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ISSN : 1225-0562(Print)
ISSN : 2287-7258(Online)
Korean Journal of Materials Research Vol.31 No.6 pp.367-374
DOI : https://doi.org/10.3740/MRSK.2021.31.6.367

Gentamicin/CTMA/Montmorillonite as Slow-Released Antibacterial Agent

Is Fatimah1, Habibi Hidayat1, Gani Purwiandono1, Saddam Husein2, Won-Chun Oh3
1Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Islam Indonesia, Kampus Terpadu UII, Jl. Kaliurang Km 14, Sleman, Yogyakarta, Indonesia
2Laboratory of Materials for Energy and Environemnt, Department of Chemistry, Universitas Islam Indonesia, Kampus Terpadu UII, Jl. Kaliurang Km 14, Sleman, Yogyakarta, Indonesia
3Department of Advanced Materials Science and Engineering, Hanseo University, Seosan-si, Chungnam 356-706, South Korea
Corresponding author E-Mail : isfatimah@uii.ac.id (I. Fatimah, Univ. Islam Indonesia)
April 21, 2021 April 21, 2021 June 8, 2021

Abstract


This paper presents the characteristics of gentamicin-loaded into cetyl trimethyl ammonium intercalated montmorillonite (GtM/CTMA/Mt) as a hybrid composite for a slow-released antibacterial delivery systems. The work describes the successful immobilization of gentamicin into the interlayers of surfactant-modified montmorillonite. Physicochemical characterization of the material is carried out by means of X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and Fourier transform infrared spectroscopy. The kinetics of the gentamicin release is investigated by in vitro study and analyzed based on UV–Vis spectrometry. In addition, antibacterial study is performed towards Klebsiella pneumoniae Staphylococcus aureus, Escherichia coli, and Streptococcus pyogenes. The results show that the gentamicin loading into CTMA/ Mt increases the effectiveness of the antibacterial activity, as shown by the higher inhibition zone for all tested bacteria, compared to gentamicin as a positive control. The kinetics study suggests that the gentamicin release obeys the modified Korsmeyer–Peppas model. The physicochemical study and activity test demonstrate the feasibility of the GtM/CTMA/Mt for practical applications.



초록


    © Materials Research Society of Korea. All rights reserved.

    This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    1. Introduction

    Development of slow-release drugs to control the effectiveness and to prevent the allergic reaction risks is gained focus and attention recently. Within these schemes, the immobilized antibacterial agent in an inorganic material for providing delivery system with the extentrelease is a strategy for the many applications, such as the preparation of biocompatible material for tissue engineering and antimicrobial film.1) Clay minerals are hydrous phyllosilicates materials having capability to host and distribute active organic compounds.1) Montmorillonite, a smectite class of clay has been investigated to have potency as a drug carrier due to its capability either to adsorb and desorb active organic molecules, or being kinetically modified in releasing active agents.2) Some montmorillonitebased composites using surfactant, polymers, metal and metal oxide nanoparticles have been investigated for antibacterial purposes.3-5) In wider range, the composites can be designed in tissue engineering, food packaging, sustainable building, and veterinary and environmental materials.6,7)

    The composites of surfactant/montmorillonite and antibacterial/ antibiotics agent-intercalated montmorillonite were reported to be effective as the controlled-release hybrid materials. Aminoglycoside antibiotics are broadspectrum type of antibiotics which have been extensively used in many fields such as veterinary, sanitizing and agriculture fields.8) The loaded-gentamicin using inorganic matrices such as silica, hydroxyapatite, polymers were reported,8-11) one of the aminoglycosides, with clay mineral such as smectite class of clay minerals is a good composite candidate. Such studies releaved the antibacterial activity of smectite clays such as saponite and montmorillonite.12-14) The presence of cationic in in the smectite layers leads to influence the cell membrane of bacteria for furthermore cell lysis will be occurred. In another scheme, surface modifications using surfactant to form organoclay including the use of quaternary ammonium have been reported to be promising hybrid materials with antibacterial properties.3,15) Organoclays inhibit the growth of Escherichia coli and Staphylococcus aureus via adsorbing the bacteria and influence the electronic states of the cells.3) The hydrophobic properties from the surface modification of clay structure can effectively increase the adsorptive capability towards antibiotics and pharmaceutical agents in clay support.4,5,16) Based on the synergistic and supportive properties for antibacterial and adsorption capability, the combination of organoclay and antibiotic modification was proposed to give better effectiveness in preparation of antibacterial nanocomposite. Aim of this research is to study the physicochemical character and antibacterial activity of organoclay/antibiotic/clay nanocomposite using cetyl trimethyl ammonium (CTMA), gentamicin (GtM) and montmorillonite (Mt).

    2. Materials and Method

    2.1 Materials

    Materials used in this research consist of montmorillonite K-10 (Mt), gentamicin and CTMA were purchased from Sigma-Aldrich (Germany).

    2.2 Method

    Preparation of the nanocomposite was conducted by two steps; CTMA intercalation to form organoclay of CTMA-intercalated montmorillonite (CTMA/Mt), and gentamicin loading to the organoclay. The CTMA intercalation was performed by mixing Mt suspension in water (10 wt%) at the CTMA: Mt is 5 % wt. followed by stirring overnight. Into the suspension, gentamicin was added at the mass percentage of 10 % wt. The mixture was undergoes stirred for overnight, and then frozen. The spray drying was performed to get GtM/CTMA/Mt powder. As comparison, CTMA intercalation without gentamicin loading and the gentamicin loading to the Mt suspension were engaged to form CTMA/Mt and GtM/Mt. respectively. The prepared materials were characterized by using Fourier- Transform Infra-Red (FTIR), x-ray diffraction (XRD, Shimadzu), scanning electron microscopy (SEM, PhenomX, Singapore), and transmission electron microscopy (TEM, JEM 2100, Jeol Ltd., Japan). A Shimadzu diffractometer using monochromatic CuKα radiation (λ = 0.154 nm) was employed operating at 40 kV and 50 mA over a 2θ range from 4° to 70°.

    Antibacterial activity of the materials was evaluated against Klebsiella pneumoniae (ATCC 13883), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 11303), and Streptococcus pyogenes (ATCC 19615) by disc diffusion method. The powders were dispersed in double distilled water at the concentration of 5 % (w/v). The nutrient media were prepared by suspending nutrient agar in distilled water and autoclaved before use. Bacterial culture was evenly spread throughout the petri plate and a 6 mm sterile filter disc loaded with 5 μL of the dispersion solution.

    3. Results and Discussion

    Fig. 1 exhibits the XRD patterns of materials. As shown in the figure, all samples demonstrate the characteristics reflections of montmorillonite structure at the 2θ = 6.63°; 19.89° and 35.6°, which correspond to (001), (211) and (004) reflections, respectively.17,18) Using the Braggs law, d001-spacing is about 1.01 nm. The d001 basal spacing is increased as the CTMA intercalation performed as shown by the shift of (001) reflection into smaller angle, which corresponding to the d001 = 1.42 nm. The increasing space is referred to the replacement of native cations from Mt with the bigger molecular size in between the silica-alumina interlayer regions. However, the value of increasing is relatively less than similar intercalation conducted by previous works.19) Beside of the less amount of the CTMA, the cation exchange capacity of the Mt is the influencing factor for the swelling ability of the smectite layers. Furthermore, after gentamicin loading, the basal spacing d001 of Mt structure decreased to the insignificant different with the d001 of Mt.

    The phenomenon of the change of d001 basal spacing is confirmed by the pore distribution from the gas sorption analysis data presented in Table 1 and Fig. 2. The CTMA intercalation and GtM/CTMA/Mt formation led to the decreasing specific surface area. In addition, the value of external surface area of GtM/CTMA/Mt is smaller compared to the total specific surface area, meanwhile the both external and total specific surface area of Mt and CTMA/Mt are in the same values. These data represent that the incorporated gentamicin molecule fulfill the interlayer and pore region of the clay structure.

    These identifications are associated with the TEM profile in Fig. 3. The GtM molecule loading into the CTMA/Mt support is significantly appeared from the image represents that the layers of Mt structure was being more compact. The molecular size of gentamicin is approximately 5 Å in width and 10 Å in length, which can fulfil the the layers and pores filling. This description is in line with the decreasing external surface area respect to the total specific surface area of GtM/CTMA/Mt. The change in TEM profile is similar with the gentamicin loading into calcium sulfate cement porous TiO2 microsphere composites, which reflected the decreasing porosity of the support.20)

    The loaded gentamicin in the nanocomposite is confirmed by FTIR analysis (Fig. 4). As shown in the Figure, the samples exhibit some peaks: 459.2 cm−1 peak demonstrates Si-O bending vibration (in plane) group; 523.1 cm−1 peak illustrates Si-O-Al vibration peak indicates Mg-O-Si or Fe-O-Si groups; 799.3 cm−1 peak shows AlMgOH vibration group; 1,044.3 cm−1 peak reveals Si- O stretching vibration (in plane) group; 1651.1 and 3423 cm−1 peaks depict OH bending and stretching vibration groups, and finally, 3627.7 cm−1 peak represents OH stretching group. It can be summarized that there is no obvious difference among the spectra for all samples, except the presence of additional peaks at around 2920 cm−1 with minor intensity. The peaks represent symmetric and asymmetric vibrations of C-H stretching related with the CTMA in CTMA/Mt and GtM/CTMA/Mt structures.21,22)

    The SEM analyses presented in Fig. 5 provided the important information for the Mt, CTMA/Mt and the nanocomposite surface and the composition. The SEM image represents that the formed GtM/CTMA/Mt composite showed agglomeration on surface, meanwhile the modification of Mt into CTMA/Mt created the rougher surface.

    The kinetics of gentamicin release were performed in vitro under room temperature. About 200 mg of composite was soaked in 15 mL phosphate buffered saline, and the sampling was conducted by collecting supernatant of the sample media at regular time intervals (1 ~ 12 days). The kinetics of gentamicin release was obtained from the colorimetric measurement refer to reference.23,24)

    Three kinetics models: first order, Higuchi and Korsmeyer– Peppas models were applied for the kinetics of drug release evaluation.25,26) The equations of models are as follow:

    • First order model: Ct = 1 − exp(− K1t)

    • Higuchi model: Ct = KHt0.5, and

    • Modified Korsmeyer–Peppas model: Ct = atn + b

    Where Ct is amount of dissolved gentamicin, K1 is firstorder kinetics constant, KH is Higuchi release constants, t is the time of release, and n is the release exponent.26)

    From the kinetics of gentamicin release in Fig. 6, it is seen that the released antbiotic is about 24 % at first day, and reach constantly around 70 % in 12 days. The data suggests the potency of material to be slow-release composite since the antibiotics are dropped frequently to treat the bacterial infection.

    The kinetics calculation of gentamicin release is presented in Table 2.

    From the simulated models, kinetics data meet with the modified Korsmeyer-Peppas model, which constructed based on the assumption that the mechanism of drug release is controlled by diffusion and dissolution mechanisms from the matrix.27) Refer to previous studies of drug release experiments, the Korsmeyer–Peppas compromises with the polymerix matrix of drug delivery system.28,29) The value of release exponent, n is 3.22 (> 0.5) suggests the non-Fickian diffusion mechanism, and implies that the mass transport of gentamicin release is dominantly occurred in the releasing process [9].

    The releasing gentamicin is in line with the antibacterial activity test presented in Fig. 7, and some figures from the test are presented in Fig. 8.

    The antibacterial test was conducted by using dispersed powder in water (1 mg/mL) on disc method. S. aureus and S. pneumoniae bacteria were chosen as representative of the gram positive bacteria, meanwhile E. coli and K. aerogenes were as gram negative. The Pores and a unique periplasmic space found in gram-negative bacteria are not found in gram-positive bacteria, determine the intracellular accumulation and bactericidal effect.

    As can be seen from the data, it is revealed that the GtM/CTMA/Mt exhibits the excellent antibacterial activity. Compared gentamicin as the positive control, Mt, GtM/ Mt and CTMA/Mt samples, the composite form shows highest inhibition zone. Not only the highest inhibition zone appeared by the composite, the zone increased along the increasing time, as an indication that the composite is not only inhibit the bacteria living, but also kill the bacteria. On the other hand, CTMA/Mt and GtM/Mt also show the influence on the growth inhibition, but less active compared to GtM/CTMA/Mt. The pattern of inhibition zone by using CTMA/Mt and Mt is similar, which the zone maintained after 24 h. It was suggested that both Mt and CTMA/Mt are the bacteriostatic effect which inhibit the bacteria growth. In addition, as the positive control GtM showed less active, the data represented the synergistic effect among CTMA/Mt as antibacterial support. The data are associated with the kinetics of GtM release which represent the gradual GtM desorption from the nanocomposite. Compared to similar nanocomposite, the GtM/CTMA/Mt represent an excellent activity as can be seen from Table 3. With similar percentage of gentamicin release during first 24 h (~25 %), the GtM/CTMA/Mt inhibits more effective compared to gentamicin-loaded in poly(lactide-co-glycolide) nanoparticles, which showed the inhibition zone at around 32 ~ 39 nm towards S. aureus. Similarly, the antibacterial against E. coli is better compared to gentamicine loaded in nanoparticles entrapped nanofibers (MSNs-PCL) which showed less inhibition zone (22.39 ± 0.99 mm), meanwhile the released gentamicin at 24 h was same.30)

    From varied tested bacteria and the values of the inhibition zone, it can be concluded that the nanocomposite has stronger bactericidal effect on the gram-negative strains rather than on the gram-positive strains. Similar phenomena were reported on the use of Ag nanoparticlesbased antibacterial agents, which the gram-negative bacteria have a cytoplasmic membrane, containing peptidoglycan and lipopolysaccharide layers. The membrane consists of carboxyl, phosphate, and amino groups from phospholipid structure which lead to the negative charges, and can easily being penetrated by the ionic charge of the nanocomposites. 33-35)

    In general, the results and data of this study indicate high applicability of the combination of alkylammonium (CTMA) and clay mineral for further development in drug delivery, slow-released antibiotics and surgical support.

    4. Conclusion

    The nanocomposite of organoclay/antibiotic/clay (GtM/ CTMA/Mt) has been successfully synthesized with potential applications for slow-released gentamicin system. Physicochemical character of the nanocomposite shows the effective loaded gentamicin as shown by XRD, FTIR, gas sorption analysis, TEM and SEM-EDX analyses. The gentamicin release obeys the modified Korsmeyer–Peppas kinetics model with the release exponent suggesting the non-Fickian diffusion mechanism. The antibacterial activity tests show that the composite has high potential to inhibit S. aureus, S. pneumoniae, E. coli and K. aerogenes. Overall, it is identified that GtM/CTMA/Mt exhibited a high applicability for further development drug delivery, slow-released antibiotics and surgical support.

    Figure

    MRSK-31-6-367_F1.gif

    XRD pattern of materials.

    MRSK-31-6-367_F2.gif

    (a) Adsorption-desorption isotherm of materials, (b) Pore size distribution of materials.

    MRSK-31-6-367_F3.gif

    TEM profile of materials.

    MRSK-31-6-367_F4.gif

    FTIR spectra of materials.

    MRSK-31-6-367_F5.gif

    SEM profile of materials.

    MRSK-31-6-367_F6.gif

    (a) Kinetics of gentamicin release, (b) Modified Korsmeyer–Peppas plot of gentamicin release.

    MRSK-31-6-367_F7.gif

    Inhibition zone measurement in antibacterial test.

    MRSK-31-6-367_F8.gif

    Some images from antibacterial test.

    Table

    Surface parameters from gas sorption analysis.

    Kinetics model, parameter and equation of gentamicin release of GtM/CTMA/Mt.

    Comparison of antibacterial activity of GtM/CTMA/Mt with similar nanocomposites.

    Reference

    1. P. Gao, X. Nie, M. Zou, Y. Shi and G. Cheng, J. Antibiot., 64, 625 (2011).
    2. Y. He, Z. Wu, L. Tu, Y. Han, G. Zhang and C. Li, Appl. Clay Sci., 109–110, 68 (2015).
    3. H. Bujdakova, V. Bujdakova, H. Majekova-Koscova, B. Gaalova, V. Bizovska, P. Bohac and J. Bujdak, Appl. Clay Sci., 158, 21 (2018).
    4. M. Anggraini, A. Kurniawan, L. K. Ong, M. A. Martin, J. C. Liu, F. E. Soetaredjo, N. Indraswati and S. Ismadji, RSC Adv., 4, 16298 (2014).
    5. T. Saitoh and T. Shibayama, J. Hazard. Mater., 317, 677 (2016).
    6. P. Herrera, R. C. Burghardt and T. D. Phillips, Vet. Microbiol., 74, 259 (2000).
    7. M. R. Virto, P. Frutos, S. Torrado and G. Frutos, Biomaterials, 24, 79 (2003).
    8. S. Perni, K. Martini-Gilching and P. Prokopovich, Colloids Surfaces A Physicochem. Eng. Asp., 541, 212 (2018).
    9. M. Stevanovic, M. Dosic, A. Jankovic, V. Kojic, M. Vukasinovic-Sekulic, J. Stojanovic, J. Odovic, M. C. Sakac, K. Y. Rhee and V. Miskovic-Stankovic, ACS Biomater. Sci. Eng., 4, 3994 (2018).
    10. S. Tang, B. Tian, Q. F. Ke, Z. A. Zhu and Y. P. Guo, RSC Adv., 40, 41500 (2014).
    11. D. A. Mosselhy, Y. Ge, M. Gasik, K. Nordstrom, O. Natri and S. P. Hannula, Materials (Basel)., 9, 1 (2016).
    12. L. Zarate-Reyes, C. Lopez-Pacheco, A. Nieto-Camacho, E. Palacios, V. Gomez-Vidales, S. Kaufhold, K. Ufer, E. G. Zepeda and J. Cervini-Silva, J. Hazard. Mater., 342, 625 (2018).
    13. A. S. Maryan, M. Montazer, A. Rashidi and M. K. Rahimi, Asian J. Chem., 25, 2889 (2013).
    14. G. Lv, C. W. Pearce, A. Gleason, L. Liao, M. P. MacWilliams and Z. Li, J. Asian Earth Sci., 77, 281 (2013).
    15. L. Zhang, J. Chen, W. Yu, Q. Zhao and J. Liu, J. Nanomater., 2018, 6190251 (2018).
    16. X. Yuan, J. Zhang, R. Zhang, J. Liu, W. Wang and H. Hou, Materials (Basel)., 12, 4148 (2019).
    17. M. Honarmand, M. Golmohammadi and A. Naeimi, Mater. Chem. Phys., 241, 122416 (2020).
    18. I. Fatimah, D. Rubiyanto, I. Sahroni, R. S. Putra, R. Nurillahi and J. Nugraha, Appl. Clay Sci., 193, 105671 (2020).
    19. I. Fatimah and T. Huda, Appl. Clay Sci., 74, 115 (2013).
    20. W. Luo, Z. Geng, Z. Li, S. Wu, Z. Cui, S. Zhu, Y. Liang and X. Yang, Int. J. Nanomedicine, 13, 7491 (2018).
    21. R. Batul, M. Bhave, P. J. Mahon and A. Yu, Molecules, 25, 1 (2020).
    22. A. Bayoumi, M. T. Sarg, T. Y. A. Fahmy, N. F. Mohamed and W. K. El-Zawawy, Arab. J. Chem., 13, 8920 (2020).
    23. A. Rapacz-Kmita, E. Stodolak-Zych, M. Dudek, M. Gajek, M. Ziąbka, J. Therm. Anal. Calorim., 127, 871 (2017).
    24. Y. Liu, P. Ji, H. Lv, Y. Qin and L. Deng, Int. J. Biol. Macromol., 98, 550 (2017).
    25. M. Karthikeyan, M. K. Deepa, E. Bassim, C. S. Rahna and K. R. S. Raj, J. Pharm. Innov., Research Article (2020).
    26. D. Wojcik-Pastuszka, J. Krzak, B. Macikowski, R. Berkowski and B. Osinski, Materials (Basel)., 12, 1202 (2019).
    27. I. Y. Wu, S. Bala, N. Skalko-Basnet and M. P. D. Cagno, Eur. J. Pharm. Sci., 138, 105026 (2019).
    28. G. Arora, K. Malik and I. Singh, Polim. Med., 41, 23 (2011).
    29. R. Gouda, H. Baishya and Z. Qing, J. Dev. Drugs., 06, 1000171 (2017).
    30. X. Chen, C. Xu and H. He, Biophys. Res. Commun., 516, 1085 (2019).
    31. U. Posadowska and M. Brzychczy-Włoch, Acta Bioeng. Biomech., 17, 41 (2015).
    32. M. Stigter, J. Bezemer, K. D. Groot and P. Layrolle, J. Control. Release, 99, 127 (2004).
    33. G. Franci, A. Falanga, S. Galdiero, L. Palomba, M. Rai, G. Morelliiano and M. Galdiero, Molecules, 20, 8856 (2015).
    34. D. Garibo, H. A. Borbon-Nunez, J. N. D. D. Leon, E. G. Mendoza, I. Estrada, Y. Toledano-Magana, H. Tiznado, M. Ovalle-Marroquin, A. G. Soto-Ramos, A. Blanco, J. A. Rodriguez, O. A. Romo, L. A. Chavez-Almazan and A. Susarrey-Arce, Sci. Rep., 10, 12805 (2020).
    35. Y. Y. Loo, Y. Rukayadi, M. A. R. Nor-Khaizura, C. H. Kuan, B. W. Chieng, M. Nishibuchi and S. Radu, Front. Microbiol., 9, 1 (2018).