Fluorescent Tissue Site-Selective Lanthanide ... - ACS Publications

Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas ... Designed Chemicals R&D, The Dow Chemical Company, Freeport, Texas ...
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Anal. Chem. 1999, 71, 2607-2615

Fluorescent Tissue Site-Selective Lanthanide Chelate, Tb-PCTMB for Enhanced Imaging of Cancer Darryl J. Bornhop,* Darren S. Hubbard, Michael P. Houlne, and Chris Adair

Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061, and Southwest Cancer Center, University Medical Center, Lubbock, Texas 79415 Garry E. Kiefer

Designed Chemicals R&D, The Dow Chemical Company, Freeport, Texas 77541-3257 Barbara C. Pence† and David L. Morgan

Department of Pathology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, and Southwest Cancer Center, University Medical Center, Lubbock, Texas 79415

In-vivo and in-vitro investigations indicate that a newly developed polyazamacrocyclic chelate of Tb(III) has superior properties for use as an abnormal tissue marker. In addition to tissue selectivity, this molecule is unique because of its low toxicity, attractive fluorescent properties, rapid pharmokinetics, and relatively high water solubility. The complex Tb-3,6,9-tris(methylene phosphonic acid n-butyl ester)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene (Tb-PCTMB) has also been shown to exhibit strongly shifted emission (∆λ - 280 nm), moving the detection frequency away from autofluorescence backgrounds, and good quantum efficiencies (Φ ) 0.51), providing high brightness. Fluorescence imaging was used to quantify Tb-PCTMB at the picomolar level in tissues and to show the significant difference in affinity for the chelate by adenocarcinoma cells HT-29 versus normal epithelial cells (IEC-6). Topical application, or lavage introduction, under endoscopy was used to instill a millimolar aqueous solution of Tb-PCTMB into a dimethylhydrizene-treated Sprague Dawley rat large intestine containing a suspect growth. Subsequent in vitro fluorescence detection and standard histological evaluation confirmed enhanced uptake by adenocarcinoma tissue. Semiquantitative signal interrogation was employed to show the potential for using Tb-PCTMB as a contrast enhancement marker for disease detection. Contrast enhancement markers are critical to the identification and diagnosis of tissue abnormalities.1-4 Generally, these markers are used after biopsy or resection of the suspect tissue. In-vivo * Corresponding Author: (tel.) (806) 742-3142; (e-mail) [email protected]. † Southwest Cancer Center. (1) Pearse, A. G. E. Histochemistry, Theoretical and Applied, 3rd ed.; Churchill: London, 1968. (2) Sternberg, Stephen S., Ed. Intestinal Neoplasms. In Diagnostic Surgical Pathology, 2nd ed.; Raven Press: New York, 1994; pp 1371-1417. 10.1021/ac981208u CCC: $18.00 Published on Web 05/29/1999

© 1999 American Chemical Society

diagnostic and identification procedures have greatly expanded with the advent of high-quality imaging technology.1-9 In fact, using endoscopy, it might be possible to use native tissue spectroscopic properties to perform optical diagnosis.10-17 Yet, these methods normally measure the loss or reduction of fluorescence intensity from abnormal tissue, e.g., they measure the small change in a large background signal. The result of such a measurement might be good for diagnosing later-stage disease, but low signal-to-noise ratio (S/N) and low contrast for abnormal versus normal tissue make these techniques less attractive for early detection. (3) Copenhaven, W. M.; Keely, D. E.; Wood, R. L. Histology of the Intestine, In Bailey’s Textbook of Histology, 17th ed.; The Williams & Wilkins Co.: Baltimore, MD, 1978; pp 495-509. (4) Haugland, R. P.; Minta A.; Satir, B. H., Eds. Design and Application of Indicator Dyes. In Noninvasive Techniques in Cell Biology; Wiley-Liss, New York: 1990; pp 1-21. (5) Cotton, P. B. Practical Gastrointestinal Endoscopy, 3rd ed.; Boston Blackwell Scientific: Oxford, 1990. (6) Oguro, Y. Endoscopic Approaches to Cancer Diagnosis and Treatment; Japan Scientific Societies Press: Tokyo and Taylor & Francis, London, 1996. (7) Seidlitz, H. K.; Classen, M. Endoscopy 1989, 24, 225-228. (8) Knyrim, K.; Seidlitz, H. K.; Hagenmuller F.; Classen, M. Endoscopy 1987, 19, 156-159. (9) Knyrim, K.; Seidlitz, H.; Vakil, N.; Hagenmuller F.; Classen, M. Gastroenterology 1989, 96, 776-782. (10) Wang, T. D.; Van Dam, J.; Feld, M. S. Gastroenterology 1996, 111, 11821191. (11) Cothren, R. M.; Sivak, M. V., Jr.; Feld. M. S. Gastrointestinal Endoscopy 1996, 44, 168-176. (12) Chak, A.; Sivak, M. V. Endoscopy 1994, 26, 169-174, and references therein. (13) Schomacker, K. T.; Frisoli, J. K.; Compton, C. C. Lasers Surg. Med. 1992, 12, 63-78. (14) Marchesini, R. M.; Brambilla, E.; Pignoli, A. J. Photochem. Photobiol. 1992, 14, 219-230. (15) Cothren, R. M.; Richards-Kortum, R.; Sivak, M. V.; Fitzmaurice, M.; Rava, R. P.; Boyce, G. A.; Doxtader, M.; Blackman, R.; Ivanc, T. B.; Hayes, G. B.; Feld, M. S.; Petras, R. E. Gastrointestinal Endoscopy 1990, 36, 105-111, and references therein. (16) Lam, S., MacAulay, C.; Hung, J.; Riche, J.; Profio, A. E.; Palcic, B. J. Thorac. Cardiovasc. Surg. 1993, 105, 1035-1040. (17) Vo-Dinh, T.; Panjehpour, M.; Overholt, B. F.; Buckley, P., III Appl. Spectrosc. 1997, 51, 58-63.

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Presently, the most common white-light endoscopic indicators used to identify a potentially cancerous growth are the observation of color changes, increased vascularization, and the presence of surface structure, such as polyps.18-20 While these methods have continued to improve with the advent of higher resolution imaging devices and more sensitive cameras, flat dysplasia and early abnormalities are still most often unidentifiable by white-light endoscopy. One approach taken to improve contrast in tissue imaging has been to introduce an exogenous chemical marker.6,21-24 For example, photodynamic therapy (PDT) drugs can be introduced into the circulatory system as injectable dyes. As a result of their inherently slow exchange rate with cancerous tissue, added contrast is introduced to a suspect site.6,21,25 While conceptually attractive, various limitations have moderated the enthusiasm for the approach of using PDT compounds for contrast enhancement in ‘early’ detection of disease.26 As noted by G. A. Wagnieres et al., the drawbacks of using PDT agents for ‘detection’ are related to relatively slow pharmokinetics, poor tissue selectivity, and lightinduced toxicity. For example, it takes many hours after injection for contrast to be seen in the tissues, and visualization must be performed within a narrow window of time, otherwise the therapeutic PDT agent will have not cleared the normal tissue interstitial space. Further, since PDT agents are light-activated singlet oxygen producing compounds, if they have not cleared normal tissue, cells other than those that are diseased can be killed during imaging. After extensive study, it was concluded that, “The fluorochrome is therefore the key parameter that has to be improved in order to increase significantly the performance of cancer LIF (laser induced fluorescence) photodetection”. Another technique, which has been somewhat effective at delineating suspect sites, was termed Chromoscopy.27-29 In the work by Jaramillo et al., indigo carmine (IC) dye was sprayed through an endoscope to enhance details of the mucosal surface features. While attractive, Chromoscopy using colored dyes as visual enhancement markers has several major drawbacks. First, the IC dye has poor specificity toward abnormal tissues; second, the technique cannot perform solute quantitation at endoscopy; third, the use of dyes without fluorescence is inherently signal(18) Schomacker, K. T.; Frisoli, J. K.; Compton, C. C. Gastroenterology 1992, 102, 1155-1160. (19) Cotran, R. S., Kumar, V., Robbins, S. L., Schoen, F. J., Eds. The Gastrointestinal Tract. In Pathological Basis of Disease, 5th ed.; W. B. Saunders Co.: Philadelphia, PA, 1990; pp 809-817. (20) Melville, D. M.; Jass, J. R.; Morson, B.; Pollock, D. J.; Richman, P. I.; Shepard, N. A.; Ritchie, J. K.; Love, S. B.; Lennard-Jones, J. E. Human Pathology 1989, 20, 1008-1014. (21) Kriegmair, M.; Baumgartner, R.; Hofstetter, A. J. Urol. (Baltimore) 1996, 155, 105-109. (22) Heim, R. D.; Prasher, C.; Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12501-12504. (23) Heim, R.; Cubitt, A. B.; Tsien, R. Y. Nature (London) 1995, 373, 663-664. (24) Taylor, C. R. Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist, 2nd ed.; W. B. Saunders Co.: Philadelephia, PA, 1994. (25) Kato, H.; Imaizumi, I.; Aizawa, K. J. Photochem. Photobiol., B 1990, 6, 189196. (26) Wagnieres, G. A.; Studzinski, A. P.; Braichotte, D. R.; Monnier, P.; Deperursinge, C.; Chatelain, A.; van den Bergh, H. E. Appl. Opt. 1997, 36, 5608-5620. (27) Kudo, S. Endoscopy 1993, 25, 455-461. (28) Mitooka, H.; Fujimori, T.; Ohno, S. Gastrointestinal Endoscopy 1992, 38, 373-374. (29) Jaramillo, E.; Watanabe, M.; Slezak, P.; Rubio, C. Gastrointestinal Endoscopy 1995, 42, 114-122.

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Figure 1. Molecular structure of Tb-PCTMB.

to-noise limited (no spectral discrimination); and fourth, flat lesions often escape detection. Additionally, contrast enhancement that is primarily dependent upon diffusion differences for normal versus abnormal tissue is time consuming and is fundamentally limited by the presence of a concentration gradient (as more dye is added, all tissues become colored, reducing contrast). The most promising approach for early disease detection would be to use molecules that are fluorescent at wavelengths significantly shifted from the excitation wavelength and compounds that exhibit some level of preferential uptake by abnormal tissue. Properties, such as a broad spectral shift, can allow marker detection in the presence of otherwise-large optical background signals, while tissue specificity further improves visualization of a suspect site. Additionally, these markers should be nontoxic, be chemically stable, and exhibit reasonable quantum efficiencies. For lavage, enema, or spray-on introduction, water solubility is also attractive, allowing use in endoscopic procedures. We have been investigating a class of lanthanide complexes with many of these properties. One of the most attractive molecules in this class is Tb-3,6,9-tris(methylene phosphonic acid n-butyl ester)-3,6,9,15-tetraaza-bicyclo[9.3.1]pentadeca-1(15),11,13triene, a polyazamacrocyclic chelate of Tb[III], (Tb-PCTMB) (Figure 1).30-33 Unlike previous markers, which have been large molecular weight compounds, Tb-PCTMB has good water solubility and, like many of its analogues used in MRI image enhancement,34-38 it is chemically and thermodynamically stable.35,39 The advantageous spectroscopic properties of Tb-PCTMB include a separation between excitation and emission wavelengths of greater than 280 nm and a quantum yield of 0.51.30 Here we show, in a preliminary study using a Sprage Dawley animal model and in-vitro cell studies, that Tb-PCTMB has many of the properties needed to be a good fluorescent diseased tissue (30) Houlne, M. P.; Kiefer, G. E.; Bornhop, D. J. Applied Spectroscopy 1996, 10, 225-244. (31) Kiefer, G. E.; Bornhop, D. J. World Patent. Filed Oct. 11, 1996. (32) Houlne, M. P.; Hubbard, D. S.; Kiefer, G. E.; Bornhop, D. J. J. Biomed. Opt. 1998, 3, 145-153. (33) Hubbard, D. S.; Houlne, M. P.; Kiefer, G. E.; Janssen, H. F.; Hacker, C.; Bornhop, D. J. Lasers in Medical Science, 1998, 13, 14-21. (34) Kim, W. D.; Kiefer, G. E.; Maton, F.; McMillan, K.; Muller, R. N.; Sherry, A. D. Inorg. Chem. 1995, 34, 2233-2243. (35) Aime, S.; Botta M.; Sisti, M. J. Chem. Soc., Chem. Commun. 1995, 18, 18851886. (36) Geraldes, C. F. G. C.; Sherry, A. D.; Lazar, I.; Miseta, A.; Bogner, P.; Berenyi, E.; Sumegi, B.; Kiefer, G. E.; McMillan, K.; Matou, F.; Muller, R. N. Magn. Reson. Med. 1993, 30, 696-703. (37) Kiefer, G. E.; Simon, J.; Garlich, J. R. World Patent 93/11802, 1989. (38) Simon, J.; Garlick, J. R.; Wilson, D. A.; McMillan, K. U.S. Patent 4,882,142, 1989, and U.S. Patent 4,976,950. (39) Cahceris, W. P.; Nickel, S. K.; Sherry, D. A. Inorg. Chem. 1987, 26, 958960.

marker. First, we show that the Tb-PCTMB complex exhibits no cytotoxicity and that the complex shows a significant level of preferential uptake by adenocarcinoma cells (specificity). Second, it is shown that when an aqueous solution of Tb-PCTMB is sprayed onto tissue at endoscopy, the complex is up-taken to a much greater extent by cancerous tissue than by normal tissue. Finally, it is shown that, by using semiquantitative fluorescence imaging techniques, an analytical evaluation of the contrast enhancement facilitated by using Tb-PCTMB is possible. In short, it is demonstrated that Tb-PCTMB can be introduced topically at low concentration, is not toxic, provides contrast for disease tissue rapidly, and can be detected with a standard Xe light source. EXPERIMENTAL SECTION Instrumentation. The aqueous-phase spectroscopic investigations on the chelate were carried out on commercial spectrometers operating at ambient room temperature (recorded to be 23-25° C). The absorption spectra were acquired and recorded using a Shimadzu 265 UV-vis spectrophotometer using a standard 10mm quartz cuvette and a slit width of 1 mm. The on-board computer peak-find feature was used to identify the maximum absorbance wavelength value used in the fluorescence investigations. The emission spectrum was acquired in a standard 10-mm quartz cuvette on a SLM Aminco 4800C fluorimeter with the excitation and emission slit widths set to 4 mm. A Ziess Axioplan microscope was specially configured for UV fluorometry with a 150-W Xe arc lamp, a quartz condenser lens set, chromatically corrected (air-spaced) UV transmitting objective lenses, and a special set of excitation/emission filters. The filter set consisted of a, UV transparent Hot Mirror (ZC&R Coatings), UV transparent cutoff filter (UG-11, Melles Griot), a dichroic beam splitter (DCF 310 Chroma Tech), and a 550-nm 10 nm bandwidth interference filter (INF 550/10 Chroma Tech). A high-resolution, mega-pixel (6.8 µm wide), digital, thermoelectrically cooled CCD (Photometrics, Tucson, AZ) was affixed to the camera mount on the headpiece of the microscope. Images were collected after a 0.01-s integration time with the camera temperature set to 10 °C. Images were digitized, processed, and exported using Image Pro Plus v. 3.0 image processing software. MatLab version 4.2c.1 (Mathworks, Inc.) was used to prepare the false color contour plots, where intensity values correspond directly to CCD gray scale values. Undosed substrates and microscope slides where tested to ensure that the fluorescence originating from the sample came from the lanthanide chelate. These blanks exhibited little or no fluorescence, as expected, because upon excitation at 270 nm: (a) the free Tb3+ ion emission would be quite weak due to quenching and (b) the native fluorescence (autofluorescence) from tissues would also be quite low at the measured emission wavelength of 550 nm. Endoscopic visualization of the colon was accomplished using a 2.5-mm by 3.0-m fiber scope of in-house construction.40,41 A 150-W Xe lamp was coupled through a fiber optic to the illumination fibers of the scope, providing white-light illumination to visualize the cavity. The images where then magnified by an objective lens, sensed by a single chip 1/3in CCD camera (40) Houlne, M. P.; Hubbard, D. S.; Bornhop, D. J. SPIE Proc. 1996, 2678, 464474. (41) Bornhop, D. J.; Clayton, J. B.; Freiman A. G.; Middle, G. H. U.S. Patent 5,456,245, October 10, 1995.

(Panasonic, Japan), passed through a VCR for recording, and then displayed by a high-resolution video monitor (Sony, Japan). The working lumen of the endoscope was used to introduce saline solution at the distal end of the scope to clear the field of view. This working lumen (approximately 1.0 mm in diameter) was also employed to distill the chelate to the site where the growth (tumor) was visualized. Visualization deep into the colon of the Sprague Dawley rat has thus far been problematic, requiring the use of small-diameter, flexible scopes and careful preparation of the specimen. While small-diameter scopes are still required for in-vivo rat investigations, we have recently performed an extensive methodology study42 and can now routinely inspect the entire colon, effectively identify suspect sites, introduce our chelate to a suspect site, and correlate position with pathology. Chelate Synthesis and Complexation. The chelate ligand was synthesized according to procedures described in detail elsewhere.30,37,38,43 Complexation of the ligand to the metal ion was accomplished by first making a 0.19 mmol solution from the potassium salt of 150 mg of PCTMB dissolved in 3 mL of deionized water, resulting in a solution of pH 10.5. The pH was lowered to 5.5 using 1 N HCl with continuous stirring. An aqueous solution (3 mL) of terbium chloride hexahydrate (85.5 mg, 0.23 mmol) was then added in one portion to give a solution having a pH ) 3.47. The pH was slowly raised by adding 0.1-mL aliquots of 0.1 N KOH. Addition of KOH was terminated when a pH of 6.4 was sustained. At this point, the homogeneous solution became soapy, and considerable turbidity was observed. The turbid solution was then freeze-dried and the resulting solid dissolved in chloroform/ methanol (3:1, 40 mL). This organic solution was filtered through Celite and concentrated to give a glassy solid. The solid was redissolved in water (20 mL), filtered through a 0.2-µm filter, and freeze-dried to give the complex as a flaky, snow-white solid. Materials, Cells, and Animals. For the cytotoxicity studies, HT-29 and IEC-6 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). The HT-29 cells were grown in DMEM with 10% fetal bovine serum (FBS) and the IEC-6 cells were grown in DMEM supplemented with 5% FBS, 100 µL of insulin, and 1 mL of glutamine per 100 mL. Three days prior to confluency, the cells were inoculated with 2 mL of 2 mM Tb-PCTMB and allowed to grow for 24 h. After the inoculation period, unbound Tb-PCTMB was removed with 4 washings of PBS, and the cells were removed from the flask with 3 mL of trypsin. The cell suspension was transferred to a centrifuge tube with 12 mL of growth media and spun at 1000g for 10 min to pellet the cells. The media and trypsin were removed, and the cell suspension was resuspended in 5 mL of media. For ease of counting, 100 µL of the cell suspension was mixed with 100 µL of media in a microfuge tube. Afterward, 50 µL of this dilution was mixed with 50 µL of Trypan Blue stain and loaded into the hemocytometer. Five squares of the cells were counted on the hemocytometer, using the criteria that stained cells were dead and unstained cells were alive. Multiplication of the average count per square by the dilution factor, 4, gave the number of cells × 104 per mL. The number of cells reported is the average of three individual counts. (42) Stewart, M.; Strickland, A.; Obriant S.; Bornhop, D. J., manuscript in preparation. (43) Cheng, R. C.; Fordyce, W. A.; Goeckler, W. A.; Kruper, W. J.; Baughman, S.; Garlich, J. R.; Kiefer, G. E.; McMillan, J.; Simon, J. U.S. Patent 5; Chem. Abstr. 1990, 435, 990.

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Marker uptake investigations used HT-29 and IEC-6 cells that were propagated according to established protocol so that at least >(2 × 106) cells were contained in 25.0 mL. After adequate growth, the cells were inoculated with 2 mL of 2.2 × 10-3 M Tb-PCTMB, providing 4.4 µmol of Tb-PCTMB to the cells. Both cell lines were allowed to equilibrate with the chelate for approximately 2 h and then were rinsed with medium. The time course for this incubation was chosen on the basis of a preliminary time-course study using fluorescence microscopy, which is now under more intensive investigation. To remove unbound chelate, the cells were washed with phosphate buffered saline (PBS) three times. The cells were dislodged from the T-25 cell culture flask with a 3-mL aliquot of trypsin. Once free, the cells were transferred to a 15-mL conical centrifuge tube and pelleted by centrifugation at 1000g for 10 min. The cells were resuspended in media, agitated with a Pasteur pipet, and respun. This process was repeated three times to ensure that all unbound chelate was completely removed from the cells. The pellet was resuspended in 5 mL of media. Three drops of this suspension was placed in a Shandon Cytospin Cytocentrifuge and spun, to leave a cell plug on a clear microscope slide. The cell plug required centrifugation for 15 min at 1200 rpm to complete the plug. These slides, 3 from each cell line, were viewed under a Zeiss microscope modified for UV fluorescence as described above. The camera was operated at 10 °C with an integration time of 0.1 s. Sprague Dawley rats where obtained from Hamond (Houston, TX), grown following the established protocols, and cared for as dictated by the TTU Health Sciences Center and the Animal Care and Use Committee (ACUC). Once the animals had grown to an acceptable size and weight, tumors were induced via injection of 1,2-dimethylhydrazine dihydrochloride (DMH) and the subsequent feeding of a high-fat, meat-based diet.44 Anesthetization of the animals was accomplished using a 50 mg/kg of body weight dose of pentobarbital solution (50 g/mL) by intramuscular (I. M.) injection. An aqueous solution of the fluorescent chelate (at a concentration of 1.0 × 10-3 M) was introduced to the colon via the 1.0-mm-diameter working lumen of the endoscope until fluid was noted to be draining from the anus. The solution was allowed to equilibrate in the colon for approximately 20 min; this was followed by three thorough rinsings with saline. After rinsing, the animal was killed, the colon was removed, and tissues from various sites (suspect and apparently normal) were collected. Excised tissue samples were flash frozen in liquid N2 to minimize post-collection modifications. Four frozen sections were then cut from each of the normal and suspect tissues. Two of each were stained using Hemotoxylin & Eosin (H&E) for conventional histological diagnosis. Briefly, the samples were dehydrated and fixed by four washes in alcohol baths of increasing ethanol concentration (70-100% ethanol by volume). For nuclear staining, the slides were placed in Hemotoxylin for 30 s, and excess stain was removed by four 100% ethanol washes. The cytoplasm was stained with OG-6 followed by four alcohol washes to remove excess stain. Finally, for ease of interrogation, the cytoplasm was counterstained to highlight morphology with EA50 followed by four alcohol washes. These slides were dipped in Xylenes and coverslipped with Permount. The adjacent sections (44) Pence, B. C. Texas Tech University Health Sciences Protocol no. 4201.

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Figure 2. Aqueous-phase absorption spectrum for the complex TbPCTMB.

Figure 3. Aqueous-phase emission spectrum for the complex TbPCTMB.

taken for fluorescence evaluation were unstained and fixed in 70% ethanol. RESULTS AND DISCUSSION Chelate Spectroscopy. The spectroscopic characteristics of the polyazamacrocyclic Terbium chelates are nearly ideal for tissue imaging. First, unlike the IC dye used in Chromoscopy, TbPCTMB is a clear dye that is brightly fluorescent in the greenred spectrum when illuminated with UV light. Second, as shown by comparison of Figures 2 and 3, the lanthanide chelate fluorescence exhibits a huge spectral shift. With reference to Figure 2, the excitation spectrum is somewhat narrow and has a maximum at 270 nm. Figure 3 shows the spiked emission spectra seen in the lanthanide ions with features at 490, 550, 590, and 625 nm. Because of the unique spectroscopic properties of the lanthanide chelates,45 the most intense emission peak is red shifted approximately 280 nm from the absorption maximum! This large spectral shift has several advantages, including improved rejection of nonsolute background signals emanating from the excitation source or tissue autofluorescence (UV excited autofluorescence is minimal at 550 nm) and reduced constraints on the instrumentation used to visualize the site (a good interference filter is sufficient). Third, while Tb-PCTMB has a modest molar extinction coefficient ( ≈ 3000 L‚mol-1‚cm-1), the relatively high quantum efficiencies (Φ ) 0.51) combined with the large spectral shift for emission allow for quantitation of the chelate at the subpicogram (45) Soni, E.; Lovgren, T. Crit. Rev. Anal. Chem. 1987, 18, 105-154.

level in tissues.32 Additionally, the long-lived excited states exhibited by lanthanide chelates allow simple instrumentation to be used for lifetime imaging, thus providing another selectivity modality.33 The mediating factor associated with using Tb-PCTMB in biological systems is that absorbance is in the DNA damaging region of the electromagnetic spectrum, at the UV wavelength of 270 nm. Yet, we have found that the light intensities needed to quantify these solutes in tissues are below the level required by the FDA for mutageneous testing.32 Tissue and Disease Specificity. Of the many lanthanide chelates we have prepared and evaluated by intravenous introduction,30-33 just one has been shown to concentrate in the small intestine of the Sprague Dawley rat. Upon tail-vein injection, the molecule rapidly passes the liver, is carried with bile to the lower digestive tract, subsequently traverses the small intestine, and then, after about 10 h, the injected chelate is primarily found in the epithelial layer of the colon (large intestine). While the actual distribution pathway and binding mechanism is yet to be determined for abnormal tissue, we postulate that the lipophilic nature of Tb-PCTMB, in combination with the neutral net charge for the complex, facilitates a transport process involving active and passive transport mechanisms (intra- and paracellular mechanisms). Furthermore, in our recent in-vitro cell fractionation investigations32 and cellular imaging experiments46 there is an indication that chelate transport is rapid, particularly when it is directly introduced into culture. Given these observations, we performed the experiments described here to determine if a lavage or sprayon approach to chelate introduction would allow contrast enhancement of disease in minutes (30-90 min) instead of hours (18-24 h). First, we postulated that preferential or enhanced uptake by abnormal cells of the intestine might be possible; particularly, given that intestinal tissues normally perform an absorptive function and exhibit an inherently high cellular turnover rate. To determine if there was a measurable level of marker specificity for Tb-PCTMB, a key to improving contrast for diseased tissue identification, normal epithelial (IEC-6) and human andenocarcinoma cells (HT-29) were propagated and then inoculated with a 2 × 10-3 M solution of the chelate. As noted in the Experimental Section, the cells were then washed multiple times and deposited onto a microscope slide. White-light and fluorescence microscopy was then performed. The same camera integration time were used to produce all the fluorescence images so that they could be quantitatively compared. It should be further noted that control cells, e.g., cells not inoculated with chelate, show no appreciable fluorescence above the normal background. Figures 4 and 5 present the 200X white-light images, fluorescence images, and a false color contour reconstruction for fluorescence images for the IEC-6 cells and the HT-29 cells, respectively. Both of the whitelight images show that many cells are packed in a small regions a result of how the washed cells are deposited on the slide. In the fluorescence images, there is slight, low-level emission (Gray Scale Value (GSV), ∼25) which is evident throughout the image. Using blanks, we have determined that this signal is not derived from chelate fluorescence but is a background signal coming from ambient light. To aid in visualization of the fluorescent signal, false color contour plots have been displayed in Figures 4C and 5C. (46) Stewart, M.; Adair, C. Obriant S.; Bornhop, D. J., submitted for publication.

Figure 4. (A) White-light microscopic image of IEC-6 cells after inoculation and preparation. (B) Fluorescence microscopic images of IEC-6 cells after inoculation with Tb-PCTMB. (C) False color contours of the fluorescence microscopic images of IEC-6 cells after inoculation with Tb-PCTMB, showing the relative fluorescence intensity, corresponding to the amount of chelate detected in the imaged area.

While a slight fluorescent signal is detected in the IEC-6 cells, the level of emission is quite low (1 day) will be monitored by tracking cell viability and metabolic rate (growth rate). Additionally, we have a preliminary animal study currently underway to establish that the chelate is safe to use for in-vivo applications, particularly as a spray-on contrast agent. Contrast Enhancement of Murine Andenocarcinoma. Using the small intestine site-specific uptake of Tb-PCTMB, we recently showed that excised tissues from Sprague Dawley rats31-34 can be imaged with improved contrast by fluorescence endoscopy32 allowing optical interrogation of solute distribution. Under cross-sectional imaging, we noted that the solute marker was localized at the surface or in the superficial epithelial regions of the tissue taken from the small intestine. Since there are similarities between the cells that line small and large intestine, e.g., they are both morphologically epithilial cells and they both have functionality which is absorptive in nature, we believed that the chelate might concentrate at the surface of the large intestine. Furthermore, after finding that the potential marker exhibited cellular uptake by the HT-29 colon cancer cell line, we postulated that there would be enhanced affinity for dysplastic tissue or cancer cells in the large intestine if properly administered (by direct lavage of an aqueous solution). Thus, this preferential chelate uptake, upon lavage introduction, would facilitate enhanced visualization during colonoscopy procedures by image contrast enhancement resulting from the differential uptake by tissue abnormalities as a result of the difference in respiration, physiology, and morphology of epithelial cancer cells relative to normal cells. In short, a preliminary animal study was undertaken to determine if surface abnormalities of the large intestine could be made to fluoresce for improved disease detection. As noted in the Experimental Section, using the DMH protocol, Sprague Dawley rats where made to develop colon cancers. Subject animals that potentially had colon abnormalities were identified from a larger group using a three-step process. (i) Rats that exhibited weight loss and feeding difficulties were set aside as potential candidates. (ii) These animals were inspected for signs of rectal bleeding or blackened fecal pellets, often an indication of a significant abnormal growth. (iii) Finally, using a 2.5 mm diameter, 3-m-long, flexible microendoscope,40,41 the large intestines of several animals were inspected under white-light endoscopic visualization to confirm the presence of an abnormal tissue

mass. To perform this investigation, we anesthetized the animals, washed the colons several times with a saline solute of Golightly (to remove fecal matter and debris), and then rinsed the colons (to clear the field of view) during visualization with normal saline. To the animal with an observable lesion we introduced, through the 1.0-mm working lumen of the microendoscope, an aqueous solution of the fluorescent marker (Tb-PCTMB at a concentration of 0.1 mmol/kg of animal body weight), allowing the animal to respire during this lavage. Introduction of the marker solution was made first at the site of the suspect tissue mass, followed which the additional volume necessary to fill the entire large intestine was introduced. The marker solution was allowed to reside in the large intestine for 20 min before the rat was sacrificed and dissected the rat. Upon dissection of the large intestine, a large occlusive mass approximately 4.5 × 5.0 cm was found approximately 15 cm from the rectum (coinciding with the location determined by endoscopy). Normal tissue samples were collected from the intestine about 10 and 15 cm from the suspect mass. Tissue samples from the suspect mass and from the normal region of the intestine were then placed in liquid N2 and immediately transported for further histological preparation. Four frozen sections were prepared of all samples. Four slides from each tissue region were first left unstained for fluorescence microscopy imaging as described below and then were stained with H&E. Two pathologists used standard histological criteria for diagnosis.2,3,49,50 The result was that the tissue taken from a location spatially removed from the abnormality was determined to be normal (Figures 6A,B,C), and the tissue taken from the suspect site was determined by to be adenocarcinoma (Figures 7A,B,C). Microscopic images of the regions in question, stained using the H&E methodology, are shown in Figures 6A and 7A. Using an in-house modified microscope, as described in the Experimental Section, samples from the suspect and remote regions of the animal were imaged and then subsequently stained with H&E for histological evaluation (as noted above). The fluorescence images for the suspect region and that for the remote colon section are presented in Figures 6B and 7B. In Figures 6C and 7C the false color enhanced fluorescence images of the identical regions of the same tissues are shown. Remembering that false color contours in the fluorescence image correspond to the emission intensity in gray scale values and represent the relative amount of chelate entrained in the tissue, it is clear that a significant difference in chelate quantity exists between the two samples (Figures 6C and 7C). As seen in the in-vitro study, there is a measurable tendency toward enhanced or preferential distribution of the chelate in the adenocarcinoma or the tissue containing carcinoma cells. This observation is indicated by the large difference in fluorescence intensity for normal versus neoplastic tissue. It is now thought that the mechanism leading to enhanced chelate uptake/association in tissues is possibly more complicated than that seen in cultured cells and likely involves several transport processes. Recent observations from a larger animal study51 suggest that, in animals, M-cells (antigen-transporting epithelial cells that are often associated with disease) aid in (49) Sternberg, S. S., Ed. Histology of the Colon. In Histology for Pathologists; Raven Press: New York, 1992; pp 573-592. (50) Pozharisski, K. M. Tumors of the Intestines. In Pathology of Tumors in the Laboratory Animals; Turosov, E. V., Ed.; IARC: Lyon, 1973; pp 119-140.

Figure 6. (A) Standard microscopic image of H&E stained tissue taken from a site in the colon removed from the suspect mass. Histologically judged to be normal. (B) Fluorescence microscopic image of unstained, Tb-PCTMB dosed tissue microscopic image of H&E stained tissue taken from a site in the colon removed from the suspect mass (normal). (C) Color contour enhanced, fluorescence microscopic image of unstained, Tb-PCTMB dosed tissue microscopic image of H&E stained tissue taken from a site in the colon removed from the suspect mass (normal).

the transport of the Tb-PCTMB chelate and that mucins overexpressed by cancer cells bind the chelate, holding it up in the interstitial space.51-53 Semiquantitative Evaluation. While a great deal of contrast is observed for the colon cancer, as shown in Figure 6C some fluorescence signal is also detected in the normal tissue, and it is likely that the chelate uptake is not explicitly preferential. Assuming fluorescence is attributable to chelate tissue adhesion (51) Bornhop, D. J.; Stewart, M. S.; Morgan, D. L.; Pence, B. C.; Obriant, S.; Kiefer, G. E. In Preparation for Gastroenterology (April 1999). (52) Shimamoto, F.; Vollmer, E. J. Cancer Res. Clin. Oncol. 1987, 113, 41-50. (53) Savidge, T. C. Trends in Microbiology 1996, 4, 308-317.

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(the GSVs’ amplitudes do not correspond to the background fluorescence or a false positive signal) and by simply using GSV differences, we find that the contrast enhancement for normal tissue is about a factor of 3. A 3-fold signal increase is substantial and would certainly improve the endoscopist’s ability to identify suspect sites and possibly help with demarkation for surgical excision. Knowing the mass of solute detected in a tissue sample is of great value and has important implications toward biomedical imaging for molecular biopsy. By performing a semiquantitative evaluation of the signal amplitude for both tissues, insight is gained regarding the relative amount of solute bound to each tissue sample. Our experimental conditions (magnification, camera settings, etc.) were essentially identical to those used previously,32 allowing us to estimate the quantity of Tb-PCTMB found in our tissue sections (Figures 6C and 7C) by generating a relationship between GSV and solute mass. The linear plot (GSV ) fluorescence intensity {I}) is produced with

Qavg. ) 2.98 × 10-16 I - 1.62 × 10-14

(1)

where Qavg. is the moles of analyte per pixel and I is the fluorescence signal/pixel. Using eq 1, a standard blob analysis as provided by Image Pro Plus software, and an estimate of the fluorescent signal that represents the blank noise, the total mass of Tb-PCTMB chelate in the colon tissue imaged here can be estimated. In fact, the actual quantity of dye, in either sample, is very low. First we take a conservative approach and assume a 2σ detection threshold using 50 GSV for the blank or σ. In other words, we consider any GSV greater than 100 to be a fluorescent signal from the chelate. Using this criteria, a blob analysis indicates that, for the normal tissue (Figure 7B), in the region with pixels above the threshold, the average signal level observed is GSV ) 125 corresponding to approximately 2.11 × 10-14 moles/ pixel. In the sampled region, the blob with a GSVave > 100, there are about 2.3 × 105 pixels, making the total fluorescence signal produced by the normal tissue correspond to about 4.84 pmol of solute. The signal intensity for the fluorescent regions of the abnormal tissue have a GSVave ) 235 (many are near the camera saturation level), thus using the calibration curve there are 5.38 × 10-14 mol/pixel. The corresponding amount of chelate in the tissue, 6.1 × 105 sampled fluorescent pixels, would then be 32.8 picomoles. There is roughly a factor of 7 more chelate marker found in optical probe volume of the two tissue samples (cancer vs normal). It should be noted that these values are only semiquantitative estimates, and error can be introduced from several sources. First, quantification of fluorescent solutes in highly scattering media is difficult because fluence losses are hard to quantify. Second, sampling only the pixels with an average GSV value greater than a somewhat arbitrary value (2σ) introduces error. Third, the optical properties of the calibration substrate do not exactly match those of the tissue. In any event, the values obtained are reasonable and demonstrate the potential of using lanthanide chelate contrast markers with fluorescence imaging for improved detection of tissue types. 2614 Analytical Chemistry, Vol. 71, No. 14, July 15, 1999

Figure 7. (A) Standard microscopic image of H&E stained tissue taken from the suspect colon mass. Histologically judged to be adenocarcinoma. (B) Fluorescence microscopic image of unstained, Tb-PCTMB dosed tissue, taken from the suspect colon mass (adenocarcinoma). (C) Color contour enhanced fluorescence microscopic image of unstained, Tb-PCTMB dosed tissue taken from the suspect colon mass (adenocarcinoma).

Given the validity of the above estimation, the sensitivity of our technique appears to be quite good, allowing low doses of chelate and modest light levels to be used. Here, we used a lavage solution containing a total of 5.76 µmol of solute and imaged the tissue at 400X magnification using 0.1-s camera integration time. Thus, it is reasonable to believe that our chelate dyes can be used in communion with technology like that described by B. Palcic et al., US Patent, 5,507,287 (1996) or that currently under European clinical evaluation and marketed by Carl Zeiss or Richard Wolf (Photodynamic Diagnosis (PDD) systems), for disease detection. In this case, low doses of the marker dye Tb-PCTMB, modest excitation light levels (100 W Xe lamp before extensive filtering and losses to collection optics), and short camera integration times would be used. Therefore, either upon lavage introduction as

described here or by IV injection as demonstrated elsewhere,30-33 it should be possible to use Tb-PCTMB as a disease marker. Summary. While more evidence is needed, we believe that the observed difference in fluorescent signals exhibited by cells and tissues suggests some preferential or enhanced uptake of TbPCTMB by abnormal colon tissue. We postulate that such differences in fluorescence signal will allow for contrast enhancement for cancers of the colon, leading to improved detection. Many of the drawbacks associated with using PDT agents are circumvented, since preferential marker uptake occurs rapidly, during a low-concentration, aqueous lavage application. Introduction through an endoscope working lumen should make it possible to use such a technique for contrast enhancement during colonoscopy using a sigmoidoscope modified for fluorescence imaging. Ongoing investigations include expanded animal and cell studies to define

the mechanism for specificity that the chelate exhibits and to demonstrate the utility of using it as a spray-on contrast enhancement marker for disease of the colon. ACKNOWLEDGMENT This research was supported partially by grants from the Whitaker Foundation, by the Texas Tech University Research Enhancement Fund, and by The Dow Chemical Company. CRA was supported in part by a Howard Hughes Medical Institute Grant through the Undergraduate Biological Sciences Education Program to Texas Tech University. Received for review November 4, 1998. Accepted April 9, 1999. AC981208U

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