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Recent Advances in Cryo-TEM Imaging of Soft Lipid Nanoparticles

  • Received: 17 February 2015 Accepted: 05 May 2015 Published: 12 May 2015
  • Cryo-transmission electron microscopy (Cryo-TEM), and its technological variations thereof, have become a powerful tool for detailed morphological characterization and 3D tomography of soft lipid and polymeric nanoparticles as well as biological materials such as viruses and DNA without chemical fixation. Here, we review and discuss recent advances in Cryo-TEM analysis of lipid-based drug nanocarriers with particular emphasis on morphological and internal nanostructure characterization of lyotropic liquid crystalline nanoparticles such as cubosomes and hexosomes.

    Citation: Shen Helvig, Intan D. M. Azmi, Seyed M. Moghimi, Anan Yaghmur. Recent Advances in Cryo-TEM Imaging of Soft Lipid Nanoparticles[J]. AIMS Biophysics, 2015, 2(2): 116-130. doi: 10.3934/biophy.2015.2.116

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  • Cryo-transmission electron microscopy (Cryo-TEM), and its technological variations thereof, have become a powerful tool for detailed morphological characterization and 3D tomography of soft lipid and polymeric nanoparticles as well as biological materials such as viruses and DNA without chemical fixation. Here, we review and discuss recent advances in Cryo-TEM analysis of lipid-based drug nanocarriers with particular emphasis on morphological and internal nanostructure characterization of lyotropic liquid crystalline nanoparticles such as cubosomes and hexosomes.

    [1] Moghimi SM, Hunter AC, Murray JC (2005) Nanomedicine: current status and future prospects. FASEB J 19: 311-330. doi: 10.1096/fj.04-2747rev
    [2] Grazú V, Moros M, Sánchez-Espinel C (2012) Nanobiotechnology - Inorganic Nanoparticles vs Organic Nanoparticles. Front Nanosci 4: 337-440. doi: 10.1016/B978-0-12-415769-9.00014-5
    [3] Lim SB, Banerjee A, Önyüksel H (2012) Improvement of drug safety by the use of lipid-based nanocarriers. J Control Release 163: 34-45. doi: 10.1016/j.jconrel.2012.06.002
    [4] Nel AE, Maedler L, Velegol D, et al. (2009) Understanding biophysicochemical interactions at the nano-bio interface. Nat Mater 8: 543-557. doi: 10.1038/nmat2442
    [5] Couvreur P, Vauthier C (2006) Nanotechnology: intelligent design to treat complex disease. Pharm Res 23: 1417-1450. doi: 10.1007/s11095-006-0284-8
    [6] Jin S-E, Jin H-E, Hong S-S (2014) Targeted delivery system of nanobiomaterials in anticancer therapy: from cells to clinics. BioMed Res Int 2014: 814208.
    [7] Moghimi SM, Peer D, Langer R (2011) Reshaping the future of nanopharmaceuticals: adiudicium. ACS Nano 5: 8454-8458. doi: 10.1021/nn2038252
    [8] Klang V, Valenta C, Matsko NB (2013) Electron microscopy of pharmaceutical systems. Micron 44: 45-74. doi: 10.1016/j.micron.2012.07.008
    [9] Danino D (2012) Cryo-TEM of soft molecular assemblies. Curr Opin Colloid Interface Sci 17: 316-329. doi: 10.1016/j.cocis.2012.10.003
    [10] Danino D (2001) Digital cryogenic transmission electron microscopy: an advanced tool for direct imaging of complex fluids. Colloids Surf A 183-185: 113-122.
    [11] Burrows ND, Penn RL (2013) Cryogenic Transmission Electron Microscopy: Aqueous Suspensions of Nanoscale Objects. Microsc Microanal 19: 1542-1553. doi: 10.1017/S1431927613013354
    [12] Cho EJ, Holback H, Liu KC, et al. (2013) Nanoparticle characterization: State of the art, challenges, and emerging technologies. Mol Pharm 10: 2093-2110. doi: 10.1021/mp300697h
    [13] McNeil SE (2011) Challenges for nanoparticle characterization. Methods Mol Biol 697: 9-15. doi: 10.1007/978-1-60327-198-1_2
    [14] Friedrich H, Frederik PM, de With G, et al. (2010) Imaging of self-assembled structures: interpretation of TEM and cryo-TEM images. Angew Chem Int Ed Engl 49: 7850-7858. doi: 10.1002/anie.201001493
    [15] Kuntsche J, Horst JC, Bunjes H (2011) Cryogenic transmission electron microscopy (cryo-TEM) for studying the morphology of colloidal drug delivery systems. Int J Pharm 417: 120-137. doi: 10.1016/j.ijpharm.2011.02.001
    [16] Rubino S, Akhtar S, Melin P, et al. (2012) A site-specific focused-ion-beam lift-out method for cryo Transmission Electron Microscopy. J Struct Biol 180: 572-576. doi: 10.1016/j.jsb.2012.08.012
    [17] Frederik PM, Stuart MC, Bomans PH, et al. (1991) Perspective and limitations of cryo-electron microscopy. From model systems to biological specimens. J Microsc 161: 253-262.
    [18] Mielanczyk L, Matysiak N, Michalski M, et al. (2014) Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples. Folia Histochem Cytobiol 52: 1-17.
    [19] Pilhofer M, Ladinsky MS, McDowall AW, et al. (2010) Bacterial TEM: new insights from cryo-microscopy. Methods Cell Biol 96: 21-45. doi: 10.1016/S0091-679X(10)96002-0
    [20] De Carlo S, Harris JR (2011) Negative staining and cryo-negative staining of macromolecules and viruses for TEM. Micron 42: 117-131. doi: 10.1016/j.micron.2010.06.003
    [21] Adrian M, Dubochet J, Lepault J, et al. (1984) Cryo-electron microscopy of viruses. Nature 308: 32-36. doi: 10.1038/308032a0
    [22] Fatouros DG, Müllertz A (2013) Development of Self-Emulsifying Drug Delivery Systems (SEDDS) for Oral Bioavailability Enhancement of Poorly Soluble Drugs. Drug Delivery Strategies for Poorly Water-Soluble Drugs: John Wiley & Sons Ltd. pp. 225-245.
    [23] Talmon Y (2007) Seeing giant micelles by cryogenic-temperature transmission electron microscopy (cryo-TEM). Surfactant Sci Ser 140: 163.
    [24] Spernath L, Regev O, Levi-Kalisman Y, et al. (2009) Phase transitions in O/W lauryl acrylate emulsions during phase inversion, studied by light microscopy and cryo-TEM. Colloids Surf A 332: 19-25. doi: 10.1016/j.colsurfa.2008.08.026
    [25] Almgren M, Edwards K, Karlsson G (2000) Cryo transmission electron microscopy of liposomes and related structures. Colloids Surf A 174: 3-21. doi: 10.1016/S0927-7757(00)00516-1
    [26] Ferreira DA, Bentley MVLB, Karlsson G, et al. (2006) Cryo-TEM investigation of phase behaviour and aggregate structure in dilute dispersions of monoolein and oleic acid. Int J Pharm 310: 203-212. doi: 10.1016/j.ijpharm.2005.11.028
    [27] Wytrwal M, Bednar J, Nowakowska M, et al. (2014) Interactions of serum with polyelectrolyte-stabilized liposomes: Cryo-TEM studies. Colloids Surf B 120: 152-159. doi: 10.1016/j.colsurfb.2014.02.040
    [28] Fatouros DG, Bergenstahl B, Mullertz A (2007) Morphological observations on a lipid-based drug delivery system during in vitro digestion. Eur J Pharm Sci 31: 85-94. doi: 10.1016/j.ejps.2007.02.009
    [29] Basáñez G, Ruiz-Argüello MB, Alonso A, et al. (1997) Morphological changes induced by phospholipase C and by sphingomyelinase on large unilamellar vesicles: a cryo-transmission electron microscopy study of liposome fusion. Biophys J 72: 2630-2637. doi: 10.1016/S0006-3495(97)78906-9
    [30] Phan S, Hawley A, Mulet X, et al. (2013) Structural aspects of digestion of medium chain triglycerides studied in real time using sSAXS and Cryo-TEM. Pharm Res 30: 3088-3100. doi: 10.1007/s11095-013-1108-2
    [31] Alfredsson V (2005) Cryo-TEM studies of DNA and DNA-lipid structures. Curr Opin Colloid Interface Sci 10: 269-273. doi: 10.1016/j.cocis.2005.09.005
    [32] Abraham SA, Edwards K, Karlsson G, et al. (2004) An evaluation of transmembrane ion gradient-mediated encapsulation of topotecan within liposomes. J Control Release 96: 449-461. doi: 10.1016/j.jconrel.2004.02.017
    [33] Johansson E, Lundquist A, Zuo S, et al. (2007) Nanosized bilayer disks: attractive model membranes for drug partition studies. Biochim Biophys Acta 1768: 1518-1525. doi: 10.1016/j.bbamem.2007.03.006
    [34] Yaghmur A, Glatter O (2009) Characterization and potential applications of nanostructured aqueous dispersions. Adv Colloid Interface Sci 147-148: 333-342.
    [35] Yaghmur A, Glatter O (2012) Self-Assembly in Lipidic Particles. Self-Assembled Supramolecular Architectures: Lyotropic Liquid Crystals: John Wiley & Sons pp. 129-155.
    [36] Yaghmur A, Rappolt M (2012) Structural characterization of lipidic systems under nonequilibrium conditions. Eur Biophys J 41: 831-840. doi: 10.1007/s00249-012-0815-7
    [37] Nilsson C, Edwards K, Eriksson J, et al. (2012) Characterization of oil-free and oil-loaded liquid-crystalline particles stabilized by negatively charged stabilizer citrem. Langmuir 28: 11755-11766. doi: 10.1021/la3021244
    [38] Hedegaard SF, Nilsson C, Laurinmaki P, et al. (2013) Nanostructured aqueous dispersions of citrem interacting with lipids and PEGylated lipids. RSC Adv 3: 24576-24585. doi: 10.1039/c3ra44583f
    [39] Chang DP, Jankunec M, Barauskas J, et al. (2012) Adsorption of lipid liquid crystalline nanoparticles: effects of particle composition, internal structure, and phase behavior. Langmuir 28: 10688-10696. doi: 10.1021/la301579g
    [40] Siegel DP, Burns JL, Chestnut MH, et al. (1989) Intermediates in membrane fusion and bilayer/nonbilayer phase transitions imaged by time-resolved cryo-transmission electron microscopy. Biophys J 56: 161-169. doi: 10.1016/S0006-3495(89)82661-X
    [41] Barauskas J, Johnsson M, Tiberg F (2005) Self-assembled lipid superstructures: beyond vesicles and liposomes. Nano Lett 5: 1615-1619. doi: 10.1021/nl050678i
    [42] Janiak J, Bayati S, Galantini L, et al. (2012) Nanoparticles with a bicontinuous cubic internal structure formed by cationic and non-ionic surfactants and an anionic polyelectrolyte. Langmuir 28: 16536-16546. doi: 10.1021/la303938k
    [43] Yaghmur A, De Campo L, Sagalowicz L, et al. (2005) Emulsified microemulsions and oil-containing liquid crystalline phases. Langmuir 21: 569-577. doi: 10.1021/la0482711
    [44] Yaghmur A, de Campo L, Salentinig S, et al. (2006) Oil-loaded monolinolein-based particles with confined inverse discontinuous cubic structure (Fd3m). Langmuir 22: 517-521. doi: 10.1021/la052109w
    [45] de Campo L, Yaghmur A, Sagalowicz L, et al. (2004) Reversible Phase Transitions in Emulsified Nanostructured Lipid Systems. Langmuir 20: 5254-5261. doi: 10.1021/la0499416
    [46] Nilsson C, Østergaard J, Larsen SW, et al. (2014) PEGylation of phytantriol-based lyotropic liquid crystalline particles-the effect of lipid composition, PEG chain length, and temperature on the internal nanostructure. Langmuir 30: 6398-6407. doi: 10.1021/la501411w
    [47] Sagalowicz L, Michel M, Adrian M, et al. (2006) Crystallography of dispersed liquid crystalline phases studied by cryo-transmission electron microscopy. J Microsc 221: 110-121. doi: 10.1111/j.1365-2818.2006.01544.x
    [48] Gao M, Kim Y-K, Zhang C, et al. (2014) Direct observation of liquid crystals using cryo-TEM: specimen preparation and low-dose imaging. Microsc Res Tech 77: 754-772. doi: 10.1002/jemt.22397
    [49] Angelova A, Angelov B, Drechsler M, et al. (2013) Protein entrapment in PEGylated lipid nanoparticles. Int J Pharm 454: 625-632. doi: 10.1016/j.ijpharm.2013.06.006
    [50] Sagalowicz L, Mezzenga R, Leser ME (2006) Investigating reversed liquid crystalline mesophases. Curr Opin Colloid Interface Sci 11: 224-229. doi: 10.1016/j.cocis.2006.07.002
    [51] Driever CD, Mulet X, Waddington LJ, et al. (2013) Layer-by-layer polymer coating on discrete particles of cubic lyotropic liquid crystalline dispersions (cubosomes). Langmuir 29: 12891-12900. doi: 10.1021/la401660h
    [52] Oliveira IMSC, Silva JPN, Feitosa E, et al. (2012) Aggregation behavior of aqueous dioctadecyldimethylammonium bromide/monoolein mixtures: a multitechnique investigation on the influence of composition and temperature. J Colloid Interface Sci 374: 206-217. doi: 10.1016/j.jcis.2012.01.053
    [53] Bode JC, Kuntsche J, Funari SS, et al. (2013) Interaction of dispersed cubic phases with blood components. Int J Pharm 448: 87-95. doi: 10.1016/j.ijpharm.2013.03.016
    [54] Azmi IDM, Wu L, Wibroe PP, et al. (2015) Modulatory effect of human plasma on the internal nanostructure and size characteristics of liquid crystalline nanocarriers. Langmuir 31: 5042-5049. doi: 10.1021/acs.langmuir.5b00830
    [55] Nilsson C, Barrios-Lopez B, Kallinen A, et al. (2013) SPECT/CT imaging of radiolabeled cubosomes and hexosomes for potential theranostic applications. Biomaterials 34: 8491-8503. doi: 10.1016/j.biomaterials.2013.07.055
    [56] Angelov B, Angelova A, Filippov SK, et al. (2014) Multicompartment lipid cubic nanoparticles with high protein upload: millisecond dynamics of formation. ACS Nano 8: 5216-5226. doi: 10.1021/nn5012946
    [57] Yaghmur A, Laggner P, Almgren M, et al. (2008) Self-assembly in monoelaidin aqueous dispersions: direct vesicles to cubosomes transition. PLoS ONE 3: e3747. doi: 10.1371/journal.pone.0003747
    [58] Rizwan SB, Dong Y-D, Boyd BJ, et al. (2007) Characterisation of bicontinuous cubic liquid crystalline systems of phytantriol and water using cryo field emission scanning electron microscopy (cryo FESEM). Micron 38:478-485. doi: 10.1016/j.micron.2006.08.003
    [59] Sagalowicz L, Acquistapace S, Watzke HJ, et al. (2007) Study of liquid crystal space groups using controlled tilting with cryogenic transmission electron microscopy. Langmuir 23: 12003-12009. doi: 10.1021/la701410n
    [60] Percec V, Wilson DA, Leowanawat P, et al. (2010) Self-assembly of Janus dendrimers into uniform dendrimersomes and other complex architectures. Science 328: 1009-1014. doi: 10.1126/science.1185547
    [61] Siegel DP, Epand RM (1997) The mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms. Biophys J 73: 3089-3111. doi: 10.1016/S0006-3495(97)78336-X
    [62] Siegel DP, Green WJ, Talmon Y (1994) The mechanism of lamellar-to-inverted hexagonal phase transitions: a study using temperature-jump cryo-electron microscopy. Biophys J 66: 402-414. doi: 10.1016/S0006-3495(94)80790-8
    [63] Ansari MJ, Kohli K, Dixit N (2008) Microemulsions as potential drug delivery systems: a review. PDA J Pharm Sci Technol 62: 66-79.
    [64] Lawrence MJ, Rees GD (2000) Microemulsion-based media as novel drug delivery systems. Adv Drug Delivery Rev 45: 89-121. doi: 10.1016/S0169-409X(00)00103-4
    [65] Chatzidaki MD, Mitsou E, Yaghmur A, et al. (2015) Formulation and characterization of food-grade microemulsions as carriers of natural phenolic antioxidants. Colloid Surf A Doi: 10.1016/j.colsurfa.2015.03.060.
    [66] Yaghmur A., Aserin A, Garti N (2002) Phase behavior of microemulsions based on food-grade nonionic surfactants: effect of polyols and short-chain alcohols. Colloids Surf A 209:71-81. doi: 10.1016/S0927-7757(02)00168-1
    [67] Chevalier Y, Zemb T (1990) The Structure of Micelles and Microemulsions. Rep Prog Phys 53: 279-371. doi: 10.1088/0034-4885/53/3/002
    [68] Strey R (1994) Microemulsion microstructure and interfacial curvature. Colloid Polym Sci 272: 1005-1019. doi: 10.1007/BF00658900
    [69] Gradzielski M (2008) Recent developments in the characterisation of microemulsions. Curr Opin Colloid Interface Sci 13: 263-269. doi: 10.1016/j.cocis.2007.10.006
    [70] Belkoura L, Stubenrauch C, Strey R (2004) Freeze fracture direct imaging: A new freeze fracture method for specimen preparation in cryo-transmission electron microscopy. Langmuir 20: 4391-4399. doi: 10.1021/la0303411
    [71] Won YY, Brannan AK, Davis HT, et al. (2002) Cryogenic transmission electron microscopy (cryo-TEM) of micelles and vesicles formed in water by polyethylene oxide-based block copolymers. J Phys Chem B 106: 3354-3364. doi: 10.1021/jp013639d
    [72] Subramaniam S, Milne JL (2004) Three-dimensional electron microscopy at molecular resolution. Annu Rev Biophys Biomol Struct 33: 141-155. doi: 10.1146/annurev.biophys.33.110502.140339
    [73] Milne JLS, Borgnia MJ, Bartesaghi A, et al. (2013) Cryo-electron microscopy--a primer for the non-microscopist. FEBS J 280: 28-45. doi: 10.1111/febs.12078
    [74] Lengyel J, Milne JLS, Subramaniam S (2008) Electron tomography in nanoparticle imaging and analysis. Nanomedicine 3: 125-131. doi: 10.2217/17435889.3.1.125
    [75] Hoenger A, Bouchet-Marquis C (2011) Cellular tomography. Adv Protein Chem Struct Biol 82: 67-90. doi: 10.1016/B978-0-12-386507-6.00003-8
    [76] Hurbain I, Sachse M (2011) The future is cold: cryo-preparation methods for transmission electron microscopy of cells. Biol Cell 103: 405-420. doi: 10.1042/BC20110015
    [77] Milne JL, Subramaniam S (2009) Cryo-electron tomography of bacteria: progress, challenges and future prospects. Nat Rev Microbiol 7: 666-675. doi: 10.1038/nrmicro2183
    [78] Tocheva EI, Li Z, Jensen GJ (2010) Electron cryotomography. Cold Spring Harb Perspect Biol 2: a003442.
    [79] Pierson J, Vos M, McIntosh JR, et al. (2011) Perspectives on electron cryo-tomography of vitreous cryo-sections. J Electron Microsc (Tokyo) 60 Suppl 1: S93-100.
    [80] Subramaniam S, Bartesaghi A, Liu J, et al. (2007) Electron tomography of viruses. Curr Opin Struct Biol 17: 596-602. doi: 10.1016/
    [81] Yahav T, Maimon T, Grossman E, et al. (2011) Cryo-electron tomography: gaining insight into cellular processes by structural approaches. Curr Opin Struct Biol 21: 670-677. doi: 10.1016/
    [82] Wisedchaisri G, Reichow SL, Gonen T (2011) Advances in structural and functional analysis of membrane proteins by electron crystallography. Structure 19: 1381-1393. doi: 10.1016/j.str.2011.09.001
    [83] Orlova EV, Saibil HR (2011) Structural analysis of macromolecular assemblies by electron microscopy. Chem Rev 111: 7710-7748. doi: 10.1021/cr100353t
    [84] Frank J (2009) Single-particle reconstruction of biological macromolecules in electron microscopy—30 years. Q Rev Biophys 42: 139-158. doi: 10.1017/S0033583509990059
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