Anal. Chem. 1998, 70, 4853-4859
Dye-Encapsulating Liposomes as Fluorescence-Based Oxygen Nanosensors Kerry P. McNamara and Zeev Rosenzweig*
Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148
Nanometer-sized liposomes containing the oxygen-sensitive indicator tris(1,10-phenanthroline)ruthenium chloride (Ru(phen)3) in their internal compartment have been prepared and tested for their oxygen-sensing capabilities in aqueous solution. A standard injection technique, where a lipid mixture consisting of dimyristoylphospatidylcholine, cholesterol, and dihexadecyl phosphate (molar ratio 5:4:1) all dissolved in dry 2-propanol injected into an aqueous solution of 10 mM Ru(phen)3 under vortexing, is used to prepare the liposomes. A high uniformity of the liposomes is realized by extruding them back and forth through a 100-nm pore size polycarbonate membrane. TEM images of the liposomes, stained with uranyl acetate, show that the liposomes are unilamellar, round in shape, maintain high structural integrity, and average 70 nm in diameter. Dynamic light-scattering measurements support this observation. Under our experimental conditions, the entrapment efficiency of Ru(phen)3, defined as the ratio between the concentration of dye molecules encapsulated in the liposomes and the dye concentration in the solution used for liposome formation, is ∼1%. The liposomes show high stability with respect to dye leaking at room temperature for 8 days and high photostability when exposed to the excitation light. Individual liposomes are used to monitor the enzymatic oxidation of glucose by glucose oxidase in their vicinity. The newly prepared oxygen-sensitive liposomes can be applied for noninvasive oxygen analysis in tissues and single biological cells. The development of fluorescence-based chemical sensors and their environmental and biomedical applications have attracted the attention of researchers over the last 30 years. With the recent increased interest in the areas of nanotechnology and nanophase materials, there is a growing interest in the development of nanosized chemical and biochemical sensors. Sensors of such miniaturized dimensions may find use in biological applications such as cell biology, biotechnology, immunology, and bacteriology where chemical manipulations are routinely performed on individual cells.1,2 These sensors may also be used to measure the kinetics of chemical processes in narrow-bore capillaries and microsized pores and may contribute to further understanding the dynamics of chemical reactions in confined dimensions. (1) Giuliano, K. A.; Taylor, D. L. Methods Neurosci. 1995, 27, 1-16. (2) Frank, A. J.; Proctor, S. J.; Tilby, M. J. Blood 1996, 88, 977-984. 10.1021/ac9803232 CCC: $15.00 Published on Web 10/15/1998
© 1998 American Chemical Society
In recent years, studies in the area of submicrometer-sized sensors have focused on growing the sensing element at the distal end of a pulled micropipet or an optical fiber tip.3-13 In both experimental geometries, a beam size below the diffraction limit (λ/2) is realized in the near field of the tip.3,4 In the fiber-optic geometry, a Gaussian laser light is transmitted through the tip and used to grow a submicrometer-sized polymer sensing element at the end of a pulled optical fiber via controlled photopolymerization.5 The size of the sensing element is controlled by the light intensity and the polymerization time. Using this technique, fiberoptic chemical sensors with a size of ∼0.2 µm have been fabricated and applied for pH measurements in the yolk sacs of rat embryos at different days of gestation6,7 and for measurements of the level of dissolved oxygen in aqueous solutions.8 Similarly, micrometersized biosensors for glucose9,10 have also been developed. Another advancement in this area has been the development of miniaturized sensing arrays where micrometer-sized sensing tips are fabricated on the surface of a fiber-optic imaging bundle.11,12 A different approach to fabricate submicrometer-sized sensors has been the immobilization of a fluorescence-sensing indicator in a sol-gel matrix rather than in a polymer support.13 Sol-gel supportive matrixes may increase the stability and lifetime of submicrometer-sized sensors. However, it is more difficult to control the size of these sensors in a consistent manner because a dip-coating technique is used to grow the sensing element on the pulled tip. Submicrometer-sized optochemical sensors offer significant improvements in their absolute detection limit, response time, and spatial resolution compared to conventional micrometer- or millimeter-sized chemical sensors14 and biosensors.15 However, the use of an optical fiber or a micropipet tip as a support limits the applicability of the method in biological research, particularly in (3) Betzig, E.; Trautman, J. K.; Harris, T. D.; Weiner, J. S.; Kostenak, R. L. Science 1991, 251, 1468-1470. (4) Betzig, E.; Trautman, J. K. Science 1992, 257, 189-195. (5) Walt, D. R.; Munkholm, C.; Milanovich, F. P.; Klainer, S. M. Anal. Chem. 1986, 58, 1427-1430. (6) Tan, W.; Shi, Z. Y.; Kopelman, R. Anal. Chem. 1992, 64, 2985-2990. (7) Tan, W.; Shi, Z. Y.; Smith, S.; Birenbaum, D.; Kopelman, R. Science 1992, 258, 778-781. (8) Rosenzweig, Z.; Kopelman, R. Anal. Chem. 1995, 67, 2650-2654. (9) Rosenzweig, Z.; Kopelman, R. Anal. Chem. 1996, 68, 1408-1413. (10) Rosenzweig, Z.; Kopelman, R. Sens. Actuators 1996, B35-36, 475-483. (11) Healey, B. G.; Li, L.; Walt, D. R. Biosens. Bioelectron. 1997, 12, 521-529. (12) Healey, B. G.; Walt, D. R. Anal. Chem. 1997, 69, 2213-2216. (13) Shalom, S.; Strinkovski, A.; Peleg, G.; Druckmann, S.; Krauss, A.; Lewis, A.; Linial, M.; Ottolenghi, M. Anal. Biochem. 1997, 244, 256-259. (14) Shakhsher, Z.; Seitz, W. R.; Legg, K. D. Anal. Chem. 1994, 66, 1731-1735. (15) Eggins, B. Biosensors: an introduction; Wiley-Teubner: New York, 1996.
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the field of single-cell analysis where the sensor has to be inserted into a single cell through the cell membrane. While the measurement technique is not as invasive as conventional cellular fluorescence labeling,16 the damage to the cell membrane may affect cellular properties. Furthermore, the advantage of using fiber optics for remote detection in intracellular measurements is only theoretical, since the low signal level does not permit remote signal collection through the pulled tip. In addition, this approach is not feasible when a large population of cells needs to be examined for statistical purposes. To overcome these problems, we introduce a unique approach for the fabrication of nanosized optochemical probes in which the fluorescent dye is entrapped in nanosized phospholipid vesicles. Upon dispersion in aqueous solution, entropic and hydrophobic effects cause phospholipids to aggregate into arranged bilayers and nearly spherical emulsions, known as liposomes, encapsulating a certain volume of the surrounding media.17-23 These vesicles may be unilamellar or multilamellar, the difference dramatically affecting the volume of the entrapped media. The size of liposomes and their size distribution greatly depend on the method used for the liposome preparation.18-20 Unilamellar vesicles have been prepared by a variety of techniques including direct injection/rapid vortexing, sonication, and simple slow mixing. The entrapment efficiency of fluorescent dyes in liposomes is between 1 and 10%.24 However, once entrapped, these dyes exhibit a wide variation in leaking, depending on the composition of the liposome membrane, the environmental conditions such as pH and temperature, and the hydrophobicity or hydrophilicity of the encapsulated dye molecules. Under ideal storage conditions, physiological pH and 4 °C, a liposome sample may be stable for up to 14 days. There are variations in the stability of liposomes in a cellular environment as well. For example, Weinstein et al. have shown an extreme case of ∼90% leakage of 6-carboxyfluorescein from phosphatidylcholine (PC)-based liposomes when the liposomes diffuse into frog retinas and human lymphocytes.25 On the other hand, Verkman et al. reported less than a 10% leakage of the Cl- -sensitive dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) from PC-based liposomes under similar conditions.26 Our study focuses on the fabrication of a nanosized liposome-based optochemical probe for dissolved oxygen. Molecules of the fluorescence dye tris(1,10phenanthroline)ruthenium chloride (Ru(phen)3) are encapsulated (16) Wang, X. F.; Herman, B. Fluorescence Imaging Spectroscopy and Microscopy; John Wiley: New York, 1996. (17) Roberts, M. A.; Locascio-Brown, L.; MacCrehan, W. A.; Durst, R. A. Anal. Chem. 1996, 68, 3434-3440. (18) Lim, F. Biomedical Applications of Microencapsulation; CRC Press: Boca Raton, FL, 1984. (19) Gregoriadis, G. Liposome Technology, Volume I: Preparation of Liposomes; CRC Press: Boca Raton, FL, 1984. (20) Park, K. Ed. Controlled Drug Delivery: Challenges and Strategies; American Chemical Society: Washington, DC, 1997. (21) Batzri, S.; Korn, E. D. Biochim. Biophys. Acta 1973, 298, 1015-1019. (22) Kremer, J. M. H.; Esker, M. W. J.; Pathmamanoharan, C.; Wiersema, P. H. Biochemistry 1977, 16, 3932-3935. (23) Hope, M. J.; Bally, M. B.; Webb, G.; Cullis, P. R. Biochim. Biophys. Acta 1985, 812, 55-65. (24) New, R. Liposomes: a practical approach; Oxford University Press: Oxford, U.K., 1990. (25) Weinstein, J. N.; Yoshikami, S.; Henkart, P.; Blumenthal, R.; Hagins, W. A. Science 1977, 195, 489-492. (26) Verkman, A. S.; Takla, R.; Sefton, B.; Basbaum, C.; Widdicombe, J. H. Biochemistry 1989, 28, 4240-4244.
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in unilamellar liposomes with an average size of 70 nm. The liposomes are prepared using a standard injection technique followed by extrusion through a 100-nm pore size polycarbonate membrane to ensure true nanosized liposomes. The luminescence properties of Ru(phen)3 and other ruthenium diimine complexes have been studied extensively by Demas et al.27-30 These dyes exhibit high fluorescence quantum yields (Φ ∼ 0.1-0.6), high photostability, large Stokes shift, and long excited-state lifetimes in the microseconds time scale. The fluorescence of Ru(phen)3 is readily quenched by molecular oxygen. The variation in the fluorescence intensity of the Ru(phen)3 as a function of the dissolved oxygen concentration is given by the Stern-Volmer equation:
I0/Ic ) 1 + Ksv[O2]
(1)
where I0 is the fluorescence intensity of the dye in a nitrogenated solution, Ic is the fluorescence intensity of the dye in a solution of a given oxygen concentration [O2], and Ksv is the Stern-Volmer quenching constant. Capitalizing on great advancements in liposome technology over the last 30 years, we have prepared for the first time nanosized oxygen-sensitive fluorescent liposomes. We have demonstrated the feasibility of oxygen measurement in confined dimensions using individual nanosized liposomes and investigated the analytical capabilities of this new and promising optochemical sensing technique. EXPERIMENTAL SECTION Digital Fluorescence Imaging Microscopy. The detection system used to measure the fluorescence of the oxygen-detecting liposomes in aqueous solution is shown in Figure 1. The system consists of an inverted fluorescence microscope (Olympus IX70) equipped with a 100-W mercury lamp as a light source. The fluorescence image of the liposomes is collected by a 20× microscope objective with NA ) 0.5. A 450-nm narrow-band excitation filter, a 500-nm dichroic mirror, and a 550-nm long-pass emission filter are used to ensure spectral imaging purity. A slowscan high-performance CCD camera (Princeton Instruments, model 512-TKM1) is employed for digital imaging of the liposomes. Typically, an exposure time of 100 ms is used for image collection. A PC microcomputer (Gateway 2000, Pentium 120 MHz) is employed for data acquisition using the Princeton Instrument software WinView 1.4.3. The software Adobe PhotoShop v3.0 is used for the enhancement of presented images. Fluorescence Spectroscopy Measurements. Excitation and emission spectra, as well as kinetic measurements are carried out using a PTI International (model QM-1) fluorometer, equipped with a 75-W continuous Xe arc lamp as a light source. Transmission Electron Microscopy (TEM) Measurements. TEM images of the dye-encapsulating liposomes are obtained using a Zeiss-10C TEM microscope. A staining technique using 0.5% uranyl acetate is applied to observe the liposomes. (27) Carraway, E. R.; Demas, J. N.; DeGraff, B. A.; Bacon, J. R. Anal. Chem. 1991, 63, 337-342. (28) Demas, J. N.; DeGraff, B. A. Anal. Chem. 1991, 63, 829A-837A. (29) Sacksteder, L.; Demas, J. N.; DeGraff, B. A. Anal. Chem. 1993, 65, 34803483. (30) Xu, W.; McDonough, R. C., III; Langsdorf, B.; Demas, J. N.; DeGraff, B. A. Anal. Chem. 1993, 66, 4133-4141.
Figure 1. Digital fluorescence imaging microscopy system. The experimental setup consists of an inverted fluorescence microscope with a 20× objective (NA ) 0.5), a high-performance charge-coupled device camera (16-bit 512 × 512 chip size), and a microcomputer for image analysis. A scanning spectrograph and a photomultiplier are used to characterize the photophysics of the dyes used for optochemical sensing.
Light-Scattering Measurements. The light-scattering setup was described by Smith et al.31 It consists of a vertically polarized helium-neon laser (15 mW, Spectra Physics SB-124b), a photomultiplier tube (EMI 9863b), and an ALV-5000 correlator. The temperature of the sample is maintained at (0.01 °C using a Hart Scientific model 2100 controller. Liposome Preparation. A direct vortexing method is used to prepare the Ru(phen)3-encapsulating liposomes.20 Following Batzri and Korn,21 a 50 mM lipid stock solution is prepared with a 5:4:1 molar ratio of dimyristoylphospatidylcholine, cholesterol, and dihexadecyl phosphate in chloroform. This phospholipid composition provides maximum structural stability of liposomes stored at room temperature.19-21 The phospholipid solution is stored in a sealed vial at -10 °C until use. All reagent transfers during liposome preparation are made with a Hamilton glass syringe. A 40-µL aliquot of the stock solution is dried under nitrogen in a glass vial until all the chloroform is removed. The sample is immediately reconstituted in 50 µL of dry 2-propanol with rapid vortexing or it is sealed and stored in a desiccator until use. This solution is then injected, while vortexing, into 1 mL of a 10 mM aqueous solution of Ru(phen)3 at pH 7.2. The liposomes form spontaneously, encapsulating the Ru(phen)3 dye. Liposomes that are larger than 100 nm are removed by extruding the liposome sample back and forth several times using an extrusion device through a 100-nm pore size polycarbonate membrane (Avanti Polar Lipids, Inc.). The small size of the liposomes (
