of these pH changes can form the basis for pH sensitive drug release Again the performance of hydrogels such as its overall swelling or water intake, drug carrying capacity, uncoiling and drug delivering capabilities are known to be affected by the character of the constituent polymer chains as well as by the extent of polymerization/ crosslinking grade The greater monomer and cross-linker concentration in the reactant solution results in an increased association linking the macromolecules, resulting in a tighter gel network with less porous fragments between cross-linkage The firmness of network also gets improved with the increasing crosslinking between polymer networks and this can affect its performance in DDS [6] Neutral hydrogels such as polyacrylamide (PAM) are more suitable for DDS as they are biocompatible and not very reactive (Fig 1) PAM based hydrogels have already been used in several in vitro and in vivo studies to deliver various drugs such as ibuprofen [7], cytarabine [8], famotidine [9], citric acid [10], etc http://dx.doi.org/10.1016/j.jsamd.2017.02.003 2468-2179/© 2017 The Authors Publishing services by Elsevier B.V on behalf of Vietnam National University, Hanoi This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) 46 R Dwivedi et al / Journal of Science: Advanced Materials and Devices (2017) 45e50 O H N H2N O (a) Polyacrylamide (PAM) _ O + N N N O O O O (b) Furazodilone (Dependal-M) Fig Chemical structure of (a) Polyacrylamide (PAM) (b) Furazodilone (Dependal-M) Dependal-M drug is a combination of furazolidone and metronidazole, suggested in oral rehydration therapy for traveller's bacillary dysentery from bacterial or mixed origin amoebiasis (intestinal or extra intestinal) ailments and also in warning-less cyst passers It is an effectual antiprotozoal and antibacterial agent hostile to genre of Escherichia coli, Salmonella, etc [11] It is also used for treating beaver fever, a common cause of gastroenteritis In typhoid fever, furazolidone and metronidazole combination is given in a dose of 200 mg times a day for 14 days So, sustained release of dependal-M can be a better alternative to encourage its bio accessibility in amoebiasis and bacterial infections Neutral PAM hydrogel based drug delivery system can be supportive in such situations Dependal-M laden neutral PAM hydrogel can be a better substitute for oral or intravenous (infusion) drug administration therapy as the hydrogel can be a support for faster relief and rehydration by the release of water/electrolyte along with sustained release of antibacterial drug directly to the site of the ailment Our present work intended to study in detail the release mechanism of drug from dependal-M embedded polyacrylamide hydrogel and to test its potential as pH responsive polymeric carrier device for rehydration and for controlled release of dependal-M to intestinal protozoal or bacterial infected site Materials and methods Dependal-M the model drug in the tablet form was procured from local medical representative Acrylamide (AM) and N,N0 methylenebisacrylamide (MBA) were procured from Sisco Research Laboratory (SRL) and were used as monomer and crosslinker for forming the hydrogel matrix Potassium peroxo disulphate (KPS) (AR grade, Central Drug House, CDH) was used as a initiator for polymerisation For preparing PAM hydrogel network (H2), acrylamide monomer (0.8 g) was dissolved in 10 ml of distilled water and then 0.010 g of MBA crosslinker was added to the monomer solution with stirring Alternatively, 0.01 g of KPS was dissolved in another 10 ml of distilled water and was added dropwise to the above prepared monomerecrosslinker solution with stirring This concoction was then emptied into a cylindrical vial of dimension (1.2 cm diameter and 4.2 cm height) to form a hydrogel of the similar dimension, after which the reaction was allowed to reach to completion by leaving the mixture in the vial for h at 500  C The vial was then broken to obtain the hydrogel which was then washed several times with distilled water to remove any unreacted species Hydrogel was also prepared adopting similar procedure except for increasing the cross linker amount to 0.030 g PAM hydrogel was then loaded with the model drug dependalM via a method of soaking and saturation Amount of water required for equilibrium swelling of PAM hydrogel was determined in advance and a known quantity of the model drug dependal-M was dissolved in the water The completely dried hydrogel sample was dipped in the above mentioned drug solution (of known concentration) and left for a period of days for maximum swelling in the drug solution Drug swollen hydrogel samples were taken out of the solution after days and washed many times with double distilled water in order to wash away the drug held on to its superficial surface The supernatant liquid after taking out hydrogel was kept aside for absorption measurements so as to know the amount of unabsorbed drug remaining back in the solution Buffer solution of pH 5.8 was prepared by making up the volume of the solution of 7.5 ml glacial acetic acid and 75 g sodium acetate to 500 ml by distilled water pH of the buffer solution was made sure employing a pre calibrated pH meter Results and discussion 3.1 Fourier transform infrared spectroscopy (FTIR) FTIR spectra of the samples were recorded in transmission mode with KBr pressed pellets Spectra were recorded over the wave number ranging from 4000 to 500 cmÀ1 using Thermo Nicolet 380 infrared spectrophotometer FTIR spectrum of dependal-M drug (D), PAM hydrogels (H1 and H2) (where and distinguishes the hydrogels with different cross linker concentrations i.e 0.030 g and 0.010 g, respectively) and of drug-loaded-PAM (DH2) was analyzed (Fig 2) to find the variation in peak or peak shifting that could give an indication of bonding or association among the polymer molecules The strong intensity bands appearing around 3430 cmÀ1 in H2 and 3405.5 cmÀ1 in H1 are undoubtedly associated with the NeH stretching vibrations This difference may be related to the extent of cross linking interaction between the polymer chains wherein this NeH group must be involved These interactions may thereby be affecting the strength of intermolecular NeH hydrogen bond and hence their stretching frequency The bands at 2939.2 and 2915 cmÀ1 in the spectra of H1 and at 2930 and 2773 cmÀ1 in H2 due to eCH stretching of CH2 (methylene) group is informative regarding the extent of polymerisation in PAM These CeH vibration frequencies and band shifts are suggestive of a greater extent of polymerisation and hence tighter gel network in H1 The characteristic band at 1648.9 cmÀ1 in H1 and at 1635 cmÀ1 in H2 is due to amide group (eCONH2) of PAM (>C]O stretching) [12] The vibrational modes of amide groups are considerably affected by the involvement of these groups in hydrogen bonding The difference in the amide band in H1 and H2 is due to the difference in the strengths of intermolecular R Dwivedi et al / Journal of Science: Advanced Materials and Devices (2017) 45e50 47 units and the solution was scanned in the UV region from 200 to 500 nm and from the spectra (Fig 4a) obtained we could work out the value of lmax as 322 nm [16] More standard solutions of dependal-M were prepared with concentration ranging from 5200 ppm to 1000 ppm These solutions were also suitably diluted with distilled water and were scanned in the UV region to obtain the absorbance value at the lmax (322 nm) point A calibration curve was plotted of the absorbance values against the known concentration values (Fig 4b) 3.4 Drug release from dependal-M laden PAM hydrogel Fig Comparative FTIR spectra of dependal-M drug, hydrogel 1, hydrogel and drug laden hydrogel and intramolecular hydrogen bonds In case of DH2 (drug loaded H2), characteristic band of NeH stretching is shifted to 3435.6 cmÀ1 from 3430 cmÀ1 in the virgin H2, whereas CeH vibration frequency appears at 2941 cmÀ1 In DH2 characteristic band of >C]O stretching of amide group appears at 1647.2 cmÀ1 These shifts in the bands of drug laden hydrogel suggest an association of drug within the polymer gel network Drug occupies the pores formed between the interconnected network structure and associative interaction takes place Drug is further released off from the hydrogel network with the changing pH of the external medium in conjunction with the changing physical and chemical structure of hydrogel in the medium 3.2 Scanning electron microscopy (SEM) Morphological structure of hydrogel was examined using scanning electron microscope (SEM, Hitachi) SEM images demonstrated the dissimilarities in the surface morphology of H1 and H2 which were attained with regard to different crosslinker amounts The crosslinker plays a crucial role in the polymerization reaction, bridging two or more polymer chains together Higher amount of crosslinker resulted in a smoother surface with lesser pores (Fig 3a and b), because with an increased crosslinker amount more and more polymerisation occurred which strengthened the network of hydrogel forming a more compact structure [13] While the lesser amount of crosslinker resulted in a more porous surface (Fig 3c and d) These pores, i.e free space or region between the interconnected networks, provide available regions for the diffusion of water molecules and drug molecules Thus H2 hydrogel with lesser crosslinker amount exhibits a higher water absorption capacity, drug holding and retention capacity as there are greater free spaces between its networks [14,15] This observation goes on well with the calculated amount of drug ingrained into the hydrogel network via UVevisible studies 3.3 UVevis spectroscopy Standard solution of dependal-M (furazolidone and metronidazole) was prepared by dissolving 150 mg of drug via ultrasonication in 50 ml of distilled water as solvent to prepare a solution of concentration 3000 ppm The stock solution was suitably diluted to 100 times with distilled water so as to contain 300 ppm of dependal-M for fitting absorption limit to less than Drug release from batches of hydrogels differing in the monomer:crosslinker ratios were comprehended in a buffer solution of pH 5.8 The buffer of pH 5.8 was deliberately chosen to mimic the conditions existing in the intestine right the way through the suffering by protozoal or bacterial infection PAM hydrogel can present water/electrolyte for the rehydration and can release the drug in the intestinal tract at near neutral pH of about 5.8e6.0 Furazolidone and metronidazole combination works by penetrating into the protozoan and bacterial cell, excluding the mammalian cell and proceed directly to reduce cytotoxic 5-nitro group causing rupture of its DNA strand and eventually to the collapse of bacterial or protozoan cell Drug laden hydrogel was immersed in the buffer solution (pH ¼ 5.8) and 10 ml of the aliquot was withdrawn at regular intervals out of the drug laden hydrogel dipped buffer solution The solution after measurement was again put back to the reserved hydrogel immersed solution for further measurement The withdrawn samples were evaluated spectrophotometrically at 323 nm Calibration curve was used to determine the released drug amount and cumulative percentage of drug release versus time is presented in Fig 5a The amount of drug absorbed in each of the hydrogels was worked out by back calculating the amount of drug left behind in the solution after each of the hydrogel was swelled to equilibrium in (3000 ppm) drug solution The amount of drug absorbed was found out be 210 ppm for H1 and 250 ppm for H2 which is well in agreement of the SEM and FTIR results which predicted a more porous structure for H2 (it should be H1) and hence a greater drug and water absorption capacity to the free spaces in its interpenetrating network structure These results specify that drug loading will be on the same wavelength (proportional to) as the porosity and swelling properties of the hydrogels Similarly, the release contour of the hydrogel entity above all is a function of interactions of the drug within the polymeric network, the solubility of the drug, and swelling profile of the hydrogel in the suspension standard The release pattern of all the hydrogels was governed by a heavy pour in the beginning caused by the existence of the drug on the shell of the hydrogels, followed by a prolonged release of drug from the core of the hydrogel Higher concentration gradient through the bulk of the hydrogel may be a reason for the opening heavy pour of the drug, followed by reduction in the release rate, attributable to the diffusion difficulty meant for drug, covering more distance within the thicker core of hydrogel for simultaneous release The amount of dependal-M released from the hydrogel matrix in relation to time engaged is summarized for different hydrogels (HI and H2) in Table These hydrogels differ in the amount of crosslinker linking the macromolecular polymer chains The amount of dependal-M released from hydrogel (with lesser cross linker) is quite significant in the beginning in comparison to a lesser initial release in hydrogel with higher crosslinking ratio The slower and lower amount of dependal M drug released from H1 with higher cross-linker amount is because of the more rigid structure of this hydrogel formed due to the lessening of the pores verified by SEM micrographs and also this initial release 48 R Dwivedi et al / Journal of Science: Advanced Materials and Devices (2017) 45e50 Fig SEM micrographs of (a and b) hydrogel and (c and d) are of hydrogel of drug is because of the drug near the hydrogel surface (greater loading of 250 ppm in H2 as compared to 210 ppm in H1) which can easily diffuse out However, later a greater slowdown in the release rate was seen in case of H2 as compared to H1 This occurrence can be attributed to a more attractive association between the drug and polymer matrix in H2 and hence diminishing release rate of drug thereafter from the matrix while such an effect can be neglected in H1 which already must have balanced the charges on its groups by more evident crosslinking interactions So, the leftover reactive groups in the weakly crosslinked polymer matrix (H2) can be a driving force for greater association with drug forming some weak bonds therein among themselves and hence slowing down the release rate 3.5 Drug release kinetics For studying the drug release kinetics and mechanism involved in detail, the drug release data was fitted into various kinetic models such as zero order, first order, Higuchi's model, KorsmeyerePeppas model using the equations given underneath [17,18] Zero order : Q t =Q ¼ K0 t First order : In Q t =Q ¼ K1 t Haguchi0 s model : Q =Q ẳ 2Dt =pị ẳ KHt 1=2 KorsmeyerePeppas model : Q t =Q ¼ Ktn Fig a UVevisible absorption spectra of dependal-M drug in the range of 200e500 nm b Calibration curve for different standard solutions of dependal-M drug where Qt is the amount of drug released at time t, Q0 is the original drug concentration in the gel, D is the diffusion coefficient of a diffusant, n is release exponent and K is the release rate constant Correlation coefficient values (r2) were calculated for different kinetic models and is summarized in Table along with the rate constant predicted for these models Comparison of these r2 values suggest diffusion as the preferred mechanism of release for dependal-M from H1 and H2 with r2 value of 0.9958 for H1 and R Dwivedi et al / Journal of Science: Advanced Materials and Devices (2017) 45e50 49 Fig a Drug release profile of two dependal-M laden hydrogel matrices b Plot of drug release amount versus square root of time engaged with inset showing logarithmic plot of cumulative drug release as a function of log of engaged time Table Summarized report of amount of drug released from both the hydrogels H1 and H2 with time Hydrogel Hydrogel Time (min) Absorbance Concentration (ppm) Time (min) Absorbance Concentration (ppm) 15 30 45 60 75 90 1.4185 1.7678 2.0544 2.2695 2.4472 2.5677 24 27 30 32 34 35 15 30 45 60 75 90 2.4746 2.7137 2.8750 2.9824 3.1126 3.1814 34 37 38 39 40 41 Table Drug release kinetics and correlation coefficient values from different kinetic models Hydrogel identification Hydrogel (H1) Hydrogel (H2) Correlation coefficient (r2) Zero order First order Higuchi's model KorsmeyerePeppas model 0.9728 0.9382 0.9753 0.9407 0.9958 0.9779 0.9950 0.9966 0.9779 for H2 from best fit Higuchi model So, Higuchi equation is followed in preference to zero or first order release kinetics evident from the r2 value of H1 (signifying linearity in equation) While in case of H2 release mechanism follows KorsmeyerePeppas kinetics Release exponent ‘n’ from KorsmeyerePeppas model fit Rate constant KH Higuchi's model Rate constant KK from Korsmeyer model 0.217 0.099 7.4716 3.6539 1.1834 1.1955 perceived by a higher r2 value from data fit to this model Contradiction in the values of rate constant predicted of the two methods for H1 and H2 is because of the different release kinetics followed i.e H1 follows release according to 3rd equation while the release in 50 R Dwivedi et al / Journal of Science: Advanced Materials and Devices (2017) 45e50 H2 is according to 4th equation of KorsmeyerePeppas The plot of released drug amount records versus square root of time engaged (Fig 5b) was studied to calculate the diffusion coefficient D [19] By means of the slope of above plot, we could work out the diffusion coefficient of both the hydrogels The value of the diffusion coefficient can clearly verify the different release kinetics of the hydrogels The diffusion coefficient (D) was found out to be 2.57 for H1 and 1.799 for H2 To further make sure of the diffusion and not erosion or dissolution as the prime and preferred mechanism of release, release exponent value was calculated for H1 and H2 using the KorsmeyerePeppas equation and plot of log Qt/Q0 versus log t shown in Fig 5b For both H1 and H2 value of ‘n’ came out to be less than 0.45 indicative of Fickian diffusion of drug from hydrogel matrix [20] Conclusion This study highlighted the potential of dependal-M loaded hydrogel to be used in the rehydration therapy for relief from bacillary dysentery originating from protozoal or bacterial infection in intestinal tract Water and drug swollen PAM hydrogel can present faster relief for the rehydration by providing water/electrolyte and release the drug at the intestinal tract ailment in the prevailing near neutral pH (5.8e6.0) condition The drug release experiments conducted using two hydrogels with varying crosslinker ratios revealed that hydrogel with lesser crosslinker amount has a higher drug loading capacity The release mechanism was found to be diffusion controlled and not accompanied by dissolution of matrix Drug release pattern was complicated in view of the associative interaction of drug within the polymeric network The release kinetics in H1 (higher crosslinker) follow Higuchi's model and H2 (lesser crosslinker) followed KorsmeyerePeppas model Unpredictably, the slow release of drug after the opening pour from hydrogel with lesser crosslinker amount was attributed to some associative interaction between the drug and matrix, slowing down the release rate Acknowledgements Authors are thankful to the Management of Surajmal Memorial Education Society, Janakpuri and Management of HMRITM, Hamidpur for providing the healthy and supportive environment for research work Authors extend their thanks to Delhi Technological University, Delhi for providing the characterization facilities References [1] F Ganji, E.V Farahani, Hydrogels in controlled drug delivery systems, Iran Polym J 18 (2009) 63e88 [2] T.R Hoare, D.S Kohane, Hydrogels in drug delivery: progress and challenges, Polymer 49 (2008) 1993e2007 [3] P Bonina, Ts Petrova, N Manolova, pH-sensitive hydrogels composed of chitosan and polyacrylamide e preparation and properties, J Bioact Compat Polym 19 (2004) 101e116 [4] E Mah, R Ghosh, Thermo-responsive hydrogels for stimuli-responsive membranes, Processes (2013) 238e262 [5] J Fallingborg, Intraluminal pH of the human gastrointestinal tract, Dan Med Bull 46 (1999) 183e196 [6] D Calvet, J.Y Wong, S Giasson, Rheological monitoring of polyacrylamide gelation: importance of cross-link density and temperature, Macromolecules 37 (2004) 7762e7771 [7] M.D Hussain, J.A Rogers, R Mehvar, G.K Vudathala, Preparation and release of ibuprofen from polyacrylamide gels, Drug Dev Ind Pharm 25 (1999) 265e271 [8] C Gomez, M.D Blanco, M.V Bernardo, R.L Sastre, J.M Teijon, Poly(acrylamidecomonoethylitaconate) hydrogels as devices for cytarabine release in rats, J Pharm Pharmacol 50 (1998) 703e712 [9] R Tripathi, B Mishra, Development and evaluation of sodium alginateepolyacrylamide grafteco-polymer-based stomach targeted hydrogels of famotidine, AAPS PharmSciTech 13 (2012) 1091e1102 [10] E Karadag, D Saraydin, N Sahiner, O Guven, Radiation induced acrylamide/ citric acid hydrogels and their swelling behaviours, J Macromol Sci Pure Appl Chem 11 (2001) 1105e1121 [11] M.L.M Gonzales, L.F Dans, E.G Martinez, Antiamoebic drugs for treating amoebic colitis (review), Cochrane Database Syst Rev (2009), http:// dx.doi.org/10.1002/14651858 John Wiley & Sons, Ltd [12] R Murugan, S Mohan, A Bigotto, FTIR and polarised Raman spectra of acrylamide and polyacrylamide, J Korean Phys Soc 32 (1998) 505e512 [13] R Singhal, S Sachan, J.S.P Rai, Study of cure kinetics of polyacrylamide hydrogels by differential scanning calorimetry, Iran Polym J 11 (2002) 143e149 [14] M Pandey, M.C.I.M Amin, N Ahmad, M.M Abeer, Rapid synthesis of superabsorbent smart-swelling bacterial cellulose/acrylamide-based hydrogels for drug delivery, Int J Polym Sci (2013), http://dx.doi.org/10.1155/2013/ 905471 [15] M.Y Kizilay, O Okay, Effect of hydrolysis on spatial inhomogeneity in poly(acrylamide) gels of various crosslink densities, Polymer 44 (2003) 5239e5250 [16] A.A Kale, A.B Rasane, A.K Bhatiyani, Validated derivative spectrophotometric method for estimation of metronidazole and furazolidone in pure and in tablet dosage form, Int J Pharm Technol (2012) 4805e4813 [17] S Bhalla, M Nagpal, Comparison of various generations of superporous hydrogels based on chitosan-acrylamide and in vitro drug release, ISRN Pharm (2013), http://dx.doi.org/10.1155/2013/624841 [18] B.N Nalluri, D Jyotheermayi, D Anusha, K.M Maheswari, Controlled release tablet formulations of carvedilol, J Chem Pharm Res (2012) 4266e4274 [19] E Anwar, K.S.S Putri, S Surini, Novel excipient for matrix of floating tablet containing Nile tilapia gelatine, chitosan and cellulose derivatives, J Med Sci 11 (2011) 203e207 [20] R Parhi, S.K Bhupati, P Suresh, V Kumar, Formulation and evaluation of bilayer sustained release tablet of zolpidem tartarate, Indones J Pharm 24 (2013) 291e300  ... SEM micrographs and also this initial release 48 R Dwivedi et al / Journal of Science: Advanced Materials and Devices (2017) 45e50 Fig SEM micrographs of (a and b) hydrogel and (c and d) are of. .. plot of cumulative drug release as a function of log of engaged time Table Summarized report of amount of drug released from both the hydrogels H1 and H2 with time Hydrogel Hydrogel Time (min)... site Materials and methods Dependal- M the model drug in the tablet form was procured from local medical representative Acrylamide (AM) and N,N0 methylenebisacrylamide (MBA) were procured from Sisco