Research article Received: 26 February 2014,
Revised: 14 June 2014,
Accepted: 16 June 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2740
The interaction of CuInS2/ZnS/TGA quantum dots with tyrosine kinase inhibitor and its application Shenghua Liao,a,b Yu. Huang,a,b Jiaxin Zuoa,b and Zhengyu Yana,b* ABSTRACT: The interactions between thioglycolic acid-capped-CuInS2/ZnS quantum dots (CuInS2/ZnS/TGA QDs) and tyrosine kinase inhibitor (TKI) were investigated using fluorescence, ultraviolet–visible spectrometry and Fourier transform infrared spectrometry. The results indicated that the fluorescence intensity of CuInS2/ZnS/TGA could be quenched by imatinib, dasatinib, nilotinib, gefitinib and erlotinib, which hinted that CuInS2/ZnS/TGA QDs could be used in the detection of TKI in active pharmaceutical ingredients (API). Calibration curves showed good linear correlation and low detection limits. The average recovery was between 98 and 102%. Moreover, the nature of the fluorescence quenching mechanism of CuInS2/ ZnS/TGA QDs by TKI was discussed. A ground state complex was formed by hydrogen bonding between the carboxyl group of CuInS2/ZnS/TGA QDs and the amino group of TKI. This led to an increase in non-radiative transition and fluorescence quenching of CuInS2/ZnS/TGA QDs. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: thioglycolic acid-capped-CuInS2/ZnS quantum dots; tyrosine kinase inhibitor; interaction; content determination; intermolecular hydrogen bond
Introduction Quantum dots (QDs) have attracted considerable attention in the chemistry, biology and medicine fields with many unique photo-electronic characteristics, such as broad absorption spectra, narrow symmetric emission bands and high photostability. Recently, the surface modifier of QD bonding with certain drugs has been shown to affect fluorescence intensity, which made it applicable for the determination and tracing of drugs based on a linear or exponential relationship between concentration and fluorescence intensity. For example, Liang et al. detected spironolactone based on the fluorescence quenching of CdSe QDs by spironolactone (1) and Liao et al. established a determination method for doxorubicin using a thioglycolic acid (TGA)-functionalized CdS QD fluorescence probe (2). However, these QDs containing Cd pose potential risks to human health and the environment. In order to apply low toxicity QDs in drug assays, I–III–VI2 semiconductor nanocrystals were developed. Su et al. synthesized mercapto propionic acid (MPA)-capped CuInS2 ternary QDs for the detection of ascorbic acid and folic acid (3). Recently, they have prepared mercaptopropionic acid-capped CuInS2 QDs as a near-infrared fluorescence probe for the determination of dopamine, as well as L-cysteine modified CuInS2 QDs for heparin and heparinase determination (4,5). The advantages of the high fluorescence intensity and low toxicity of ternary QDs have encouraged the development of fluorescence probes for drug content determination. Tyrosine kinase inhibitor (TKI) is mainly used for targeted therapy of tumors. According to the protein molecules or gene fragments in the tumor cells, targeted drugs are designed at the cellular and molecular level. These drugs can bind to carcinogenic sites specifically so the tumor cells are killed while the peripheral normal tissues remain intact (6). The possible mechanisms are as follows: inhibition of tumor cell repairing, blocking of cell
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division in the G1 phase, induction and maintenance of apoptosis, formation of anti-angiogenesis, etc. Currently, the main analytical methods for TKI are isotopic analysis, fluorescence polarization immunoassay, fluorescence resonance energy transfer, microbead technology, microfluidic technology, enzyme-linked immunosorbent assay and dissociation enhanced lanthanide fluorescence immunoassay (7). High performance liquid chromatography and high performance liquid chromatography– mass spectrometry are also used for determination (8–10). Although these methods are of high sensitivity, the use of complex operations and expensive equipment hinders their popularization. The detection of TKI using CuInS2/ZnS/TGA QDs based on fluorescence quenching has not yet been reported. This method was applied to determine TKI for the first time with satisfactory results. Under physiological pH conditions, this method encompases high accuracy and sensitivity, a low detection limit, a wide linear range and a strong anti-interference ability. In addition, the interaction between CuInS2/ZnS/TGA QDs and TKI was investigated and a possible mechanism was discussed.
* Correspondence to: Z. Yan, Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, Nanjing 211198, China. Tel: +86 13512503905; Fax: +86 25 86185170. E-mail: [email protected] a
Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, Nanjing 211198, China
b
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 211198, China Abbreviations: API, active pharmaceutical ingredient; QD, quantom dots; RSD, relative standard deviation; TKI, tyrosine kinase inhibitor.
Copyright © 2014 John Wiley & Sons, Ltd.
S. Liao et al.
Experimental Apparatus and agents The absorption spectrum was acquired using a UV2100 UV–vis spectrometer (Shimadzu, Japan). The fluorescence measurements were made with a RF-5301 spectrofluorophotometer (Shimadzu). All pH measurements were made using a Model pH S-25 (Leici Equipment Factory, Shanghai, China). All infrared measurements were acquired by Fourier transform infrared spectroscopy (Shimadzu FTIR-8400S). AgI (99%), InI3 (99.999%), bis(trimethylsilyl)sulfide [(Me3Si)2S] (98%), mercaptoacetic acid (TGA) and tri-n-octylphosphine (TOP) (90%) were purchased from Alfa Aesar. Oleyl amine (OLA) (80–90%) was purchased from Acros Organics. These chemicals were used without further purification. Methanol and ethanol were purchased from Jiangsu Hanbang Technology Co. (Jiangsu, China). Trihydroxymethyl aminomethan and anhydrous sodium acetate were all purchased from Sinopharm Chemical Reagent Co. Ltd. Disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Nanjing Chemical Reagent Co. (Nanjing, China). Hydrochloric acid was purchased from Nanjing Chemical Reagent Co. (Nanjing, China). Among the above, methanol was of chromatography reagent grade, trihydroxymethyl aminomethane was biochemical reagent, other reagents were of analytical reagent grade. Imatinib, dasatinib, nilotinib, gefitinib and erlotinib were provided by the Department of Analytical Chemistry, China Pharmaceutical University.
Experimental procedures The CuInS2/ZnS/TGA QDs were prepared as follows: 0.05 mM CuI in 1.5 mL OLA and 0.10 mM InI3 in 3mL TOP were prepared under vacuum and loaded into a degassed three-necked flask. The mixture was heated at 150°C under magnetic stirring for several minutes until transparent. Subsequently, 0.15 mM (Me3Si)2S in 2 mL TOP was injected swiftly and reacted for 3 min at the temperature. The resulting solution was quickly cooled down to 80°C and the ZnS precursor was injected drop by drop, and had been prepared by mixing zinc stearate (0.8 mM) in 1.5 mL OLA. Then the solution was heated to 180°C and maintained for 1 h. The thus-prepared CuInS2/ZnS QDs were precipitated by adding methanol and n-butanol and centrifuged. The supernatant containing unreacted material was discarded and the remaining precipitates were dispersed in chloroform. To form a –COOH functional group, CuInS2/ZnS QDs and TGA (5mM) were mixed at the ratio of 1:1 and vortexed for 3 min followed by the addition of 0.5 mL NaOH solution and another vortex. Finally, the supernatant was precipitated by adding ethanol. TKI stock solutions were prepared as follows: appropriate amounts of imatinib, dasatinib, nilotinib, gefitinib and erlotinib were weighed, respectively. Imatinib and the other four reagents were dissolved in a small amount of water and methanol, respectively. All solutions were ultrasounded for a few minutes and cooled to room temperature. Five stock solutions at a concentration of 5.00 × 104 mol L1 were prepared by diluting to volume with the corresponding solvent. The CuInS2/ZnS/TGA QDs that precipitated after centrifugation were dissolved to a concentration of 7.81 × 105 mol L1 with trihydroxymethyl aminomethane–HCl solution at pH 7.40 (the concentration of QDs were calculated by the concentration
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of Cu2+); 3.00 mL CuInS2/ZnS/TGA QDs were pipetted precisely. The fluorescence intensity F0 of QDs was recorded with a 1 cm quartz cell. Then, 10 μL of imatinib, dasatinib, nilotinib, gefitinib and erlotinib stock solutions were added to the CuInS2/ZnS/TGA QDs successively and respectively to measure the fluorescence intensity (F) with the following settings on the fluorescence spectrophotometer (excitation wavelength (ex) 350 nm, slit width 5 nm and scan range is 420–680 nm).
Results and discussion Fluorescence quenching In accordance with this method, a designated amount of imatinib, dasatinib, nilotinib, gefitinib and erlotinib reference solutions were added successively and respectively to CuInS2/ZnS /TGA QD solution at certain concentrations and under optimum conditions. A linear relationship between the quenched fluorescence intensity of the CuInS2/ZnS /TGA QDs and the concentration of TKI was observed (Fig. 1 and Table 1). The correlation coefficient was close to 1 and the limit of detection was low within the measured concentration range. Therefore, the proposed method was proved to be sensitive.
Detection of TKI in API Three different concentrations of imatinib, dasatinib, nilotinib, gefitinib and erlotinib solutions were prepared for the determination of the sample concentration. Then the same amounts of the reference solutions were added respectively six times. The recovery and relative standard deviation (RSD) of each drug were calculated. From Table 2 it can be seen that the average recovery is between 98 and 102% and the RSD is less than 3%, which met the requirement for microanalysis. Therefore, the proposed method was proved to be reliable.
Interaction between CuInS2/ZnS/TGA QDs and TKI Interaction force There are many p–π and π–π conjugated systems in TKI. The stretching vibrations of –NH– were very obvious in the infrared spectra. It can be inferred that the interaction from the intermolecular hydrogen bond was formed by the interaction of –NH– and –COOH in thioglycolic acid.
Fluorescence quenching mechanism UV–visible absorption spectra. Interactions between CuInS2/ ZnS/TGA QDs and TKI were investigated under optimum conditions with UV–visible absorption spectra. As shown in Fig. 2, the absorbance values changed when the drugs were mixed with QDs. For imatinib, dasatinib, nilotinib, gefitinib and erlotinib, the red shift was 11 nm ( from 254 nm to 265 nm), 12 nm (from 316 nm to 328 nm), 13nm (from 260 nm to 273 nm), 12 nm (from 331 nm to 343 nm) and 10 nm (from 334 nm to 344 nm), respectively. These results indicated that drugs interacted with the QDs. The red shift of the absorption spectra demonstrated that a new compound was generated (11).
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CuInS2/ZnS/TGA quantum dots and TKI
Figure 1. The fluorescence spectra of CuInS2/ZnS/TGA quantum dots (QDs) in the presence of various concentrations of imatinib (A), dasatinib (B), nilotinib (C), gefitinib (D) 5 1 and erlotinib (E). The concentration of CuInS2/ZnS /TGA QDs was 7.81 × 10 mol L , from top to bottom, the concentrations of imatinib were 0.000, 0.125, 0.500, 2.50, 20.0, 6 1 6 1 50.0, 100, 125 × 10 mol L , the concentrations of dasatinib were 0.000, 0.150, 0.600, 3.00, 24.0, 60.0, 90.0, 120 × 10 mol L , the concentrations of nilotinib were 0.000, 6 1 6 1 0.875, 1.75, 3.50, 17.5, 35.0, 70.0, 100 × 10 mol L , the concentrations of gefitinib were 0.000, 0.150, 0.600, 3.00, 12.0, 30.0, 60.0, 100 × 10 mol L , the concentrations of 6 1 erlotinib were 0.000, 0.250, 0.500, 2.50, 10.0, 50.0, 100, 120 × 10 mol L , respectively. The inset shows the plot F0/F – 1 versus concentration of imatinib (A), dasatinib (B), nilotinib (C), gefitinib (D) and erlotinib (E). Error bars represent standard deviations from three measurements and relative standard deviations (RSD) of tyrosine kinase inhibitor (TKIs).
Table 1. Linear regression equations for determination of tyrosine kinase inhibitors Tyrosine kinase inhibitors
Linear regression equations (106 mol L1)
Imatinib Dasatinib Nilotinib Gefitinib Erlotinib
F0/F – F0/F – F0/F – F0/F – F0/F –
1 = 0.0119c + 0.1215 1 = 0.0274c + 0.1954 1 = 0.0354c + 0.0534 1 = 0.0579c + 0.1274 1 = 0.0662c + 0.1864
Correlation coefficient (r)
Linear range (106 mol L1)
Limit of detectiona (106 mol L1)
0.9994 0.9977 0.9981 0.9989 0.9991
0.125–125 0.150–120 0.875–100 0.150–100 0.250–120
1.800 × 103 3.300 × 103 6.500 × 103 3.700 × 103 4.300 × 103
Limit of detection is defined by the equation LOD = 3σ/k, where σ is the standard deviation of the measurement of blank measurements (n = 10) and k is the slope of the calibration graph.
a
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S. Liao et al. Table 2. Recovery rate (n = 6) for the determination of tyrosine kinase inhibitor Name Imatinib
Dasatinib
Nilotinib
Gefitinib
Erlotinib
Sample
Found value (106 mol L1)
Added reference solution (106 mol L1)
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
3.04 4.74 7.15 2.87 4.98 6.92 3.11 4.95 7.03 3.02 5.39 6.62 3.07 5.16 6.77
3.10 3.10 3.10 2.98 2.98 2.98 3.00 3.00 3.00 2.75 2.75 2.75 3.26 3.26 3.26
Found total value (106 mol L1) 6.10 7.82 10.22 5.83 7.93 9.89 6.06 7.92 9.99 5.72 8.09 9.35 6.29 8.44 10.02
RSD% (n = 6) 1.5 1.8 2.2 1.2 1.7 1.9 1.1 1.4 2.6 1.3 2.1 2.6 1.6 2.1 2.7
Recovery (%) 98.7 99.4 99.0 99.3 98.9 99.7 98.3 99.0 98.7 98.2 98.1 99.3 98.8 100.6 99.7
a
Figure 2. UV–visible absorption spectra of imatinib (A), dasatinib (B), nilotinib (C), gefitinib (D) and erlotinib (E). UV–visible absorption spectra of mixture of quantum dots b c d (QDs) and drugs. UV–visible absorption spectra of QDs. UV–visible absorption spectra of mixture of QDs and drugs (QDs as the blank solution). UV–visible absorption spectra of drugs.
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CuInS2/ZnS/TGA quantum dots and TKI Fourier transform infrared spectrometry. Infrared spectroscopies of CuInS2/ZnS/TGA QDs and five drugs as well as mixtures of QDs and drugs were observed to further confirm the structural changes. Figure 3(A–E) are infrared absorption spectrums of imatinib, dasatinib, nilotinib, gefitinib and erlotinib, respectively. And (a), (b) and (c) represent the infrared spectra of CuInS2/ZnS/TGA QDs, mixtures and drugs, respectively. Compared with (b) and (c), it can be speculated that peaks on spectra (c) at 3435 cm1 and 3339 cm1 (Fig. 3A), 3409 cm1 and 3246 cm1 (Fig. 3B), 3435 cm1 and 3259 cm1 (Fig. 3C) , 3385 cm1 (Fig. 3D) and 3259 cm1 (Fig. 3E) result from the stretching vibration of –NH–. However, there is only one peak at 3500 cm1 in spectra (b). The possible cause is the formation of the intermolecular hydrogen bond by interaction of –NH– with –COOH in thioglycolic acid around CuInS2/ZnS/TGA QDs. Hydrogen bonding also affects
vibration frequency and electron density, which leads to the reduction of drug absorption spectra in fingerprinting area. Theoretical basis. The different mechanisms of fluorescence quenching are usually classified as either dynamic quenching or static quenching (12). Dynamic quenching refers to the interaction process between the excited molecules of the fluorescent substance and the quencher. The quenching constant increases when the temperature rises. The decrease in intensity is described by the well known Stern–Volmer equation (13):
F0 ¼ 1 þ K q τ 0 ½C Q ¼ 1 þ K sv ½C Q F
(1)
a
Figure 3. Fourier transform infrared spectrometry of imatinib (A), dasatinib (B), nilotinib (C), gefitinib (D) and erlotinib (E). Fourier transform infrared spectrometry of quantum b c dots (QDs). Fourier transform infrared spectrometry of mixture of QDs and drugs. Fourier transform infrared spectrometry of drugs.
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S. Liao et al. F0 and F are the fluorescence intensities of CuInS2/ZnS/TGA QDs in the absence and presence of the quencher (TKI), respectively. [CQ] is the quencher concentration. KSV is the Stern-Volmer quenching constant reflecting the dose–effect relationship of dynamic equilibrium achieved by diffusion and collision between the fluorescent molecules and quenchers. Kq is the bimolecular quenching constant. Generally speaking, diffusion quenching constant is 1010 L mol1 s1 orders of magnitude (14). τ 0 is the unquenched lifetime and the average lifetime is usually 108 s. Static quenching refers to the complex reaction process of fluorescent molecules and quencher through intermolecular force. A nonluminous compound is generated in the ground state, which results in a decrease in fluorescence intensity. The quenching constant increases when the temperature declines. The Lineweaver–Burk double reciprocal equation can be used to describe this process (15): ðF0 – FÞ–1 ¼ F0 –1 þ ½CQ –1 F0 –1 ½CQ –1
(2)
5.0 298K
4.0
308K
F0/F
3.5
F0/F
Effect of temperature. The type of fluorescence quenching can be judged by the variation in temperature and fluorescence quenching constant. Dynamic quenching is caused by the interaction between quencher and excited-state molecules. The effective collision increases and the intermolecular electron transfers when the temperature rises. Thus the quenching constant increases, whilst static quenching is caused by complex reactions between the fluorescent molecular and the quencher. The generated compound is less stable and the quenching constant decreases when the temperature increases. The fluorescence quenching of CuInS2/ZnS/TGA QDs caused by TKI was obtained at 288 K, 298 K and 308 K (Fig. 4 and Table 3). The fluorescence quenching constant decreases when temperature rises. This is consistent with static quenching.
A
288K
4.5
KLB is the complex formation constant in static quenching process. It reflects the dose–effect relationship of equilibrium through the binding reaction between quencher and excitedstate molecules.
3.0 2.5 2.0 1.5 1.0 0
2
4
6
8
10
12
14
3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0
16
0
2
3.0
8
10
12
14
16
D
288K 298K 308K
4.5 4.0 3.5
2.2
F0/F
F0/F
2.4
6
5.0
C
288K 298K 308K
2.6
4
CDasatinib (X10-6mol/L)
CImatinib (X10-6mol/L)
2.8
B
288K 298K 308K
2.0 1.8
3.0 2.5 2.0
1.6
1.5
1.4
1.0
1.2 0
2
4
6
8
10
12
14
16
0
2
4
6
4.0
F0/F
3.0
10
12
14
16
18
E
288K 298K 308K
3.5
8
CGefitinib (X10-6mol/L)
CNilotinib (X10-6mol/L)
2.5 2.0 1.5 1.0 0
2
4 6 8 10 12 14 CErlotinib (X10-6mol/L)
16
18
Figure 4. Stern-Volmer equation at three different temperatures for imatinib (A), dasatinib (B), nilotinib (C), gefitinib (D) and erlotinib (E) and the concentration of CuInS2/ 5 1 ZnS/TGA quantum dots (QDs) is 7.81 × 10 mol L . Error bars represent standard deviations from three measurements and relative standard deviations (RSD) of tyrosine kinase inhibitors (TKIs).
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CuInS2/ZnS/TGA quantum dots and TKI Table 3. Linear equations and related parameters of fluorescence quenching by various drugs at different temperatures The pH
Drug
7.40
Imatinib
Temperature/K 288 298 308 288 298 308 288 298 308 288 298 308 288 298 308
Dasatinib
Nilotinib
Gefitinib
Erlotinib
Linear equation
Quenching Quenching rate Related constant Lmol–1 constant Lmol–1 s–1 parameters R2
F0/F – 1 = 2.0957 × 105[CQ] + 0.4383 F0/F – 1 = 1.5721 × 105[CQ] + 0.1256 F0/F – 1 = 1.0526 × 105[CQ] + 0.3338 F0/F – 1 = 1.1400 × 105[CQ] + 0.0310 F0/F – 1 = 8.6000 × 104[CQ] + 0.0420 F0/F – 1 = 3.1000 × 104[CQ] + 0.0880 F0/F – 1 = 8.8660 × 104[CQ] + 0.4967 F0/F – 1 = 6.8080 × 104[CQ] + 0.3626 F0/F – 1 = 4.8750 × 104[CQ] + 0.2560 F0/F – 1 = 2.2757 × 105[CQ] – 0.2900 F0/F – 1 = 1.6349 × 105[CQ] + 0.0114 F0/F – 1 = 4.3990 × 104[CQ] + 0.2497 F0/F – 1 = 1.6415 × 105[CQ] – 0.2362 F0/F – 1 = 1.0427 × 105[CQ] – 0.1506 F0/F – 1 = 6.9040 × 104[CQ] + 0.2111
2.0957 × 105 1.5721 × 105 1.0526 × 105 1.1400 × 105 8.6000 × 104 3.1000 × 104 8.8660 × 104 6.8080 × 104 4.8750 × 104 2.2757 × 105 1.6349 × 105 4.3990 × 104 1.6415 × 105 1.0427 × 105 6.9040 × 104
2.0957 × 1013 1.5721 × 1013 1.0526 × 1013 1.1400 × 1013 8.6000 × 1012 3.1000 × 1012 8.8660 × 1012 6.8080 × 1012 4.8750 × 1012 2.2757 × 1013 1.6349 × 1013 4.3990 × 1012 1.6415 × 1013 1.0427 × 1013 6.9040 × 1012
0.9972 0.9935 0.9813 0.9957 0.9980 0.9878 0.9979 0.9927 0.9859 0.9953 0.9966 0.9817 0.9879 0.9894 0.9821
the interaction between CuInS2/ZnS/TGA QDs and TKI. It is probable that an intermolecular hydrogen bond is formed by the –NH– in TKI and the –COOH in thioglycolic acid.
Fluorescence quenching mechanism clarification We clarified the quenching mechanism of the reaction as static quenching according to the above information. In general, the diffusion quenching constant is 1010 L mol1 s1. As can be seen from Table 4, the fluorescence quenching rate constant is above 1012 at physiological pH conditions. The fluorescence quenching constant decreased with increasing temperature. The ultraviolet–visible spectrum and differential spectrum also illustrate that the reaction belongs to static quenching. Typically, the mechanism of action between the QDs and drugs may be electrostatic interaction, hydrophobic interaction, energy transfer and the formation of new compounds (16). The ultraviolet–visible spectrum and the Fourier transform infrared spectra indicated that a new complex was formed by
Banding constant KA and binding sites n The binding constants and number of binding sites can be determined by modifying the equation of the static quenching process (17): lg
F0 F F
¼ lgK A þ n lgð½C Q Þ
(3)
KA is the binding constant of CuInS2/ZnS/TGA QDs with TKI and n is the number of binding sites per tyrosine kinase inhibitor
Table 4. Binding constants, the number of binding sites and thermodynamic parameters of interaction between CuInS2/ZnS/TGA QDs and TKI Drug
Temperature/K
Imatinib
Dasatinib
Nilotinib
Gefitinib
Erlotinib
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288 298 308 288 298 308 288 298 308 288 298 308 288 298 308
Binding constant/L mol–1 4.9000 × 105 2.2077 × 105 5.8247 × 104 5.3551 × 105 3.0249 × 105 8.5094 × 104 5.6576 × 105 1.7164 × 105 4.7242 × 104 2.1414 × 105 8.2054 × 104 1.3539 × 104 1.8056 × 105 8.6596 × 104 4.3122 × 104
Number of binding sites/n 1.1436 1.0938 1.0471 1.1928 1.1286 1.0679 1.1817 1.1321 1.0479 1.2504 1.1231 1.0968 1.2103 1.1912 1.0985
ΔH KJ mol–1 – –56.889 –101.677 – –40.755 –96.782 – –85.109 –98.448 – –68.447 –137.494 – –52.432 –53.204
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ΔS J mol–1 K–1 –88.601 –88.601 –238.896 –31.840 –31.842 –219.851 –185.392 –185.389 –230.153 –135.615 –135.614 –367.315 –81.424 –81.423 –84.016
ΔG KJ mol–1 –31.372 –30.486 –28.097 –31.585 –31.266 –29.068 –31.716 –29.863 –27.561 –29.390 –28.034 –24.361 –28.982 –28.168 –27.327
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S. Liao et al. molecule. The intercept and slope can be obtained by plotting a double logarithm regression curve of log [(F0 F)/F] versus log [Q] at different temperatures. Thus the number of binding sites n and binding constant KA can be calculated. As can be seen from Table 4, binding constants KA in the reaction between CuInS2/ZnS/TGA QDs and TKI are 104 and 105 orders of magnitude at 288 K, 298 K and 310 K, which indicates that the binding capacity of the above QDs and drugs is strong. Moreover, the binding constant KA decreases significantly with increasing temperature, which implies that the binding force between the CuInS2/ZnS/TGA QDs and TKI weakens as temperature increases. The number of binding sites n in Table 4 shows that there is only one binding site for the interaction between CuInS2/ZnS/TGA QDs and TKI. The main binding forces among the molecules are van der Waals force, electrostatic force, hydrophobic interaction and hydrogen bonding. From a thermodynamic point of view, the free energy change ΔG of the system determines the occurrence of spontaneous binding reaction. The increase in the entropy change ΔS or decrease in the enthalpy change ΔH, or both, are able to promote the binding reaction spontaneously (18,19). According to the relationship between the thermodynamic parameters and binding constant at different temperatures, ΔG, ΔS and ΔH can be calculated: ln ðk2 =k1 Þ ¼ ΔHð1=T1 – 1=T2 Þ=R ΔG ¼ RT lnk
Conclusion (4)
ΔS ¼ ðΔG– ΔHÞ=T
When the temperature changes slightly, the enthalpy ΔH can be regarded as a constant. Free energy change ΔG, entropy change ΔS and enthalpy change ΔH can be determined according to the relationship among thermodynamic parameters (Table 4). Ross et al. (20) hold the opinion: when ΔH > 0 and ΔS > 0, the force between molecules is hydrophobic interaction; when ΔH ≤ 0 and ΔS > 0, the force between molecules is electrostatic force; when ΔH < 0 and ΔS < 0, the force between molecules is van der Waals forces or hydrogen bonding. The free energy change of the interaction between CuInS2/ZnS/ TGA QDs and TKI is below zero based on the above equation. It indicates that the binding process is spontaneous. Due to the fact that ΔH < 0 and ΔS < 0, it can be inferred the force between CuInS2/ZnS/TGA QDs and TKI is mainly hydrogen bonding. In addition, the binding constants of imatinib, dasatinib and nilotinib are greater than that of gefitinib and erlotinib at the same temperature. There are two amino groups in imatinib, dasatinib, and nilotinib, one of which is the –NH– in the amide. However, there is only one amino group in gefitinib and erlotinib. With only one binding site, it is probable that a hydrogen bond is formed by the interaction between –NH– in the amide and carboxyl in the thioglycolic acid. All the results suggest that the electron-withdrawing ability of the oxygen atom is stronger than that of the nitrogen atom, and that the conjugation around the –NH– in amide is weaker than that in the non-amide. Thus electron density of the hydrogen atom of –NH– in the amide is weak and the hydrogen atom is easy to be exposed. Then the proton tends to form an intermolecular hydrogen bond with the carboxyl in the thioglycolic acid. The mechanism is shown in Fig. 5.
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Figure 5. A possible mechanism of interaction between CuInS2/ZnS/TGA quana b c tum dots (QDs) and tyrosine kinase inhibitor. Imatinib Dasatinib Nilotinib d e Gefitinib Erlotinib
A novel fluorescence method has been developed based on the selective quenching phenomenon of CuInS2/ZnS/TGA QDs induced by TKI. This method with good linearity, low detection limit and satisfactory recovery can be applied to the determination of TKI in API. As the APIs were of high purity, investigation of the additives’ influence was not included. Based on spectroscopic methods, it can be determined that a ground state compound is formed by CuInS2/ZnS/TGA QDs and TKI. Then the increased non-radiative transition of QDs leads to fluorescence quenching. The quenching constant decreases with increasing temperature, which is consistent with static quenching process. Calculated thermodynamic parameters infer that CuInS2/ZnS/ TGA QDs interact with TKI by hydrogen bonding force. Moreover, it is easy for the –NH– in amide of imatinib, dasatinib and nilotinib, rather than the –NH– in the non-amide of gefitinib and erlotinib to form a hydrogen bond with the carboxyl in the thioglycolic acid. This work is important to widen the investigation and application of CuInS2/ZnS/TGA QDs for drug analysis.
References 1. Liang JG, Huang S, Zeng DY, He Z, Ji X, Ai X, et al. CdSe quantum dots as luminescent probes for spironolactone determination. Talanta 2006;69:126–30. 2. Liao QG, Li YF, Huang CZ. CdS quantum dots as fluorescence probes for detection of adriamycin hydrochloride. Chem Res Chinese Univ 2007;23:138–42. 3. Liu SY, Hua JJ, Su XG. Detection of ascorbic acid and folic acid based on water-soluble CuInS2 quantum dots. Analyst 2012;137:4598–604. 4. Liu SY, Shi FP, Zhao XJ, Chen L, Su XG. 3-Aminophenyl boronic acidfunctionalized CuInS2 quantum dots as a near-infrared fluorescence probe for the determination of dopamine. Biosens Bioelectron 2013;47:379–84. 5. Liu ZP, Ma Q, Wang XY, Lin ZH, Zhang H, Liu LL, et al. A novel fluorescent nanosensor for detection of heparin and heparinase based on CuInS2 quantum dots. Biosens Bioelectron 2014;54:617–22.
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CuInS2/ZnS/TGA quantum dots and TKI 6. Qiao HC. Establishment of gefitinib tolerance PC9 cell line and correlation study of EGFR pathway signaling molecule phosphorylation level. Zhengzhou University 2012;1–2. 7. Liu ZK, Ai J, Geng MY. Research methods progress in field of tyrosine kinase inhibitor for antineoplastic. Prog Mod Biomed 2010;16:3134–71. 8. Bianchi F, Caffarri E, Cavalli S, Lagrasta C, Musci M, Quaini F, et al. Development and validation of an high performance liquid chromatography–tandem mass spectrometry method for the determination of imatinib in rat tissues. J Pharm Biomed 2013;73:103–7. 9. Vadera N, Subramanian G, Musmade P. Stability-indicating HPTLC determination of imatinib mesylate in bulk drug and pharmaceutical dosage form. J Pharm Biomed Anal 2007;43:722–6. 10. D’Avolio A, Simiele M, De Francia S, Ariaudo A, Baietto L, Cusato J, et al. HPLC-MS method for the simultaneous quantification of the antileukemia drugs imatinib, dasatinib and nilotinib in human peripheral blood mononuclear cell (PBMC). J Pharm Biomed Anal 2012;59:109–16. 11. Wang SS, Wang XT, Guo MM, Yu JS. Ligand effect on interaction between CdTe quantum dots and BSA selectively. J Chem 2012;33:1195–204. 12. Mátyus L, Szöllősi J, Jenei A. Steady-state fluorescence quenching applications for studying protein structure and dynamics. J Photochem Photobiol B 2006;83:223–36.
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13. Chen GZ, Huang XZ, Zhen ZZ. Fluorescence analytical methods. Beijing: Science Press 1990; 201 pp. in Chinese] 14. Qi ZD, Zhou B, Qi X, Chuan S, Liu Y, Dai J. Interaction of rofecoxib with human serum albumin: determination of binding constants and the binding site by spectroscopic methods. Photochem Photobiol A 2008;193:81–8. 15. Luo KL, Zhang Q, Ren LY. Study on the interaction of nano-TiO2 and BSA with fluorescence. Anqing Teachers Coll (Nat Sci Edit) 2009;15:55–7. 16. Chan WCW, Maxwell DJ, Gao XH, Bailey RE, Han MY, Nie SM. Luminescent quantum dots for multiplexed biological detection and imaging. Curr Opin Biotechnol 2002;13:40–6. 17. Bi SY, Song DQ, Tian Y, Zhou X, Liu ZY, Zhang HQ. Molecular spectroscopic study on the interaction of tetracyclines with serum albumins. Spectrochim Acta A Mol Biomol Spectrosc 2005;61:629–36. 18. Shangguan YF, Yan CN, Feng ZY. Study on characteristics of reaction between diphenyl(2-chlorophenyl)methanol and bovine serum albumin. Instrum A 2004;23:32–5. 19. Shao S, Ma BY, Wang XJ, Zhang JJ, Li XF, Qin Q. Study on reaction between cefodizime sodium and bovine serum albumin. Acta Phys Chim Sin 2005;21:792–5. 20. Ross DP, Subramanian S. Thermodynamics of protein association reactions: contributing to stability. Biochemistry 1981;20:3096–9.
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Revised: 14 June 2014,
Accepted: 16 June 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.2740
The interaction of CuInS2/ZnS/TGA quantum dots with tyrosine kinase inhibitor and its application Shenghua Liao,a,b Yu. Huang,a,b Jiaxin Zuoa,b and Zhengyu Yana,b* ABSTRACT: The interactions between thioglycolic acid-capped-CuInS2/ZnS quantum dots (CuInS2/ZnS/TGA QDs) and tyrosine kinase inhibitor (TKI) were investigated using fluorescence, ultraviolet–visible spectrometry and Fourier transform infrared spectrometry. The results indicated that the fluorescence intensity of CuInS2/ZnS/TGA could be quenched by imatinib, dasatinib, nilotinib, gefitinib and erlotinib, which hinted that CuInS2/ZnS/TGA QDs could be used in the detection of TKI in active pharmaceutical ingredients (API). Calibration curves showed good linear correlation and low detection limits. The average recovery was between 98 and 102%. Moreover, the nature of the fluorescence quenching mechanism of CuInS2/ ZnS/TGA QDs by TKI was discussed. A ground state complex was formed by hydrogen bonding between the carboxyl group of CuInS2/ZnS/TGA QDs and the amino group of TKI. This led to an increase in non-radiative transition and fluorescence quenching of CuInS2/ZnS/TGA QDs. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: thioglycolic acid-capped-CuInS2/ZnS quantum dots; tyrosine kinase inhibitor; interaction; content determination; intermolecular hydrogen bond
Introduction Quantum dots (QDs) have attracted considerable attention in the chemistry, biology and medicine fields with many unique photo-electronic characteristics, such as broad absorption spectra, narrow symmetric emission bands and high photostability. Recently, the surface modifier of QD bonding with certain drugs has been shown to affect fluorescence intensity, which made it applicable for the determination and tracing of drugs based on a linear or exponential relationship between concentration and fluorescence intensity. For example, Liang et al. detected spironolactone based on the fluorescence quenching of CdSe QDs by spironolactone (1) and Liao et al. established a determination method for doxorubicin using a thioglycolic acid (TGA)-functionalized CdS QD fluorescence probe (2). However, these QDs containing Cd pose potential risks to human health and the environment. In order to apply low toxicity QDs in drug assays, I–III–VI2 semiconductor nanocrystals were developed. Su et al. synthesized mercapto propionic acid (MPA)-capped CuInS2 ternary QDs for the detection of ascorbic acid and folic acid (3). Recently, they have prepared mercaptopropionic acid-capped CuInS2 QDs as a near-infrared fluorescence probe for the determination of dopamine, as well as L-cysteine modified CuInS2 QDs for heparin and heparinase determination (4,5). The advantages of the high fluorescence intensity and low toxicity of ternary QDs have encouraged the development of fluorescence probes for drug content determination. Tyrosine kinase inhibitor (TKI) is mainly used for targeted therapy of tumors. According to the protein molecules or gene fragments in the tumor cells, targeted drugs are designed at the cellular and molecular level. These drugs can bind to carcinogenic sites specifically so the tumor cells are killed while the peripheral normal tissues remain intact (6). The possible mechanisms are as follows: inhibition of tumor cell repairing, blocking of cell
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division in the G1 phase, induction and maintenance of apoptosis, formation of anti-angiogenesis, etc. Currently, the main analytical methods for TKI are isotopic analysis, fluorescence polarization immunoassay, fluorescence resonance energy transfer, microbead technology, microfluidic technology, enzyme-linked immunosorbent assay and dissociation enhanced lanthanide fluorescence immunoassay (7). High performance liquid chromatography and high performance liquid chromatography– mass spectrometry are also used for determination (8–10). Although these methods are of high sensitivity, the use of complex operations and expensive equipment hinders their popularization. The detection of TKI using CuInS2/ZnS/TGA QDs based on fluorescence quenching has not yet been reported. This method was applied to determine TKI for the first time with satisfactory results. Under physiological pH conditions, this method encompases high accuracy and sensitivity, a low detection limit, a wide linear range and a strong anti-interference ability. In addition, the interaction between CuInS2/ZnS/TGA QDs and TKI was investigated and a possible mechanism was discussed.
* Correspondence to: Z. Yan, Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, Nanjing 211198, China. Tel: +86 13512503905; Fax: +86 25 86185170. E-mail: [email protected] a
Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, Nanjing 211198, China
b
Department of Analytical Chemistry, China Pharmaceutical University, Nanjing 211198, China Abbreviations: API, active pharmaceutical ingredient; QD, quantom dots; RSD, relative standard deviation; TKI, tyrosine kinase inhibitor.
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S. Liao et al.
Experimental Apparatus and agents The absorption spectrum was acquired using a UV2100 UV–vis spectrometer (Shimadzu, Japan). The fluorescence measurements were made with a RF-5301 spectrofluorophotometer (Shimadzu). All pH measurements were made using a Model pH S-25 (Leici Equipment Factory, Shanghai, China). All infrared measurements were acquired by Fourier transform infrared spectroscopy (Shimadzu FTIR-8400S). AgI (99%), InI3 (99.999%), bis(trimethylsilyl)sulfide [(Me3Si)2S] (98%), mercaptoacetic acid (TGA) and tri-n-octylphosphine (TOP) (90%) were purchased from Alfa Aesar. Oleyl amine (OLA) (80–90%) was purchased from Acros Organics. These chemicals were used without further purification. Methanol and ethanol were purchased from Jiangsu Hanbang Technology Co. (Jiangsu, China). Trihydroxymethyl aminomethan and anhydrous sodium acetate were all purchased from Sinopharm Chemical Reagent Co. Ltd. Disodium hydrogen phosphate and sodium dihydrogen phosphate were purchased from Nanjing Chemical Reagent Co. (Nanjing, China). Hydrochloric acid was purchased from Nanjing Chemical Reagent Co. (Nanjing, China). Among the above, methanol was of chromatography reagent grade, trihydroxymethyl aminomethane was biochemical reagent, other reagents were of analytical reagent grade. Imatinib, dasatinib, nilotinib, gefitinib and erlotinib were provided by the Department of Analytical Chemistry, China Pharmaceutical University.
Experimental procedures The CuInS2/ZnS/TGA QDs were prepared as follows: 0.05 mM CuI in 1.5 mL OLA and 0.10 mM InI3 in 3mL TOP were prepared under vacuum and loaded into a degassed three-necked flask. The mixture was heated at 150°C under magnetic stirring for several minutes until transparent. Subsequently, 0.15 mM (Me3Si)2S in 2 mL TOP was injected swiftly and reacted for 3 min at the temperature. The resulting solution was quickly cooled down to 80°C and the ZnS precursor was injected drop by drop, and had been prepared by mixing zinc stearate (0.8 mM) in 1.5 mL OLA. Then the solution was heated to 180°C and maintained for 1 h. The thus-prepared CuInS2/ZnS QDs were precipitated by adding methanol and n-butanol and centrifuged. The supernatant containing unreacted material was discarded and the remaining precipitates were dispersed in chloroform. To form a –COOH functional group, CuInS2/ZnS QDs and TGA (5mM) were mixed at the ratio of 1:1 and vortexed for 3 min followed by the addition of 0.5 mL NaOH solution and another vortex. Finally, the supernatant was precipitated by adding ethanol. TKI stock solutions were prepared as follows: appropriate amounts of imatinib, dasatinib, nilotinib, gefitinib and erlotinib were weighed, respectively. Imatinib and the other four reagents were dissolved in a small amount of water and methanol, respectively. All solutions were ultrasounded for a few minutes and cooled to room temperature. Five stock solutions at a concentration of 5.00 × 104 mol L1 were prepared by diluting to volume with the corresponding solvent. The CuInS2/ZnS/TGA QDs that precipitated after centrifugation were dissolved to a concentration of 7.81 × 105 mol L1 with trihydroxymethyl aminomethane–HCl solution at pH 7.40 (the concentration of QDs were calculated by the concentration
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of Cu2+); 3.00 mL CuInS2/ZnS/TGA QDs were pipetted precisely. The fluorescence intensity F0 of QDs was recorded with a 1 cm quartz cell. Then, 10 μL of imatinib, dasatinib, nilotinib, gefitinib and erlotinib stock solutions were added to the CuInS2/ZnS/TGA QDs successively and respectively to measure the fluorescence intensity (F) with the following settings on the fluorescence spectrophotometer (excitation wavelength (ex) 350 nm, slit width 5 nm and scan range is 420–680 nm).
Results and discussion Fluorescence quenching In accordance with this method, a designated amount of imatinib, dasatinib, nilotinib, gefitinib and erlotinib reference solutions were added successively and respectively to CuInS2/ZnS /TGA QD solution at certain concentrations and under optimum conditions. A linear relationship between the quenched fluorescence intensity of the CuInS2/ZnS /TGA QDs and the concentration of TKI was observed (Fig. 1 and Table 1). The correlation coefficient was close to 1 and the limit of detection was low within the measured concentration range. Therefore, the proposed method was proved to be sensitive.
Detection of TKI in API Three different concentrations of imatinib, dasatinib, nilotinib, gefitinib and erlotinib solutions were prepared for the determination of the sample concentration. Then the same amounts of the reference solutions were added respectively six times. The recovery and relative standard deviation (RSD) of each drug were calculated. From Table 2 it can be seen that the average recovery is between 98 and 102% and the RSD is less than 3%, which met the requirement for microanalysis. Therefore, the proposed method was proved to be reliable.
Interaction between CuInS2/ZnS/TGA QDs and TKI Interaction force There are many p–π and π–π conjugated systems in TKI. The stretching vibrations of –NH– were very obvious in the infrared spectra. It can be inferred that the interaction from the intermolecular hydrogen bond was formed by the interaction of –NH– and –COOH in thioglycolic acid.
Fluorescence quenching mechanism UV–visible absorption spectra. Interactions between CuInS2/ ZnS/TGA QDs and TKI were investigated under optimum conditions with UV–visible absorption spectra. As shown in Fig. 2, the absorbance values changed when the drugs were mixed with QDs. For imatinib, dasatinib, nilotinib, gefitinib and erlotinib, the red shift was 11 nm ( from 254 nm to 265 nm), 12 nm (from 316 nm to 328 nm), 13nm (from 260 nm to 273 nm), 12 nm (from 331 nm to 343 nm) and 10 nm (from 334 nm to 344 nm), respectively. These results indicated that drugs interacted with the QDs. The red shift of the absorption spectra demonstrated that a new compound was generated (11).
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CuInS2/ZnS/TGA quantum dots and TKI
Figure 1. The fluorescence spectra of CuInS2/ZnS/TGA quantum dots (QDs) in the presence of various concentrations of imatinib (A), dasatinib (B), nilotinib (C), gefitinib (D) 5 1 and erlotinib (E). The concentration of CuInS2/ZnS /TGA QDs was 7.81 × 10 mol L , from top to bottom, the concentrations of imatinib were 0.000, 0.125, 0.500, 2.50, 20.0, 6 1 6 1 50.0, 100, 125 × 10 mol L , the concentrations of dasatinib were 0.000, 0.150, 0.600, 3.00, 24.0, 60.0, 90.0, 120 × 10 mol L , the concentrations of nilotinib were 0.000, 6 1 6 1 0.875, 1.75, 3.50, 17.5, 35.0, 70.0, 100 × 10 mol L , the concentrations of gefitinib were 0.000, 0.150, 0.600, 3.00, 12.0, 30.0, 60.0, 100 × 10 mol L , the concentrations of 6 1 erlotinib were 0.000, 0.250, 0.500, 2.50, 10.0, 50.0, 100, 120 × 10 mol L , respectively. The inset shows the plot F0/F – 1 versus concentration of imatinib (A), dasatinib (B), nilotinib (C), gefitinib (D) and erlotinib (E). Error bars represent standard deviations from three measurements and relative standard deviations (RSD) of tyrosine kinase inhibitor (TKIs).
Table 1. Linear regression equations for determination of tyrosine kinase inhibitors Tyrosine kinase inhibitors
Linear regression equations (106 mol L1)
Imatinib Dasatinib Nilotinib Gefitinib Erlotinib
F0/F – F0/F – F0/F – F0/F – F0/F –
1 = 0.0119c + 0.1215 1 = 0.0274c + 0.1954 1 = 0.0354c + 0.0534 1 = 0.0579c + 0.1274 1 = 0.0662c + 0.1864
Correlation coefficient (r)
Linear range (106 mol L1)
Limit of detectiona (106 mol L1)
0.9994 0.9977 0.9981 0.9989 0.9991
0.125–125 0.150–120 0.875–100 0.150–100 0.250–120
1.800 × 103 3.300 × 103 6.500 × 103 3.700 × 103 4.300 × 103
Limit of detection is defined by the equation LOD = 3σ/k, where σ is the standard deviation of the measurement of blank measurements (n = 10) and k is the slope of the calibration graph.
a
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S. Liao et al. Table 2. Recovery rate (n = 6) for the determination of tyrosine kinase inhibitor Name Imatinib
Dasatinib
Nilotinib
Gefitinib
Erlotinib
Sample
Found value (106 mol L1)
Added reference solution (106 mol L1)
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
3.04 4.74 7.15 2.87 4.98 6.92 3.11 4.95 7.03 3.02 5.39 6.62 3.07 5.16 6.77
3.10 3.10 3.10 2.98 2.98 2.98 3.00 3.00 3.00 2.75 2.75 2.75 3.26 3.26 3.26
Found total value (106 mol L1) 6.10 7.82 10.22 5.83 7.93 9.89 6.06 7.92 9.99 5.72 8.09 9.35 6.29 8.44 10.02
RSD% (n = 6) 1.5 1.8 2.2 1.2 1.7 1.9 1.1 1.4 2.6 1.3 2.1 2.6 1.6 2.1 2.7
Recovery (%) 98.7 99.4 99.0 99.3 98.9 99.7 98.3 99.0 98.7 98.2 98.1 99.3 98.8 100.6 99.7
a
Figure 2. UV–visible absorption spectra of imatinib (A), dasatinib (B), nilotinib (C), gefitinib (D) and erlotinib (E). UV–visible absorption spectra of mixture of quantum dots b c d (QDs) and drugs. UV–visible absorption spectra of QDs. UV–visible absorption spectra of mixture of QDs and drugs (QDs as the blank solution). UV–visible absorption spectra of drugs.
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CuInS2/ZnS/TGA quantum dots and TKI Fourier transform infrared spectrometry. Infrared spectroscopies of CuInS2/ZnS/TGA QDs and five drugs as well as mixtures of QDs and drugs were observed to further confirm the structural changes. Figure 3(A–E) are infrared absorption spectrums of imatinib, dasatinib, nilotinib, gefitinib and erlotinib, respectively. And (a), (b) and (c) represent the infrared spectra of CuInS2/ZnS/TGA QDs, mixtures and drugs, respectively. Compared with (b) and (c), it can be speculated that peaks on spectra (c) at 3435 cm1 and 3339 cm1 (Fig. 3A), 3409 cm1 and 3246 cm1 (Fig. 3B), 3435 cm1 and 3259 cm1 (Fig. 3C) , 3385 cm1 (Fig. 3D) and 3259 cm1 (Fig. 3E) result from the stretching vibration of –NH–. However, there is only one peak at 3500 cm1 in spectra (b). The possible cause is the formation of the intermolecular hydrogen bond by interaction of –NH– with –COOH in thioglycolic acid around CuInS2/ZnS/TGA QDs. Hydrogen bonding also affects
vibration frequency and electron density, which leads to the reduction of drug absorption spectra in fingerprinting area. Theoretical basis. The different mechanisms of fluorescence quenching are usually classified as either dynamic quenching or static quenching (12). Dynamic quenching refers to the interaction process between the excited molecules of the fluorescent substance and the quencher. The quenching constant increases when the temperature rises. The decrease in intensity is described by the well known Stern–Volmer equation (13):
F0 ¼ 1 þ K q τ 0 ½C Q ¼ 1 þ K sv ½C Q F
(1)
a
Figure 3. Fourier transform infrared spectrometry of imatinib (A), dasatinib (B), nilotinib (C), gefitinib (D) and erlotinib (E). Fourier transform infrared spectrometry of quantum b c dots (QDs). Fourier transform infrared spectrometry of mixture of QDs and drugs. Fourier transform infrared spectrometry of drugs.
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S. Liao et al. F0 and F are the fluorescence intensities of CuInS2/ZnS/TGA QDs in the absence and presence of the quencher (TKI), respectively. [CQ] is the quencher concentration. KSV is the Stern-Volmer quenching constant reflecting the dose–effect relationship of dynamic equilibrium achieved by diffusion and collision between the fluorescent molecules and quenchers. Kq is the bimolecular quenching constant. Generally speaking, diffusion quenching constant is 1010 L mol1 s1 orders of magnitude (14). τ 0 is the unquenched lifetime and the average lifetime is usually 108 s. Static quenching refers to the complex reaction process of fluorescent molecules and quencher through intermolecular force. A nonluminous compound is generated in the ground state, which results in a decrease in fluorescence intensity. The quenching constant increases when the temperature declines. The Lineweaver–Burk double reciprocal equation can be used to describe this process (15): ðF0 – FÞ–1 ¼ F0 –1 þ ½CQ –1 F0 –1 ½CQ –1
(2)
5.0 298K
4.0
308K
F0/F
3.5
F0/F
Effect of temperature. The type of fluorescence quenching can be judged by the variation in temperature and fluorescence quenching constant. Dynamic quenching is caused by the interaction between quencher and excited-state molecules. The effective collision increases and the intermolecular electron transfers when the temperature rises. Thus the quenching constant increases, whilst static quenching is caused by complex reactions between the fluorescent molecular and the quencher. The generated compound is less stable and the quenching constant decreases when the temperature increases. The fluorescence quenching of CuInS2/ZnS/TGA QDs caused by TKI was obtained at 288 K, 298 K and 308 K (Fig. 4 and Table 3). The fluorescence quenching constant decreases when temperature rises. This is consistent with static quenching.
A
288K
4.5
KLB is the complex formation constant in static quenching process. It reflects the dose–effect relationship of equilibrium through the binding reaction between quencher and excitedstate molecules.
3.0 2.5 2.0 1.5 1.0 0
2
4
6
8
10
12
14
3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0
16
0
2
3.0
8
10
12
14
16
D
288K 298K 308K
4.5 4.0 3.5
2.2
F0/F
F0/F
2.4
6
5.0
C
288K 298K 308K
2.6
4
CDasatinib (X10-6mol/L)
CImatinib (X10-6mol/L)
2.8
B
288K 298K 308K
2.0 1.8
3.0 2.5 2.0
1.6
1.5
1.4
1.0
1.2 0
2
4
6
8
10
12
14
16
0
2
4
6
4.0
F0/F
3.0
10
12
14
16
18
E
288K 298K 308K
3.5
8
CGefitinib (X10-6mol/L)
CNilotinib (X10-6mol/L)
2.5 2.0 1.5 1.0 0
2
4 6 8 10 12 14 CErlotinib (X10-6mol/L)
16
18
Figure 4. Stern-Volmer equation at three different temperatures for imatinib (A), dasatinib (B), nilotinib (C), gefitinib (D) and erlotinib (E) and the concentration of CuInS2/ 5 1 ZnS/TGA quantum dots (QDs) is 7.81 × 10 mol L . Error bars represent standard deviations from three measurements and relative standard deviations (RSD) of tyrosine kinase inhibitors (TKIs).
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CuInS2/ZnS/TGA quantum dots and TKI Table 3. Linear equations and related parameters of fluorescence quenching by various drugs at different temperatures The pH
Drug
7.40
Imatinib
Temperature/K 288 298 308 288 298 308 288 298 308 288 298 308 288 298 308
Dasatinib
Nilotinib
Gefitinib
Erlotinib
Linear equation
Quenching Quenching rate Related constant Lmol–1 constant Lmol–1 s–1 parameters R2
F0/F – 1 = 2.0957 × 105[CQ] + 0.4383 F0/F – 1 = 1.5721 × 105[CQ] + 0.1256 F0/F – 1 = 1.0526 × 105[CQ] + 0.3338 F0/F – 1 = 1.1400 × 105[CQ] + 0.0310 F0/F – 1 = 8.6000 × 104[CQ] + 0.0420 F0/F – 1 = 3.1000 × 104[CQ] + 0.0880 F0/F – 1 = 8.8660 × 104[CQ] + 0.4967 F0/F – 1 = 6.8080 × 104[CQ] + 0.3626 F0/F – 1 = 4.8750 × 104[CQ] + 0.2560 F0/F – 1 = 2.2757 × 105[CQ] – 0.2900 F0/F – 1 = 1.6349 × 105[CQ] + 0.0114 F0/F – 1 = 4.3990 × 104[CQ] + 0.2497 F0/F – 1 = 1.6415 × 105[CQ] – 0.2362 F0/F – 1 = 1.0427 × 105[CQ] – 0.1506 F0/F – 1 = 6.9040 × 104[CQ] + 0.2111
2.0957 × 105 1.5721 × 105 1.0526 × 105 1.1400 × 105 8.6000 × 104 3.1000 × 104 8.8660 × 104 6.8080 × 104 4.8750 × 104 2.2757 × 105 1.6349 × 105 4.3990 × 104 1.6415 × 105 1.0427 × 105 6.9040 × 104
2.0957 × 1013 1.5721 × 1013 1.0526 × 1013 1.1400 × 1013 8.6000 × 1012 3.1000 × 1012 8.8660 × 1012 6.8080 × 1012 4.8750 × 1012 2.2757 × 1013 1.6349 × 1013 4.3990 × 1012 1.6415 × 1013 1.0427 × 1013 6.9040 × 1012
0.9972 0.9935 0.9813 0.9957 0.9980 0.9878 0.9979 0.9927 0.9859 0.9953 0.9966 0.9817 0.9879 0.9894 0.9821
the interaction between CuInS2/ZnS/TGA QDs and TKI. It is probable that an intermolecular hydrogen bond is formed by the –NH– in TKI and the –COOH in thioglycolic acid.
Fluorescence quenching mechanism clarification We clarified the quenching mechanism of the reaction as static quenching according to the above information. In general, the diffusion quenching constant is 1010 L mol1 s1. As can be seen from Table 4, the fluorescence quenching rate constant is above 1012 at physiological pH conditions. The fluorescence quenching constant decreased with increasing temperature. The ultraviolet–visible spectrum and differential spectrum also illustrate that the reaction belongs to static quenching. Typically, the mechanism of action between the QDs and drugs may be electrostatic interaction, hydrophobic interaction, energy transfer and the formation of new compounds (16). The ultraviolet–visible spectrum and the Fourier transform infrared spectra indicated that a new complex was formed by
Banding constant KA and binding sites n The binding constants and number of binding sites can be determined by modifying the equation of the static quenching process (17): lg
F0 F F
¼ lgK A þ n lgð½C Q Þ
(3)
KA is the binding constant of CuInS2/ZnS/TGA QDs with TKI and n is the number of binding sites per tyrosine kinase inhibitor
Table 4. Binding constants, the number of binding sites and thermodynamic parameters of interaction between CuInS2/ZnS/TGA QDs and TKI Drug
Temperature/K
Imatinib
Dasatinib
Nilotinib
Gefitinib
Erlotinib
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288 298 308 288 298 308 288 298 308 288 298 308 288 298 308
Binding constant/L mol–1 4.9000 × 105 2.2077 × 105 5.8247 × 104 5.3551 × 105 3.0249 × 105 8.5094 × 104 5.6576 × 105 1.7164 × 105 4.7242 × 104 2.1414 × 105 8.2054 × 104 1.3539 × 104 1.8056 × 105 8.6596 × 104 4.3122 × 104
Number of binding sites/n 1.1436 1.0938 1.0471 1.1928 1.1286 1.0679 1.1817 1.1321 1.0479 1.2504 1.1231 1.0968 1.2103 1.1912 1.0985
ΔH KJ mol–1 – –56.889 –101.677 – –40.755 –96.782 – –85.109 –98.448 – –68.447 –137.494 – –52.432 –53.204
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ΔS J mol–1 K–1 –88.601 –88.601 –238.896 –31.840 –31.842 –219.851 –185.392 –185.389 –230.153 –135.615 –135.614 –367.315 –81.424 –81.423 –84.016
ΔG KJ mol–1 –31.372 –30.486 –28.097 –31.585 –31.266 –29.068 –31.716 –29.863 –27.561 –29.390 –28.034 –24.361 –28.982 –28.168 –27.327
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S. Liao et al. molecule. The intercept and slope can be obtained by plotting a double logarithm regression curve of log [(F0 F)/F] versus log [Q] at different temperatures. Thus the number of binding sites n and binding constant KA can be calculated. As can be seen from Table 4, binding constants KA in the reaction between CuInS2/ZnS/TGA QDs and TKI are 104 and 105 orders of magnitude at 288 K, 298 K and 310 K, which indicates that the binding capacity of the above QDs and drugs is strong. Moreover, the binding constant KA decreases significantly with increasing temperature, which implies that the binding force between the CuInS2/ZnS/TGA QDs and TKI weakens as temperature increases. The number of binding sites n in Table 4 shows that there is only one binding site for the interaction between CuInS2/ZnS/TGA QDs and TKI. The main binding forces among the molecules are van der Waals force, electrostatic force, hydrophobic interaction and hydrogen bonding. From a thermodynamic point of view, the free energy change ΔG of the system determines the occurrence of spontaneous binding reaction. The increase in the entropy change ΔS or decrease in the enthalpy change ΔH, or both, are able to promote the binding reaction spontaneously (18,19). According to the relationship between the thermodynamic parameters and binding constant at different temperatures, ΔG, ΔS and ΔH can be calculated: ln ðk2 =k1 Þ ¼ ΔHð1=T1 – 1=T2 Þ=R ΔG ¼ RT lnk
Conclusion (4)
ΔS ¼ ðΔG– ΔHÞ=T
When the temperature changes slightly, the enthalpy ΔH can be regarded as a constant. Free energy change ΔG, entropy change ΔS and enthalpy change ΔH can be determined according to the relationship among thermodynamic parameters (Table 4). Ross et al. (20) hold the opinion: when ΔH > 0 and ΔS > 0, the force between molecules is hydrophobic interaction; when ΔH ≤ 0 and ΔS > 0, the force between molecules is electrostatic force; when ΔH < 0 and ΔS < 0, the force between molecules is van der Waals forces or hydrogen bonding. The free energy change of the interaction between CuInS2/ZnS/ TGA QDs and TKI is below zero based on the above equation. It indicates that the binding process is spontaneous. Due to the fact that ΔH < 0 and ΔS < 0, it can be inferred the force between CuInS2/ZnS/TGA QDs and TKI is mainly hydrogen bonding. In addition, the binding constants of imatinib, dasatinib and nilotinib are greater than that of gefitinib and erlotinib at the same temperature. There are two amino groups in imatinib, dasatinib, and nilotinib, one of which is the –NH– in the amide. However, there is only one amino group in gefitinib and erlotinib. With only one binding site, it is probable that a hydrogen bond is formed by the interaction between –NH– in the amide and carboxyl in the thioglycolic acid. All the results suggest that the electron-withdrawing ability of the oxygen atom is stronger than that of the nitrogen atom, and that the conjugation around the –NH– in amide is weaker than that in the non-amide. Thus electron density of the hydrogen atom of –NH– in the amide is weak and the hydrogen atom is easy to be exposed. Then the proton tends to form an intermolecular hydrogen bond with the carboxyl in the thioglycolic acid. The mechanism is shown in Fig. 5.
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Figure 5. A possible mechanism of interaction between CuInS2/ZnS/TGA quana b c tum dots (QDs) and tyrosine kinase inhibitor. Imatinib Dasatinib Nilotinib d e Gefitinib Erlotinib
A novel fluorescence method has been developed based on the selective quenching phenomenon of CuInS2/ZnS/TGA QDs induced by TKI. This method with good linearity, low detection limit and satisfactory recovery can be applied to the determination of TKI in API. As the APIs were of high purity, investigation of the additives’ influence was not included. Based on spectroscopic methods, it can be determined that a ground state compound is formed by CuInS2/ZnS/TGA QDs and TKI. Then the increased non-radiative transition of QDs leads to fluorescence quenching. The quenching constant decreases with increasing temperature, which is consistent with static quenching process. Calculated thermodynamic parameters infer that CuInS2/ZnS/ TGA QDs interact with TKI by hydrogen bonding force. Moreover, it is easy for the –NH– in amide of imatinib, dasatinib and nilotinib, rather than the –NH– in the non-amide of gefitinib and erlotinib to form a hydrogen bond with the carboxyl in the thioglycolic acid. This work is important to widen the investigation and application of CuInS2/ZnS/TGA QDs for drug analysis.
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