Pharmaceutical Research

, Volume 32, Issue 11, pp 3660–3673 | Cite as

Using Environment-Sensitive Fluorescent Probes to Characterize Liquid-Liquid Phase Separation in Supersaturated Solutions of Poorly Water Soluble Compounds

  • Shweta A. Raina
  • David E. Alonzo
  • Geoff G. Z. Zhang
  • Yi Gao
  • Lynne S. Taylor
Research Paper



Highly supersaturated aqueous solutions of poorly soluble compounds can undergo liquid-liquid phase separation (LLPS) when the concentration exceeds the “amorphous solubility”. This phenomenon has been widely observed during high throughput screening of new molecular entities as well as during the dissolution of amorphous solid dispersions. In this study, we have evaluated the use of environment-sensitive fluorescence probes to investigate the formation and properties of the non-crystalline drug-rich aggregates formed in aqueous solutions as a result of LLPS.


Six different environment-sensitive fluorophores were employed to study LLPS in highly supersaturated solutions of several model compounds, all dihydropyridine derivatives.


Each fluoroprobe exhibited a large hypsochromic shift with decreasing environment polarity. Upon drug aggregate formation, the probes partitioned into the drug-rich phase and exhibited changes in emission wavelength and intensity consistent with sensing a lower polarity environment. The LLPS onset concentrations determined using the fluorescence measurements were in good agreement with light scattering measurements as well as theoretically estimated amorphous solubility values.


Environment-sensitive fluorescence probes are useful to help understand the phase behavior of highly supersaturated aqueous solutions, which in turn is important in the context of developing enabling formulations for poorly soluble compounds.


Fluorescence Phase separation Supersaturation 



Amorphous solid dispersions


Critical micelle concentration


2-(4-(dimethylamino)styryl) -N-Ethylpyridinium Iodide


Dimethyl sulfoxide


Excited state intermolecular proton transfer


Hydroxypropylmethyl cellulose


Hydroxypropylmethyl cellulose acetate succinate


Intermolecular charge transfer


Isopropyl alcohol


Liquid-liquid phase separation


Polarized light microscopy


4-(4-(Diethylamino)styryl) -N-Methylpyridinium Iodide (4-Di-2ASP) 6-Propionyl-2-dimethylaminonaphthalene




Trifluoroacetic acid




  1. 1.
    Tachibana T, Nakamura A. A methode for preparing an aqueous colloidal dispersion of organic materials by using water-soluble polymers: dispersion ofΒ-carotene by polyvinylpyrrolidone. Kolloid-ZuZPolymere. 1965;203:130–3.CrossRefGoogle Scholar
  2. 2.
    Aisha AFA, Ismail Z, Abu-salah KM, Majid AMSA. Solid dispersions of α-mangostin improve its aqueous solubility through self-assembly of nanomicelles. J Pharm Sci. 2012;101:815–25.CrossRefPubMedGoogle Scholar
  3. 3.
    Friesen DT, Shanker R, Crew M, Smithey DT, Curatolo WJ, Nightingale JAS. Hydroxypropyl methylcellulose acetate succinate-based spray-dried dispersions: an overview. Mol Pharmaceutics. 2008;5:1003–19.CrossRefGoogle Scholar
  4. 4.
    Alonzo DE, Gao Y, Zhou D, Mo H, Zhang GG, Taylor LS. Dissolution and precipitation behavior of amorphous solid dispersions. J Pharm Sci. 2011;100:3316–31.CrossRefPubMedGoogle Scholar
  5. 5.
    Kanzer J, Hupfeld S, Vasskog T, Tho I, Hölig P, Mägerlein M, et al. In situ formation of nanoparticles upon dispersion of melt extrudate formulations in aqueous medium assessed by asymmetrical flow field-flow fractionation. J Pharm Biomed Anal. 2010;53:359–65.CrossRefPubMedGoogle Scholar
  6. 6.
    Müllertz A, Ogbonna A, Ren S, Rades T. New perspectives on lipid and surfactant based drug delivery systems for oral delivery of poorly soluble drugs. J Pharm Pharmac. 2010;62:1622–36.CrossRefGoogle Scholar
  7. 7.
    Pacheco LF, Carmona-Ribeiro AM. Effects of synthetic lipids on solubilization and colloid stability of hydrophobic drugs. J Colloid Interf Sci. 2003;258:146–54.CrossRefGoogle Scholar
  8. 8.
    Sassene PJ, Knopp MM, Hesselkilde JZ, Koradia V, Larsen A, Rades T, et al. Precipitation of a poorly soluble model drug during in vitro lipolysis: characterization and dissolution of the precipitate. J Pharm Sci. 2010;99:4982–91.CrossRefPubMedGoogle Scholar
  9. 9.
    Hsieh Y-L, Box K, Taylor LS. Assessing the impact of polymers on the pH-induced precipitation behavior of poorly water-soluble compounds using synchrotron wide angle X-ray scattering. J Pharm Sci. 2014;103:2724–35.CrossRefPubMedGoogle Scholar
  10. 10.
    Hsieh Y-L, Ilevbare G, Van Eerdenbrugh B, Box K, Sanchez-Felix M, Taylor L. pH-induced precipitation behavior of weakly basic compounds: determination of extent and duration of supersaturation using potentiometric titration and correlation to solid state properties. Pharm Res. 2012;29:2738–53.CrossRefPubMedGoogle Scholar
  11. 11.
    Coan KED, Shoichet BK. Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors. J Am Chem Soc. 2008;130:9606–12.PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Zhang F, Roosen-Runge F, Sauter A, Roth R, Skoda MWA, Jacobs R, et al. The role of cluster formation and metastable liquid-liquid phase separation in protein crystallization. Faraday Discuss. 2012;159:313–25.CrossRefGoogle Scholar
  13. 13.
    Veesler S, Lafferrère L, Garcia E, Hoff C. Phase transitions in supersaturated drug solution. Org Process Res Dev. 2003;7:983–9.CrossRefGoogle Scholar
  14. 14.
    Raina SA, Zhang GGZ, Alonzo DE, Wu J, Zhu D, Catron ND, et al. Enhancements and limits in drug membrane transport using supersaturated solutions of poorly water-soluble drugs. J Pharm Sci. 2014;103:2736–48.CrossRefPubMedGoogle Scholar
  15. 15.
    S.A. Raina, G.G.Z. Zhang, D.E. Alonzo, J. Wu, D. Zhu, N.D. Catron, Y. Gao, L.S. Taylor. Impact of Solubilizing Additives on Supersaturation and Membrane Transport of Drugs. Pharm Res (2015). doi:10.1007/s11095-015-1712-4
  16. 16.
    Raina SA, Eerdenbrugh BV, Alonzo DE, Mo H, Zhang GGZ, Gao Y, et al. Trends in the precipitation and crystallization behavior of supersaturated aqueous solutions of poorly water-soluble drugs assessed using synchrotron radiation. J Pharm Sci. 2015;104:1981–92.CrossRefPubMedGoogle Scholar
  17. 17.
    Frank KJ WU, Rosenblatt KM, Hölig P, Rosenberg J, Mägerlein M, Fricker G, et al. The amorphous solid dispersion of the poorly soluble ABT-102 forms nano/microparticulate structures in aqueous medium: impact on solubility. Int J Nanomed. 2012;7:5757–68.Google Scholar
  18. 18.
    Frank KJ, Westedt U, Rosenblatt KM, Hölig P, Rosenberg J, Mägerlein M, et al. What is the mechanism behind increased permeation rate of a poorly soluble drug from aqueous dispersions of an amorphous solid dispersion? J Pharm Sci. 2014;103:1779–86.CrossRefPubMedGoogle Scholar
  19. 19.
    Wang J, Matayoshi E. Solubility at the molecular level: development of a critical aggregation concentration (CAC) assay for estimating compound monomer solubility. Pharm Res. 2012;29:1745–54.CrossRefPubMedGoogle Scholar
  20. 20.
    Taboada P, Attwood D, Ruso JM, Garcia M, Mosquera V. Static and dynamic light scattering study on the association of some antidepressants in aqueous electrolyte solutions. Phys Chem Chem Phys. 2000;2:5175–9.CrossRefGoogle Scholar
  21. 21.
    Van Eerdenbrugh B, Alonzo DE, Taylor LS. Influence of particle size on the ultraviolet spectrum of particulate-containing solutions: implications for in-situ concentration monitoring using UV/Vis fiber-optic probes. Pharm Res. 2011;28:1643–52.CrossRefPubMedGoogle Scholar
  22. 22.
    Sugano K, Kato T, Suzuki K, Keiko K, Sujaku T, Mano T. High throughput solubility measurement with automated polarized light microscopy analysis. J Pharm Sci. 2006;95:2115–22.CrossRefPubMedGoogle Scholar
  23. 23.
    Qi S, Roser S, Edler K, Pigliacelli C, Rogerson M, Weuts I, et al. Insights into the role of polymer-surfactant complexes in drug solubilisation/stabilisation during drug release from solid dispersions. Pharm Res. 2013;30:290–302.CrossRefPubMedGoogle Scholar
  24. 24.
    Gao X, Huang Y, Makhov AM, Epperly M, Lu J, Grab S, et al. Nanoassembly of surfactants with interfacial drug-interactive motifs as tailor-designed drug carriers. Mol Pharmaceutics. 2012;10:187–98.CrossRefGoogle Scholar
  25. 25.
    Giannetti AM, Koch BD, Browner MF. Surface plasmon resonance based assay for the detection and characterization of promiscuous inhibitors. J Med Chem. 2008;51:574–80.CrossRefPubMedGoogle Scholar
  26. 26.
    Abbou Oucherif K, Raina S, Taylor LS, Litster JD. Quantitative analysis of the inhibitory effect of HPMC on felodipine crystallization kinetics using population balance modeling. CrystEngComm. 2013;15:2197–205.CrossRefGoogle Scholar
  27. 27.
    Raina SA, Alonzo DE, Zhang GGZ, Gao Y, Taylor LS. Impact of polymers on the crystallization and phase transition kinetics of amorphous nifedipine during dissolution in aqueous media. Mol Pharmaceutics. 2014;11:3565–76.CrossRefGoogle Scholar
  28. 28.
    Baird JA, Van Eerdenbrugh B, Taylor LS. A classification system to assess the crystallization tendency of organic molecules from undercooled melts. J Pharm Sci. 2010;99:3787–806.CrossRefPubMedGoogle Scholar
  29. 29.
    Ilevbare GA, Taylor LS. Liquid–liquid phase separation in highly supersaturated aqueous solutions of poorly water-soluble drugs: implications for solubility enhancing formulations. Cryst Growth Des. 2013;13:1497–509.CrossRefGoogle Scholar
  30. 30.
    Hoffman JD. Thermodynamic driving force in nucleation and growth processes. J Chem Phys. 1958;29:1192–3.CrossRefGoogle Scholar
  31. 31.
    Murdande SB, Pikal MJ, Shanker RM, Bogner RH. Solubility advantage of amorphous pharmaceuticals: I. A thermodynamic analysis. J Pharm Sci. 2010;99:1254–64.CrossRefPubMedGoogle Scholar
  32. 32.
    Murdande SB, Pikal MJ, Shanker RM, Bogner RH. Solubility advantage of amorphous pharmaceuticals, part 3: Is maximum solubility advantage experimentally attainable and sustainable? J Pharm Sci. 2011;100:4349–56.CrossRefPubMedGoogle Scholar
  33. 33.
    Murdande S, Pikal M, Shanker R, Bogner R. Solubility advantage of amorphous pharmaceuticals: II. Application of quantitative thermodynamic relationships for prediction of solubility enhancement in structurally diverse insoluble pharmaceuticals. Pharm Res. 2010;27:2704–14.CrossRefPubMedGoogle Scholar
  34. 34.
    Alonzo D, Zhang GZ, Zhou D, Gao Y, Taylor L. Understanding the behavior of amorphous pharmaceutical systems during dissolution. Pharm Res. 2010;27:608–18.CrossRefPubMedGoogle Scholar
  35. 35.
    Ndouand TT, von Wandruszka R. Pyrene fluorescence in premicellar solutions: the effects of solvents and temperature. J Lumin. 1990;46:33–8.CrossRefGoogle Scholar
  36. 36.
    Chandar P, Somasundaran P, Turro NJ. Fluorescence probe investigation of anionic polymer-cationic surfactant interactions. Macromolecules. 1988;21:950–3.CrossRefGoogle Scholar
  37. 37.
    Goddard ED, Turro NJ, Kuo PL, Ananthapadmanabhan KP. Fluorescence probes for critical micelle concentration determination. Langmuir. 1985;1:352–5.CrossRefPubMedGoogle Scholar
  38. 38.
    Zana R, Yiv S, Strazielle C, Lianos P. Effect of alcohol on the properties of micellar systems: I. Critical micellization concentration, micelle molecular weight and ionization degree, and solubility of alcohols in micellar solutions. J Colloid Interf Sci. 1981;80:208–23.CrossRefGoogle Scholar
  39. 39.
    Jensen KHR, Rekling JC. Development of a no-wash assay for mitochondrial membrane potential using the styryl Dye DASPEI. J Biomol Screen. 2010;15:1071–81.CrossRefPubMedGoogle Scholar
  40. 40.
    Lakowicz JR. Principles of fuorescence spectroscopy. New York: Springer; 2006.CrossRefGoogle Scholar
  41. 41.
    Marini A, Muñoz-Losa A, Biancardi A, Mennucci B. What is solvatochromism? J Phys Chem B. 2010;114:17128–35.CrossRefPubMedGoogle Scholar
  42. 42.
    Pávez P, Encinas MV. Photophysics and photochemical studies of 1,4-dihydropyridine derivatives. Photochem Photobiol. 2007;83:722–9.CrossRefPubMedGoogle Scholar
  43. 43.
    Fasani E, Dondi D, Ricci A, Albini A. Photochemistry of 4-(2-nitrophenyl)-1,4-dihydropyridines. Evidence for electron transfer and formation of an intermediate. Photochem Photobiol. 2006;82:225–30.CrossRefPubMedGoogle Scholar
  44. 44.
    Van Eerdenbrugh B, Raina S, Hsieh Y-L, Augustijns P, Taylor L. Classification of the crystallization behavior of amorphous active pharmaceutical ingredients in aqueous environments. Pharm Res. 2014;31:969–82.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Shweta A. Raina
    • 1
    • 3
  • David E. Alonzo
    • 2
    • 4
  • Geoff G. Z. Zhang
    • 2
  • Yi Gao
    • 2
    • 5
  • Lynne S. Taylor
    • 1
  1. 1.Department of Industrial and Physical Pharmacy, College of PharmacyPurdue UniversityWest LafayetteUSA
  2. 2.Drug Product Development, Research and Development, AbbVie IncNorth ChicagoUSA
  3. 3.Manufacturing Science and Technology, AbbVie IncNorth ChicagoUSA
  4. 4.Formulation & Process Development, Gilead Sciences, IncFoster CityUSA
  5. 5.Analytical Sciences, Manufacturing Science and Technology, AbbVie IncNorth ChicagoUSA

Personalised recommendations