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Basic Science Investigation |
1 Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts; 2 Institut fur Organische Chemie, Universität Hamburg, Hamburg, Germany; and 3 Department of Radiology, Beth Israel Deaconess Medical Center, Boston, Massachusetts
Correspondence: For correspondence or reprints contact: John V. Frangioni, MD, PhD, Division of Hematology/Oncology, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Room SL-B05, Boston, MA 02215. E-mail: jfrangio{at}bidmc.harvard.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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-ray radioscintigraphic imaging of living human prostate cancer cells.
Key Words: 99mTc-pertechnetate • S-acetylmercaptoacetyltriserine (MAS3) • solid-phase chemistry • SPECT • prostate-specific membrane antigen
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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SPECT is a nuclear medicine technique by which -ray emissions from a radioisotope are used to localize cancer and its products. The major radioisotope used clinically is 99mTc, which emits a monoenergetic, 140-keV photon, has a 6-h half-life, and has a low absorbed dose. Most important, it is readily available at most institutions and is extremely inexpensive to produce, typically $10.00 for 740 MBq (20 mCi). The major problem with 99mTc is that it is eluted using saline as sodium pertechnetate (Na99mTcO4) in the +VII oxidation state, which requires reduction to the +V oxidation state, as well as exchange chelation, for stability and subsequent chemical manipulation. Any chemistry capable of converting sodium pertechnetate to a more useful form would have immediate impact on diagnostic agent development.
Given its half-life, 99mTc is an ideal radiotracer for any targeting molecule having a total-body clearance of 
6 h, such as small molecules, peptides, and single-chain antibodies. Of these, small molecules have distinct advantages for tumor targeting due to rapid biodistribution, rapid clearance, and excellent penetration into solid masses. Our group (2) and others (3,4) have reported small molecules with high-affinity binding to prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein expressed at high levels on normal and malignant prostate epithelial cells (5,6). Our GPI (2[(3-amino-3-carboxypropyl)(hydroxy)(phosphinyl)-methyl]pentane-1,5-dioic acid) molecule is a 311-Da monomeric small molecule with an affinity of 9 nM and a net charge of –4 after conjugation to contrast agents or radiotracers.
However, 2 major problems impede the use of GPI and similar molecules in diagnostic imaging. First, in its monomeric form, the phosphate anion in serum competes with it for binding to PSMA. Second, its chemical structure resembles classic exchange ligands, such as tartaric acid, which are required to stabilize 99mTc during its reduction. Hence, 99mTc could be chelated by GPI itself and block binding to PSMA, and HPLC purification of the final product using conventional labeling strategies is extremely difficult (7). In this study, we solve both problems by developing a simple and rapid cartridge-based, solid-phase prelabeling strategy for 99mTc and also describe an adamantane-based trimeric form of GPI that retains full binding to living prostate cancer cells in the presence of serum.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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HPLC/Mass Spectrometry Platform
The HPLC/mass spectrometry platform used for purification of both nonradioactive and radioactive tumor-targeting small molecules and peptides has been described in detail previously (7). Briefly, the system is composed of a Waters model 1525 binary pump, a model 2487 ultraviolet detector (Waters), a Sedex model 75 (Richards Scientific) evaporative light scatter detector (ELSD) with the nebulizer modified to reduce band broadening at low flow rates, a model FC-3200 high-sensitivity photomultiplier tube -detector (Bioscan), and a Waters fraction collector, all housed within a Capintec hot cell equipped with a model CRC-15R (Capintec) dose calibrator. For nonradioactive reactions, column eluate was split into a Waters LCT electrospray time-of-flight (ES-TOF) mass spectrometer.
Preparative Synthesis of S-Acetylmercaptoacetyltriserine (MAS3)
Ten milligrams (36 µmol) of triserine were dissolved in 350 µL of water. One equivalent (5 µL, 36 µmol) of the base triethylamine (Et3N) was added, followed by 2 equivalents (16.6 mg, 72 µmol dissolved in 160 µL N,N-dimethylformamide [DMF]) of SATA. The reaction mixture was vortexed at room temperature for 3 h. An additional equivalent (8.3 mg, 36 µmol dissolved in 80 µL DMF) of SATA was then added, and vortexing was continued for an additional 2 h.
To confirm completion of the reaction, a 10-µL sample was analyzed by reverse-phase HPLC (RP-HPLC) using a 4.6 x 150 mm Symmetry (Waters) C18 column and a linear gradient from 0% to 15% B over 35 min, starting 2 min after injection, at 1 mL/min, where A = H2O + 0.1% formic acid and B = acetonitrile + 0.1% formic acid. MAS3 eluted at a retention time (Rt) = 11.6 min as detected by the ELSD, with its mass confirmed by ES-TOF mass spectrometry. Preparative purification was performed on an HPLC system described in detail previously (7) and equipped with a 5-mL sample loop, after dilution into a final volume of 5 mL of H2O + 0.1% trifluoroacetic acid (TFA), and using a 19 x 150 mm Symmetry C18 column. The gradient consisted of 0% B for 3.5 min, then 0%–15% B over 35 min at 7 mL/min, where A = H2O + 0.1% TFA and B = acetonitrile + 0.1% TFA. MAS3 eluted at Rt = 21.8 min using ELSD detection. Fractions containing product were pooled and lyophilized. MAS3 was obtained as a white powder in 57% isolated yield (8.0 mg, 20.5 µmol), with the expected mass confirmed by ES-TOF mass spectrometry, and a purity of 
98%.
Solid-Phase Labeling of MAS3 with 99mTc-Pertechnetate
One-hundred fifty microliters of a 50% slurry of Chelex 100 resin (Bio-Rad) in 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 5.0, were added to an empty micro Bio-Spin (Bio-Rad) chromatography column, washed once with MES buffer, and centrifuged at 3,000g for 10 s; 1.2 mg (3 µmol) of MAS3 were dissolved in 1 mL of water. One milligram (4 µmol) of stannous (II) chloride dihydrate was dissolved in 1 mL of 10 mM HCl. Then 100 µL of MAS3 and 35 µL of stannous chloride were mixed well and added to the Chelex resin. 99mTc-pertechnetate (185–370 MBq [5–10 mCi]) in 100 µL of saline, eluted directly from a 99Mo generator with saline, were added to the tube. The tube was capped, wrapped in parafilm, and boiled for 10 min in a water bath. 99mTc loading of MAS3 was monitored by RP-HPLC using a 4.6 x 75 mm Symmetry C18 column with a linear gradient from 0% to 60% B over 30 min, starting 2 min after injection, at 1 mL/min, where A = 10 mM TEAA and B = absolute MeOH. 99mTc-MAS3 eluted at Rt = 14.1 min. In separate experiments, the limits of this reaction were determined to be 370 MBq (10 mCi) 99mTc-pertechnetate and 1.3 µmol of MAS3 using 150 µL of 50% Chelex 100 slurry.
A 10-mg Oasis HLB cartridge (catalog no. 186000383) was activated using methanol, then H2O, and equilibrated with H2O, pH 4.0 (pH adjusted with 10 mM HCl). The 99mTc-MAS3 solution was diluted with 1 mL of H2O, pH 4.0, and bound to the cartridge. The cartridge was washed with 10 mL of H2O, pH 4.0, to remove 99mTc-pertechnetate, tin, and free MAS3; then it was purged completely with nitrogen. Finally, 99mTc-MAS3 was eluted with 400 µL dry DMSO or DMF.
Synthesis of [99mTc-MAS3]-NHS
After 200 µg (0.7 µmol) of TSTU powder were placed in a 1.5-mL Eppendorf tube, 185–370 MBq (5–10 mCi) of purified 99mTc-MAS3 in 400 µL DMSO or DMF and 0.5 µL (2.9 µmol) of neat (5.74 M) N,N-diisopropylethylamine (DIEA) were added. The top was sealed and the solution was incubated at 60°C for 10 min. The reaction was diluted to 1 mL final volume by the addition of 600 µL of dichloromethane:hexane (6:4). The reactants and by-products were removed by passage through Waters Oasis MCX (catalog no. 186000252) and MAX (catalog no. 186000366) cartridges attached in tandem. The flow-through, containing pure [99mTc-MAS3]-NHS ester, was concentrated by loading on an Oasis HLB cartridge, washing with of 1 mL of dichloromethane:hexane (6:4), and eluting using 500 µL of either DMF or DMSO. The purity of the compound was assessed by RP-HPLC using a 4.6 x 75 mm Symmetry C18 column with a linear gradient from 0% to 60% B over 30 min, starting 2 min after injection, at 1 mL/min, where A = 10 mM TEAA and B = absolute MeOH. [99mTc-MAS3]-NHS eluted at Rt = 23.5 min. The specific activity of [99mTc-MAS3]-NHS was estimated by conjugation to tryptophan and measurement of 280-nm absorbance of the product using the same RP-HPLC conditions as those used to assess purity.
Synthesis and Purification of Radiolabeled PSMA Ligands
Covalent conjugation of GPI derivatives with [99mTc-MAS3]-NHS was performed by the addition of 100 µL (10 µmol) of 100 mM triethylamine in dry DMSO to 10 µL (0.1 µmol) of a 10 mM solution of GPI derivative in a total of 400 µL dry DMF/DMSO, followed by the addition of 200 µL of [99mTc-MAS3]-NHS (185–259 MBq [5–7 mCi]) in dry DMSO. Constant stirring at room temperature was maintained for 40–90 min, until the reaction was completed. The radiolabeled ligands were analyzed by RP-HPLC on a 4.6 x 75 mm Symmetry C18 column using a linear gradient from 0% to 60% B over 25 min, beginning 2 min after injection, at a flow rate of 1 mL/min, where A = 10 mM TEAA, pH 7.0, and B = absolute MeOH.
Synthesis of Re-MAS3
A procedure reported previously (9) was used with slight modification. MAS3 (4.8 mg, 16 µmol) was dissolved in 1.5 mL of water. Stannous chloride dihydrate (5.4 mg, 24 µmol) in 1.5 mL of 0.1 M citrate buffer, pH 5.0, and NaReO4 (6.6 mg, 24 µmol) in 1.5 mL of H2O were added to the MAS3 solution. The reaction mixture was stirred at 90°C for 1 h. After cooling to room temperature, Re-MAS3 was purified on an Oasis HLB cartridge and eluted with DMSO. The purity was assessed by liquid chromatography/mass spectrometry (LC/MS) on a 4.6 x 150 mm Symmetry C18 column using a linear gradient from 0% to 50% B over 15 min, beginning 2 min after injection, at a flow rate of 1 mL/min, where A = H2O + 0.1% formic acid and B = acetonitrile + 0.1% formic acid. Retention time was 6.4 min.
Synthesis of [Re-MAS3]-NHS
Fifty microliters (3 µmol) of a 60 mM TSTU solution in DMSO and 200 µL (2 µmol) of a 10 mM Re-MAS3 solution in DMSO were mixed together; then 25 µL (5 µmol) of 200 mM N,N-diisopropylethylamine and 25 µL of DMSO were added. The solution was vortexed at room temperature for 40 min. After dilution with dichloromethane, the NHS ester was purified using Oasis MCX, MAX, and HLB cartridges connected in series as described earlier. The purity was assessed by LC/MS on a 4.6 x 150 mm Symmetry C18 column using a linear gradient from 0% to 50% B over 15 min, beginning 2 min after injection, at a flow rate of 1 mL/min, where A = H2O + 0.1% formic acid and B = acetonitrile + 0.1% formic acid. Retention time was 10.4 min.
Synthesis and Purification of [Re-MAS3]-Conjugates
Covalent conjugation of GPI derivatives with [Re-MAS3]-NHS was performed by the addition of 0.1 mL (10 µmol) of 100 mM triethylamine in dry DMSO to 0.1 mL (1 µmol) of a 10 mM solution of GPI derivatives in dry DMSO or DMF, followed by addition of 0.2 mL (2 µmol) of a 10 mM [Re-MAS3]-NHS solution in dry DMSO. Constant stirring at room temperature was maintained for 2 h. The conjugates were purified by preparative HPLC (Symmetry C18 column, 19 x 150 mm, 7 µm) using a linear gradient from 0% to 50% B over 27 min, starting 5 min after injection, with a flow rate of 10 mL/min. The collected fractions were lyophilized, and purity was assessed by LC/MS on a 4.6 x 150 mm Symmetry C18 column using a linear gradient from 0% to 50% B over 15 min, beginning 2 min after injection, at a flow rate of 1 mL/min, where A = H2O + 0.1% formic acid and B = acetonitrile + 0.1% formic acid.
Quantification of Serum Stability
The stabilities of [99mTc-MAS3]-GPI compounds were tested by incubation in the absence (phosphate-buffered saline [PBS] only) or presence of 100% calf serum for 4 h at 37°C. Stability and transmetallation were quantified using high-resolution chromatography. For PBS experiments, a 4.6 x 75 Symmetry C18 column was used as described earlier for radiolabeled PSMA ligands. For serum experiments, an 8 x 300 mm, 200-Å Diol (YMC, catalog no. DL20S053008WT) gel-filtration column was used with PBS as the mobile phase. Gel-filtration molecular weight (M.W.) markers (Bio-Rad) were: M1 = thyroglobulin (670 kDa), M2 = -globulin (158 kDa), M3 = ovalbumin (44 kDa), M4 = myoglobin (17 kDa), and M5 = vitamin B12 (1.3 kDa).
High-Throughput, Radioactive Live Cell Binding and Affinity Assay
Human prostate cancer cell lines, PSMA-positive LNCaP and PSMA-negative PC-3, were obtained from the American Type Culture Collection. The PSMA-negative human bladder cancer cell line TsuPR1 was a generous gift of Dr. John T. Isaacs (Johns Hopkins University, Baltimore, MD). Cell lines were cultured at 37°C under humidified 5% CO2 in RPMI 1640 medium (Mediatech Cellgro) supplemented with 10% fetal bovine serum (Gemini Bio-Products) and 5% penicillin/streptomycin (Cambrex Bioscience). Cells were split onto 96-well filter plates (model MSHAS4510; Millipore) and grown to 50% confluence (approximately 35,000 cells per well) over 48 h.
To assign absolute affinity to each compound, a homologous competition assay was used with the 99mTc-labeled version as the tracer and the relabeled version as the test compound. To avoid internalization of the radioligand due to constitutive endocytosis (2), live cell binding was performed at 4°C. Cells were washed 2 times with ice-cold Tris-buffered saline (TBS), pH 7.4, and incubated for 20 min at 4°C with 0.02 MBq (0.5 µCi) of radiotracer in the presence or absence of the test compound. Cells were then washed 3 times with TBS using a Millipore vacuum manifold (catalog no. MSVMHTS00), and the well contents was transferred directly to 12 x 75 mm plastic tubes placed in -counter racks. Transfer was accomplished using a modified (Microvideo Instruments) 96-well puncher (MAMP09608; Millipore) and disposable punch tips (MADP19650; Millipore). Well contents were counted on a model 1470 Wallac Wizard (Perkin Elmer) 10-detector -counter, and curves were fit using Prism version 4.0a (GraphPad) software.
Near-Infrared (NIR) Fluorescence and -Radioscintigraphic Imaging
To assess viability and to verify confluence, living cells were loaded with the NIR fluorophore IR-786 by adding it to the cell culture medium at 1 µM for 30 min at 37°C before the start of the experiment (10). Cells were incubated as described earlier with 3.7 MBq (100 µCi) of 99mTc-labeled compound per well, in the buffer being tested, for 20 min at 4°C, before extensive washing of the filter plate. Simultaneous white light and NIR fluorescence imaging of plates was performed as described in detail previously (11,12). -Radioscintigraphy was performed with an Isocam Technologies Research Digital Camera equipped with a 
-in. NaI crystal, 86 photomultiplier tubes, and a high-resolution, low-energy lead collimator.
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RESULTS |
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The second key to this strategy is the transfer of 99mTc-MAS3 from aqueous buffer to nonaqueous buffer, as the final NHS ester is highly susceptible to attack by the hydroxyl ion of H2O. To accomplish this, and to remove unwanted molecules, the supernatant from the Chelex 100 resin is bound to a C18 mixed-bed cartridge, washed, and then eluted with desired organic solvent (DMSO or DMF). The NHS ester of 99mTc-MAS3 is then formed rapidly using TSTU and DIEA as described. Excess reactants are removed using tandem mixed-mode anion-exchange and cation-exchange cartridges, and the final product is concentrated on a C18 mixed-bed cartridge (Fig. 2C). Using this chemical strategy, [99mTc-MAS3]-NHS is prepared in ultrapure form (>99%; Fig. 2B) in DMSO or DMF, in only 25 min, with an overall radiochemical yield of 70%–75% relative to 99mTc-pertechnetate starting material. Reaction of the final product with basic water (pH 10) results in the rapid reformation of 99mTc-MAS3, thus proving the presence of an NHS ester (Fig. 2B). The typical specific activity of [99mTc-MAS3]-NHS produced using this strategy was 4.1 x 108 MBq/mmol (1.1 x 104 Ci/mmol).
To confirm, absolutely, the chemical structures produced, as well as to prepare nonradioactive derivatives of diagnostic agents for affinity measurements, the chemistry was repeated using Re. Although the exact procedure shown in Figure 2C worked well for Re-MAS3 chelates, the yield was low (
20%), likely reflecting the difficulty others have had in preparing Re chelates (8). Hence, for preparative purification of Re-MAS3, solution-phase labeling was used, followed by formation and purification of [Re-MAS3]-NHS using our solid-phase approach. A high-resolution LC/MS HPLC system (7) was also used to verify that all cartridges performed as designed and to verify the purity of final compounds (Fig. 2D).
Solid-Phase Prelabeling of Highly Anionic PSMA Small Molecules
Using this solid-phase preloading strategy, GPI-containing molecules of increasing ligand valency were labeled with either 99mTc or Re in 1 chemical step as shown in Figure 3A. For 99mTc-labeled molecules, no further purification was necessary, as the NHS ester reaction with an amine in dry DMSO or DMF goes to completion without side reactions (Fig. 3B). For Re-labeled molecules, which were to be used for affinity measurements, LC/MS analysis with ELSD (7) was used to ensure the highest possible purity and to confirm the expected isotopic pattern (Fig. 3B).
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Bioactivity and Radioscintigraphic Imaging of PSMA-Specific Radiotracers
Using the nonradioactive Re derivatives as test compounds, 99mTc derivatives as radiotracers, and a high-throughput 96-well filter plate assay (7), the absolute affinity of each molecule for the surface of living prostate cancer cells was measured using homologous competition (Table 1). Actual raw data from the assay using monomeric [99mTc-MAS3]-GPI (Ia) as radiotracer (Fig. 3D, top) and trimeric [99mTc-MAS3]-AdamGPI (IVa) as radiotracer (Fig. 3D, bottom) confirm that both phosphate and serum compete effectively with monomeric radiotracers for the active site of PSMA but are unable to compete with trimeric radiotracers, resulting in nearly identical affinities of IVa under all physiologic conditions. The measured maximum number of binding sites (Bmax) values for compounds Ia/IIa, IIIa, and IVa were 1.6 x 105, 2.1 x 105, and 2.6 x 105 PSMA receptors per cell, respectively, which is consistent with previously published reports of 1.8 x 105 to 8.0 x 105 for PSMA-specific monoclonal antibodies (16,17).
To confirm that the synthesized radiotracers permit sensitive and specific radioscintigraphic imaging of prostate cancer cells, approximately 35,000 PSMA-positive (LNCaP) or PSMA-negative (PC-3 and TsuPR1) cancer cells were grown in each well of a 96-well filter plate and incubated at 4°C for 20 min with the indicated tracer. As shown in Figure 4, and confirming the results in Table 1 and Figure 3D, 99mTc-labeled trimeric AdamGPI (IVa) bound with high affinity to living prostate cancer cells expressing PSMA in all buffers, including 100% serum, and no binding was detectable on PSMA-negative cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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An important general limitation of NHS ester chemistry is that the hydroxyl ion of water is a potent competitor. NHS esters of fluorophores are typically used in millimolar concentrations and in excess of the targeting molecule. This is not true of radioactive NHS esters, such as [99mTc-MAS3]-NHS, which are used in nanomolar concentrations. Hence, the use of [99mTc-MAS3]-NHS in aqueous buffer will result in low labeling efficiencies. When used in dry organic solvent, however, [99mTc-MAS3]-NHS reacts rapidly and completely with primary amines. Another potential limitation is the availability of the nucleophile within the targeting ligand. Each of our GPI derivatives has been engineered to have a flexible 6-carbon spacer; however, more sterically hindered amines could be difficult to conjugate.
Even with careful chemical engineering, small molecule and peptide-targeting ligands often have poor affinity or are competed by endogenous ligands. A general approach to solving these problems was proposed by Whitesides' group (13) and involves multimerizing binding epitopes into an optimally spaced rigid structure. To accomplish this, we have created a tri-NHS adamantane derivative (14), which permits the conjugation of up to 3 (same or different) targeting ligands. The molecule also has an isolating linker and a deprotectable primary amine for subsequent conjugation to contrast agents, radiotracers, or therapeutics. Adamantane, being the core structure in diamond, has maximal rigidity. In this study, we conjugated GPI through a propionyl acid spacer and made no attempt to optimize intermolecular spacing or overall rigidity. Future studies will focus on these issues.
Nonetheless, the results to date are encouraging. First, conjugation of a single GPI monomer to a single adamantane core had only a negligible effect on affinity (Table 1; compare Ia with IIa). Second, increasing multivalency from 1 to 3 led to dramatic improvements in both affinity and competition with endogenous anions. Indeed, affinity not only increased almost 2 logarithms when measured in TBS, but as a trimer was unaffected by serum (Table 1). The results also reveal, however, that the GPI molecules are not as isolated from the effect of metal chelate as one would like. For example, when Re-MAS3 is conjugated to either GPI monomer or AdamGPI Trimer, there is a 3- to 8-fold decrease in affinity. Nevertheless, the final affinity of IVa (3 nM) is equivalent to that of previously described PSMA monoclonal antibodies (16).
Because it is known that PSMA undergoes both constitutive and inducible endocytosis (19), we were careful to perform all affinity measurements on living cells at 4°C. However, both NIR fluorescent GPI derivatives (2) and 99mTc-labeled derivatives (data not shown) are concentrated inside cells via endocytosis, which serves to "amplify" radiotracer signal over time. This important feature of PSMA will likely improve in vivo performance and also needs to be incorporated into pharmacokinetic modeling.
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CONCLUSION |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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References |
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