A Biophysical Research on the Determination of Morphological and Structural Properties of Coumarin-Loaded Liposomes

Year 2023, , 43 - 57, 01.03.2023

Cisem Altunayar-unsalan

https://doi.org/10.35414/akufemubid.1104476

Abstract

The major goal of this research was to examine how coumarin affects lipid model membranes. For this reason, liposome membranes were formed using dimyristoyl phosphatidylcholine (DMPC) as zwitterionic lipid. The influence of coumarin on the morphology, packing order, fluidity, and hydration state of lipid membranes was specifically investigated by means of microscopic (field emission scanning electron microscopy (FE-SEM)) and spectroscopic (attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy) techniques. Taken into account the results obtained with FE-SEM images and analysis, liposomes without and with coumarin have uniform structures and spherical shapes in appearance. However, coumarin-loaded liposomes are observed with an increase in size when compared to a mean diameter of unloaded-liposomes. Considering ATR-FTIR analysis, the investigation of the vibrational bands which belong to the hydrophobic and hydrophilic parts of DMPC lipid reveals that coumarin alters the physical features of the DMPC liposomes by decreasing the order and increasing the fluidity of the system and making hydrogen bonding with the interfacial and headgroup regions of zwitterionic lipid DMPC. Finally, performing more biophysical studies on the interactions of biologically active compounds with model membranes plays an important role in determining the molecular action mechanisms of these compounds in drug discovery and formulations.

Keywords

Coumarin, Liposomes, Phospholipids, ATR-FTIR, FE-SEM

Supporting Institution

Ege University Scientific Research Projects Coordination Unit

Project Number

18‒FEN‒030 and FKB‒2019‒20405

Thanks

This work was supported by Ege University Scientific Research Projects Coordination Unit. Project Number: 18‒FEN‒030 and FKB‒2019‒20405.

Kumarin Yüklü Lipozomların Morfolojik ve Yapısal Özelliklerinin Belirlenmesi Üzerine Biyofiziksel Araştırma

Abstract

Bu araştırmanın esas amacı kumarinin lipit model membranları nasıl etkilediğini incelemektir. Bu nedenle, zwitteriyonik lipit olarak dimiristoil fosfatidilkolin (DMPC) kullanılarak lipozom membranlar oluşturulmuştur. Kumarinin lipit membranların paketleme düzeni, akışkanlığı, hidrasyon durumu ve morfolojisi üzerine etkisi, özellikle mikroskobik (alan emisyonlu taramalı elektron mikroskobu (FE-SEM)) ve spektroskopik (zayıflatılmış toplam yansıma Fourier dönüşüm infrared (ATR-FTIR) spektroskopisi) teknikleri ile incelenmiştir. FE-SEM görüntüleri ve analizleri ile elde edilen sonuçlar dikkate alındığında, kumarinsiz ve kumarinli lipozomlar görünüm olarak düzgün yapılara ve küresel şekillere sahiptir. Bununla birlikte, kumarin yüklü lipozomların, yüklenmemiş lipozomların ortalama çapı ile karşılaştırıldığında boyutunda bir artış gözlenmiştir. ATR-FTIR analizleri göz önüne alındığında, DMPC lipitinin hidrofobik ve hidrofilik kısımlarına ait titreşim bantlarının incelenmesi, kumarinin sistemin düzenini azaltarak ve akışkanlığını artırarak ve zwitteriyonik lipit DMPC'nin arayüzey ve baş grup bölgeleri ile hidrojen bağı yaparak DMPC lipozomlarının fiziksel özelliklerini değiştirdiğini ortaya koymaktadır. Sonuç olarak, biyolojik olarak aktif bileşiklerin model membranlar ile etkileşimleri üzerine daha fazla biyofiziksel çalışmaların gerçekleştirilmesi, ilaç keşfi ve formülasyonlarında bu bileşiklerin moleküler etki mekanizmalarının belirlenmesinde önemli rol oynamaktadır.

Keywords

Kumarin, Lipozomlar, Fosfolipitler, ATR-FTIR, FE-SEM

Project Number

18‒FEN‒030 and FKB‒2019‒20405

References

• Akoudad, S., Darweesh, S.K.L., Leening, M.J.G., Koudstaal, P.J., Hofman, A., van der Lugt, A., Stricker, B.H., Ikram, M.A. and Vernooij, M.W., 2014. Use of coumarin anticoagulants and cerebral microbleeds in the general population. Stroke, 45, 3436–3439.
• Al-Majedy, Y.K., Kadhum, A.A.H., Al-Amiery, A.A. and Mohamad, A.B., 2017. Coumarins: The antimicrobial agents. Systematic Reviews in Pharmacy, 8, 62‒70.
• Altunayar-Unsalan, C., Unsalan, O. and Mavromoustakos, T., 2022a. Insights into molecular mechanism of action of citrus flavonoids hesperidin and naringin on lipid bilayers using spectroscopic, calorimetric, microscopic and theoretical studies. Journal of Molecular Liquids, 347, 118411.
• Altunayar-Unsalan, C., Unsalan, O. and Mavromoustakos, T., 2022b. Molecular interactions of hesperidin with DMPC/cholesterol bilayers. Chemico-Biological Interactions, 366, 110131.
• Bangham, A.D., Standish, M.M. and Watkins, J.C., 1965. Diffusion of univalent ions across the lamellae of swollen phospholipids. Journal of Molecular Biology, 13, 238‒252.
• Bangham, A.D., 1978. Properties and uses of lipid vesicles: an overview. Annals of the New York Academy of Sciences, 308, 2‒7.
• Beillerot, A., Domínguez, J.-C.R., Kirsch, G. and Bagrel, D., 2008. Synthesis and protective effects of coumarin derivatives against oxidative stress induced by doxorubicin. Bioorganic & Medicinal Chemistry Letters, 18, 1102‒1105.
• Benderitter, M., Vincent-Genod, L., Pouget, J.P. and Voisin, P., 2003. The cell membrane as a biosensor of oxidative stress induced by radiation exposure: a multiparameter investigation. Radiation Research, 159, 471–483.
• BIOVIA, Dassault Systèmes, Discovery Studio Visualizer, v20.1.0.19295, Dassault Systèmes, San Diego, 2019.
• Bin, X., Zawisza, I., Goddard, J.D. and Lipkowski, J., 2005. Electrochemical and PM-IRRAS studies of the effect of the static electric field on the structure of the DMPC bilayer supported at a Au(111) electrode surface. Langmuir, 21, 330‒347.
• Briuglia, M.L., Rotella, C., McFarlane, A. and Lamprou, D.A., 2015. Influence of cholesterol on liposome stability and on in vitro drug release. Drug Delivery and Translational Reserch, 5, 231‒242.
• Bubols, G.B., Vianna, D. da R., Medina-Remon, A., von Poser, G., Lamuela-Raventos, R.M., Eifler-Lima, V.L., Garcia, S.C., 2013. The antioxidant activity of coumarins and flavonoids. Mini-Reviews in Medicinal Chemistry, 13, 318‒334.
• Burgess, I., Li, M., Horswell, S.L., Szymanski, G., Lipkowski, J., Majewski, J. and Satija, S., 2004. Electric field-driven transformations of a supported model biological membrane—an electrochemical and neutron reflectivity study. Biophysical Journal, 86, 1763‒1776.
• Casal, H.L., Cameron, D.G., Smith, I.C.P. and Mantsch, H.H., 1980. Acholeplasma laidlawii membranes: a Fourier Transform Infrared study of the influence of protein on lipid organization and dynamics. Biochemistry, 19, 444‒451.
• Casal, H.L., Mantsch, H.H. and Hauser, H., 1989. Infrared and 31P-NMR studies of the interaction of Mg2+ with phosphatidylserines: effect of hydrocarbon chain unsaturation. Biochimica et Biophysica Acta, 982, 228‒236.
• Ćavar, S., Kovač, F. and Maksimović, M., 2012. Evaluation of the antioxidant activity of a series of 4-methylcoumarins using different testing methods. Food Chemistry, 133, 930‒937.
• Chen, L.Z., Sun, W.W., Bo, L., Wang, J.Q., Xiu, C., Tang, W.J., Shi, J.B., Zhou, H.P. and Liu, X.H., 2017. New arylpyrazoline-coumarins: synthesis and anti-inflammatory activity. European Journal of Medicinal Chemistry, 138, 170–181.
• Cieślik-Boczula, K., 2018. Influence of resveratrol on interactions between negatively charged DPPC/DPPG membranes and positively charged poly-l-lysine. Chemistry and Physics of Lipids, 214, 24‒34.
• Damodaran, K.V. and Merz, K.M. Jr., 1994. A comparison of DMPC- and DLPE-based lipid bilayers. Biophysical Journal, 66, 1076‒1087.
• de la Haba, C., Palacio, J.R., Martínez, P. and Morros, A., 2013. Effect of oxidative stress on plasma membrane fluidity of THP-1 induced macrophages. Biochimica et Biophysica Acta, 1828, 357–364.
• Demetzos, C., Angelopoulou, D., Kolocouris, A., Daliani, I. and Mavromoustakos, T., 2001. Structure elucidation, conformational analysis and thermal effects on membrane bilayers of an antimicrobial myricetin ether derivative. Journal of Heterocyclic Chemistry, 38, 703‒710.
• Derenne, A., Claessens, T., Conus, C. and Goormaghtigh, E., 2013. Infrared spectroscopy of membrane lipids. In: Roberts G.C.K. (ed.) Encyclopedia of biophysics, 2013th edn. Berlin, Heidelberg, Germany, Springer pp 1074‒1081.
• Duong, T.H., Mehler, E.L. and Weinstein, H., 1999. Molecular dynamics simulations of membranes and a transmembrane helix. Journal of Computational Physics, 151, 358‒387.
• Emami, S. and Dadashpour, S., 2015. Current developments of coumarin-based anti-cancer agents in medicinal chemistry. European Journal of Medicinal Chemistry, 102, 611‒630.
• Fadel, O., El Kirat, K. and Morandat, S., 2011. The natural antioxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidation in situ. Biochimica et Biophysica Acta, 1808, 2973–2980.
• Gericke, A. and Hühnerfuss, H., 1993. In situ investigation of saturated long-chain fatty acids at the air/water interface by external infrared reflection-absorption spectrometry. The Journal of Physical Chemistry, 97, 12899–12908.
• Hong, M., Schmidt-Rohr, K. and Zimmermann, H., 1996. Conformational constraints on the headgroup and sn-2 chain of bilayer DMPC from NMR dipolar couplings. Biochemistry, 35, 8335‒8341.
• Hull, M.C., Cambrea, L.R. and Hovis, J.S., 2005. Infrared spectroscopy of fluid lipid bilayers. Analytical Chemistry, 77, 6096‒6099.
• Hübner, W., Mantsch, H.H., Paltauf, F. and Hauser, H., 1994. Conformation of phosphatidylserine in bilayers as studied by Fourier transform infrared spectroscopy. Biochemistry, 33, 320‒326.
• Kalyanram, P., Ma, H., Marshall, S., Goudreau, C., Cartaya, A., Zimmermann, T., Stadler, I., Nangia, S. and Gupta, A., 2020. Interaction of amphiphilic coumarin with DPPC/DPPS lipid bilayer: effects of concentration and alkyl tail length. Physical Chemistry Chemical Physics, 22, 15197.
• Kaplán, P., Doval, M., Majerová, Z., Lehotský, J. and Račay, P., 2000. Iron-induced lipid peroxidation and protein modification in endoplasmic reticulum membranes. Protection by stobadine. The International Journal of Biochemistry & Cell Biology, 32, 539-547.
• Keri, R.S., Sasidhar, B.S., Nagaraja, B.M. and Santos, M.A., 2015. Recent progress in the drug development of coumarin derivatives as potent antituberculosis agents. European Journal of Medicinal Chemistry, 100, 257–269.
• Khattari, Z., Sayyed, M.I., Qashou, S.I., Fasfous, I., Al-Abdullah, T. and Maghrabi, M., 2017. Interfacial behavior of myristic acid in mixtures with DMPC and cholesterol. Chemical Physics, 490, 106–114.
• Kothekar, V., 1996. Molecular dynamics study of interaction of dimyristoyl phosphotidylcholine with water. Journal of Biosciences, 21, 577‒597.
• Koukoulitsa, C., Durdagi, S., Siapi, E., Villalonga-Barber, C., Alexi, X., Steele, B.R., Micha-Screttas, M., Alexis, M.N., Tsantili-Kakoulidou, A. and Mavromoustakos, T., 2011. Comparison of thermal effects of stilbenoid analogs in lipid bilayers using differential scanning calorimetry and molecular dynamics: correlation of thermal effects and topographical position with antioxidant activity. European Biophysics Journal, 40, 865–875.
• Lee, D.C. and Chapman, D., 1986. Infrared spectroscopic studies of biomembranes and model membranes. Bioscience Reports, 6, 235–256.
• Li, M., Chen, M., Sheepwash, E., Brosseau, C.L., Li, H., Pettinger, B., Gruler, H. and Lipkowski, J., 2008. AFM studies of solid-supported lipid bilayers formed at a Au(111) electrode surface using vesicle fusion and a combination of Langmuir-Blodgett and Langmuir-Schaefer techniques. Langmuir, 24, 10313‒10323.
• Lin, H.-C., Tsai, S.-H., Chen, C.-S., Chang, Y.-C., Lee, C.-M., Lai, Z.-Y. and Lin, C.-M., 2008. Structure–activity relationship of coumarin derivatives on xanthine oxidase-inhibiting and free radical-scavenging activities. Biochemical Pharmacology, 75, 1416‒1425.
• Liu, Y.-P., Yan, G., Xie, Y.-T., Lin, T.-C., Zhang, W., Li, J., Wu, Y.-J., Zhou, J.-Y. and Fu, Y.-H., 2020. Bioactive prenylated coumarins as potential anti-inflammatory and anti-HIV agents from Clausena lenis. Bioorganic Chemistry, 97, 103699.
• Lončar, M., Jakovljević, M., Šubarić, D., Pavlić, M., Služek, V.B., Cindrić, I. and Molnar, M., 2020. Coumarins in food and methods of their determination. Foods, 9, 645.
• Longo, E., Ciuchi, F., Guzzi, R., Rizzuti, B. and Bartucci, R., 2016. Resveratrol induces chain interdigitation in DPPC cell membrane model systems. Colloids and Surfaces B: Biointerfaces, 148, 615–621.
• López-García, F., Micol, V., Villalaín, J. and Gómez-Fernández, J.C., 1993. Infrared spectroscopic study of the interaction of diacylglycerol with phosphatidylserine in the presence of calcium. Biochimica et Biophysica Acta, 1169, 264‒272.
• Mahajan, S. and Mahajan, R.K., 2013. Interactions of phenothiazine drugs with surfactants: a detailed physicochemical overview. Advances in Colloid and Interface Science, 199‒200, 1‒14.
• Mantsch, H.H., 1984. Biological applications of Fourier transform infrared spectroscopy. A study of phase transitions in biomembranes. Journal of Molecular Structure, 113, 201‒212.
• Marsan, M.P., Muller, I., Ramos, C., Rodriguez, F., Dufourc, E.J., Czaplicki, J. and Milon, A., 1999. Cholesterol orientation and dynamics in dimyristoylphosphatidylcholine bilayers: a solid state deuterium NMR analysis. Biophysical Journal, 76, 351‒359.
• Matos, M.J., Pérez-Cruz, F., Vazquez-Rodriguez, S., Uriarte, E., Santana, L., Borges, F. and Olea-Azar, C., 2013. Remarkable antioxidant properties of a series of hydroxy-3-arylcoumarins. Bioorganic & Medicinal Chemistry, 21, 3900‒3906.
• Robledo-O’Ryan, N., Matos, M.J., Vazquez-Rodriguez, S., Santana, L., Uriarte, E., Moncada-Basualto, M., Mura, F., Lapier, M., Maya, J.D. and Olea-Azar, C., 2017. Synthesis, antioxidant and antichagasic properties of a selected series of hydroxy-3-arylcoumarins. Bioorganic & Medicinal Chemistry, 25, 621‒-632.
• Mavromoustakos, T., Yang, D.P., Charalambous, A., Herbette, L.G. and Makriyannis, A., 1990. Study of the topography of cannabinoids in model membranes using X-ray diffraction. Biochimica et Biophysica Acta, 1024, 336‒344.
• Mirunalini, S. and Krishnaveni, M., 2011. Coumarin: a plant derived polyphenol with wide biomedical applications. International Journal of PharmTech Research, 3, 1693‒1696.
• Mishra, S., Pandey, A. and Manvati, S., 2020. Coumarin: An emerging antiviral agent. Heliyon, 6, e03217.
• Miskowiec, A., Buck, Z.N., Hansen, F.Y., Kaiser, H., Taub, H., Tyagi, M., Diallo, S.O., Mamontov, E. and Herwig, K.W., 2017. On the structure and dynamics of water associated with single-supported zwitterionic and anionic membranes. Journal of Chemical Physics, 146, 125102.
• Niaz, T., Shabbir, S., Noor, T., Rahman, A. Bokhari, H. and Imran, M., 2018. Potential of polymer stabilized nano-liposomes to enhance antimicrobial activity of nisin Z against foodborne pathogens. LWT - Food Science and Technology, 96, 98‒110.
• Oteiza, P.I., Erlejman, A.G., Verstraeten, S.V., Keen, C.L. and Fraga, C.G., 2005 Flavonoid membrane interactions: a protective role of flavonoids at the membrane surface?. Clinical and Developmental Immunology. 12, 19–25.
• Pasenkiewicz-Gierula, M., Takaoka, Y., Miyagawa, H., Kitamura, K. and Kusumi, A., 1999. Charge pairing of headgroups in phosphatidylcholine membranes: A molecular dynamics simulation study. Biophysical Journal, 76, 1228‒1240.
• Pereira-Leite, C., Nunes, C. and Reis, S., 2013. Interaction of nonsteroidal anti-inflammatory drugs with membranes: In vitro assessment and relevance for their biological actions. Progress in Lipid Research, 52, 571‒584.
• Pérez-Cruz, K., Moncada-Basualto, M., Morales-Valenzuela, J., Barriga-González, G., Navarrete-Encina, P., Núñez-Vergara, L., Squella, J.A. and Olea-Azar, C., 2018. Synthesis and antioxidant study of new polyphenolic hybrid-coumarins. Arabian Journal of Chemistry, 11, 525‒537.
• Petrache, H.I., Tristram-Nagle, S. and Nagle, J.F., 1998. Fluid phase structure of EPC and DMPC bilayers. Chemistry and Physics of Lipids, 95, 83‒94.
• Prenner, E.J., Lewis, R.N., Kondejewski, L.H., Hodges, R.S. and McElhaney, R.N., 1999. Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes. Biochimica et Biophysica Acta, 1417, 211‒223.
• Robson, A.L., Dastoor, P.C., Flynn, J., Palmer, W., Martin, A., Smith, D.W., Woldu, A. and Hua, S., 2018. Advantages and limitations of current imaging techniques for characterizing liposome morphology. Frontiers in Pharmacology, 9, 80.
• Sadžak, A., Mravljak, J., Maltar-Strmečki, N., Arsov, Z., Baranović, G., Erceg, I., Kriechbaum, M., Strasser, V., Přibyl, J. and Šegota, S., 2020. The structural integrity of the model lipid membrane during induced lipid peroxidation: the role of flavonols in the inhibition of lipid peroxidation. Antioxidants, 9, 430.
• Saija, A., Bonina, F., Trombetta, D., Tomaino, A., Montenegro, L., Smeriglio, P. and Castelli, F., 1995. Flavonoid-biomembrane interactions: A calorimetric study on dipalmitoylphosphatidylcholine vesicles. International Journal of Pharmaceutics, 124, 1‒8.
• Sarpietro, M.G., Giuffrida, M.C., Ottimo, S., Micieli, D. and Castelli, F., 2011. Evaluation of the interaction of coumarins with biomembrane models studied by differential scanning calorimetry and Langmuir-Blodgett techniques. Journal of Natural Products, 74, 790‒795.
• Scheibe, C. and Hauser, K., 2018. Orientation of lipids in solid supported lipid bilayers studied by polarized ATR-FTIR spectroscopy. Biomedical Spectroscopy and Imaging, 7, 17‒24.
• Schneider, C.A., Rasband, W.S., and Eliceiri, K.W., 2012. NIH Image to ImageJ: 25 years of image analysis. Nature Methods, 9, 671–675.
• Solans, R., Motta, C., Solá, R., La Ville, A.E., Lima, J., Simeón, P., Montellà, N., Armadans-Gil, L., Fonollosa, V. and Vilardell, M., 2000. Abnormalities of erythrocyte membrane fluidity, lipid composition, and lipid peroxidation in systemic sclerosis: evidence of free radical-mediated injury. Arthritis & Rheumatology, 43, 894–900.
• Sorkin, R., Kampf, N., Dror, Y., Shimoni, E. and Klein, J., 2013. Origins of extreme boundary lubrication by phosphatidylcholine liposomes. Biomaterials, 34, 5465‒5475.
• Sreekanth, V. and Bajaj, A., 2013. Fluorescence (fluidity/hydration) and calorimetric studies of interactions of bile acid–drug conjugates with model membranes. The Journal of Physical Chemistry B, 117, 2123–2133. Trucillo, P., Campardelli, R. and Reverchon, E., 2018. Production of liposomes loaded with antioxidants using a supercritical CO2 assisted process. Powder Technology, 323, 155‒162.
• Ueda, I., Chiou, J.S., Krishna, P.R. and Kamaya, H., 1994. Local anesthetics destabilize lipid membranes by breaking hydration shell: infrared and calorimetry studies. Biochimica et Biophysica Acta, 1190, 421‒429.
• Weisz, K., Gröbner, G., Mayer, C., Stohrer, J. and Kothe, G., 1992. Deuteron nuclear magnetic resonance study of the dynamic organization of phospholipid/cholesterol bilayer membranes: molecular properties and viscoelastic behavior. Biochemistry, 31, 1100‒1112.
• Witaicenis, A., Seito, L.N., da Silveira Chagas, A., de Almeida Jr, L.D., Luchini, A.C., Rodrigues-Orsi, P., Cestari, S.H. and Di Stasi, L.C., 2014. Antioxidant and intestinal anti-inflammatory effects of plant-derived coumarin derivatives. Phytomedicine, 21, 240‒246.
• Wood, M.H., Milan, D.C., Nichols, R.J., Casford, M.T.L. and Horswell, S.L., 2021. A quantitative determination of lipid bilayer deposition efficiency using AFM. RSC Advances, 11, 19768.
• Wong, P.T.T. and Mantsch, H.H., 1988. High pressure infrared spectroscopic evidence of water binding sites in 1,2-diacyl phospholipids. Chemistry and Physics of Lipids, 46, 213‒224.
• Zawisza, I., Bin, X. and Lipkowski, J., 2004. Spectroelectrochemical studies of bilayers of phospholipids in gel and liquid state on Au(111) electrode surface. Bioelectrochemistry, 63, 137‒147.
• Zhang, H.-Y. and Wang, L.-F., 2004. Theoretical elucidation of structure–activity relationship for coumarins to scavenge peroxyl radical. Journal of Molecular Structure: THEOCHEM, 673, 199‒202.
• Zhang, Y., Pu, C., Tang, W., Wang, S. and Sun, Q., 2020. Effects of four polyphenols loading on the attributes of lipid bilayers. Journal of Food Engineering, 282, 110008.
• Zhao, L. and Feng, S.S., 2006. Effects of cholesterol component on molecular interactions between paclitaxel and phospholipid within the lipid monolayer at the air-water interface. Journal of Colloid and Interface Science, 300, 314‒326.
• Zubrzycki, I.Z., Xu, Y., Madrid, M. and Tang, P., 2000. Molecular dynamics simulations of a fully hydrated dimyristoylphosphatidylcholine membrane in liquid– crystalline phase. Journal of Chemical Physics, 112, 3437‒3441.