Accepted Manuscript Fluorescence resonance energy transfer between ZnSe-ZnS quantum dots and bovine serum albumin in bioaffinity assays of anticancer drugs Chang Shu, Li Ding, Wenying Zhong PII: DOI: Reference:

S1386-1425(14)00583-6 http://dx.doi.org/10.1016/j.saa.2014.04.029 SAA 11993

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

20 December 2013 23 March 2014 6 April 2014

Please cite this article as: C. Shu, L. Ding, W. Zhong, Fluorescence resonance energy transfer between ZnSe-ZnS quantum dots and bovine serum albumin in bioaffinity assays of anticancer drugs, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.04.029

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1

Fluorescence resonance energy transfer between ZnSe-ZnS quantum dots and bovine serum

2

albumin in bioaffinity assays of anticancer drugs

3

Chang Shua, Li Ding b, Wenying Zhong

4

a

5

Pharmaceutical University, Nanjing, 210009, PR China

＊

＊a

Department of Analytical Chemistry,

b

Department of Pharmaceutical Analysis, China

6 7

ABSTRACT:

8

In the current work, using ZnSe-ZnS quantum dots (QDs) as representative nanoparticles, the

9

affinities of seven anticancer drugs for bovine serum albumin (BSA) were studied using

10

fluorescence resonance energy transfer (FRET). The FRET efficiency of BSA-QD conjugates can

11

reach as high as 24.87% by electrostatic interaction. The higher binding constant (3.63 × 107 L

12

mol-1) and number of binding sites (1.75) between ZnSe-ZnS QDs and BSA demonstrated that the

13

QDs could easily associate to plasma proteins and enhance the transport efficacy of drugs. The

14

magnitude of binding constants (103–106 L mol-1), in the presence of QDs, was between

15

drugs–BSA and drugs–QDs in agreement with common affinities of drugs for serum albumins

16

(104–106 L mol-1) in vivo. ZnSe-ZnS QDs significantly increased the affinities for BSA of

17

Vorinostat （ SAHA ） , Docetaxel (DOC), Carmustine (BCNU), Doxorubicin (Dox ） and

18

10-Hydroxycamptothecin（HCPT）. However, they slightly reduced the affinities of Vincristine

19

(VCR) and Methotrexate (MTX) for BSA. The recent work will not only provide useful

20

information for appropriately understanding the binding affinity and binding mechanism at the

21

molecular level, but also illustrate the ZnSe-ZnS QDs are perfect candidates for nanoscal drug

22

delivery system (DDS).

23

Keywords: ZnSe-ZnS quantum dot; Anticancer drug; Bovine serum albumin; Fluorescence

24

resonance energy transfer; Bioaffinity

25 26

1. Introduction

27

Recently nanoscale drug delivery has attracted increasing international attention owing to

a

b

Corresponding author. Tel.: +86 025-86185217; fax: +86 025-86185517. E-mail address: [email protected].(W.Y. Zhong) Corresponding author. Tel.: +86 025-83271289; fax: +86 025-83271289. E-mail address:[email protected] (L. Ding).

1

1

their biological application [1]. Due to their relatively large surface area, they have significant

2

capacities to attach to other molecules such as drugs, polymers and proteins by physical action and

3

chemical action [2]. Quantum dots(QDs), excellent luminescent nanoparticles, could potentially be

4

used for photovoltaic drug devices and visual pharmaceutical analysis in vivo [3]. One of the most

5

important advantages of QDs in nanomedicine over current techniques is their ability to easily

6

track cells in vivo without the need to sacrifice animals. Most previous works about QDs had been

7

focused on their candidates for biomolecule or drug delivery applications by some research groups

8

based on their versatile properties, fluorescence tracing [4], nanoparticles size modification [5],

9

cross-linking and self-assembly [6], cellular uptake enhance [7], fluorescence resonance energy

10

transfer (FRET) and so on. FRET mentioned above occurs when the electronic excitation energy

11

of a donor chromophore is transferred to an acceptor molecule nearby via a through-space

12

dipole–dipole interaction between the donor–acceptor pair [8]. The FRET process is more efficient

13

when there is an appreciable overlap between the emission spectrum of the donor and the

14

absorption spectrum of the acceptor. The strong distance dependence of the FRET efficiency has

15

been widely exploited in studying the structure and dynamics of proteins and nucleic acids, in the

16

detection and visualization of intermolecular association, in the development of intermolecular

17

binding assays, and in monitoring absorption and action site of drugs [9-11]. Compared with

18

conventional chemical analysis, FRET-based analytical method has higher sensitivity and more

19

simplicity in detection of ligand–receptor binding by observing merely the enhanced fluorescence

20

of the acceptor [12].

21

The interaction between drugs and serum albumins has important effects on the absorption,

22

distribution, free concentration, and metabolism of drugs in blood [13]. The degree of binding to

23

albumin may have consequence for the drugs delivery and distribution to the cells and tissues.

24

Reversible drug-protein interactions are expected to modulate the bioavailability and

25

pharmacological effects of drugs. If a molecule is weakly bound to plasma proteins, the transport

26

of drug in blood may be significantly reduced and the drug efficacy consequently weak [14].

27

Determining the level of drugs bound with plasma proteins in the presence and absence of

28

nanoparticles is critical because it will directly correlate with carrier efficacy [15]. However, very

29

little information has focused on whether or not the nanoparticles in vivo affect the interaction

30

between small drugs molecules and serum albumin in blood. 2

1

Using ZnSe-ZnS QDs as nanoparticles, seven anticancer drugs were studied for their

2

affinities for bovine serum albumin (BSA) in the presence and absence of ZnSe-ZnS QDs based

3

on FRET theory. The recent work has focused on fluorescence quenching effects in Drug-QDs,

4

QDs-BSA and Drug-QDs/BSA assemblies. Results got with solution phase showed that the

5

excited-state dipole–dipole coupling between QDs and BSA results in an excitation energy

6

transfer from BSA to QDs, i.e., fluorescence resonance energy transfer. The FRET process

7

between ZnSe-ZnS QDs and BSA was confirmed by the changes of their fluorescence spectra.

8

The FRET efficiency (E) and the Stern–Volmer quench constant (Ksv) were calculated. Moreover,

9

the QDs carrier could facilely associate to plasma proteins and enhance the bioavailability efficacy

10

of anticancer drugs based on the FRET process were studied in some detail. Specifically,

11

ZnSe-ZnS QDs could enhance the affinity of SAHA, DOC, BUCN, DOX and HCPT for BSA, but

12

showed negative cooperativity of MTX and VCR for BSA. ZnSe-ZnS QDs in blood will improve

13

the transporting ability of serum albumin for some drugs, which may enhance the pharmacological

14

effects of some compounds. From this viewpoint, ZnSe-ZnS QDs are perfect candidates for drug

15

delivery applications (DDS).

16 17

2. Materials and methods

18

2.1. Materials and Reagents

19

Doxorubicin (Dox), 10-Hydroxycamptothecin (HCPT), Methotrexate (MTX)，Docetaxel

20

(DOC)，Carmustine (BCNU), Vincristine (VCR) and Suberoylanilide hydroxamic acid (SAHA)

21

were obtained commercially from DaLian Meilun Biotech Corporation, Ltd (Dalian, China). The

22

solutions of anticancer drugs (1.0 × 10-3 mol L-1) were firstly prepared by ultrasonic dissolving

23

drugs with 500 µl methanol, then with phosphate buffered saline (PBS) diluting to the required

24

concentration (1.0 × 10-3 mol L-1). Bovine Serum Albumin(BSA，fraction V) was purchased from

25

Aladdin Reagent Corporation (Shanghai, China). The solution of BSA (1.0 × 10-5 mol L-1) was

26

prepared in PBS buffer (pH 7.4, 1.0 × 10-3 mol L-1) and stored in a refrigerator (4℃) until use. All

27

other reagents and solvents were analytical grade and all aqueous solutions were prepared using

28

double-distilled water.

3

1 2 3

Doxorubicin（Dox）

Docetaxel (DOC)

Vincristine (VCR)

Methotrexate

(MTX) O H N OH

N H O

4 5

10-Hydroxycamptothecin（HCPT）

6

(BCNU)

Vorinostat（SAHA）

Carmustine

7 8

2.2. Preparation of water-soluble ZnSe-ZnS QDs

9

The ZnSe-ZnS QDs were prepared by our group following the method described previously

10

[16]. In a three-necked flask (25 mL), 0.10 mmol selenium powder and 500 µL water were

11

deaerated with nitrogen for 5 min. Then slightly excessive NaBH4 was added and the resulting

12

suspension was cooled to 0 ℃ under vigorous stirring and nitrogen bubbling. During 40min, the

13

colored suspension changed its characteristic color from dark to colorless. Stopped stiring to

14

terminate the reaction for 10 min, fresh Nansen solution was obtained. In a three-necked flask

15

(250 mL), Zn (OAc) 2·2H2O (0.4 mmol) and L-glutathione (GSH, 0.5 mmol) were dissolved in

16

100 mL deionized water. The solution was adjusted to pH 10.5 by drop wise addition of 1.0 M

17

NaOH solution with stirring. Then 0.1 mmol NaHSe solution was injected through asyringe under

18

stirring and nitrogen bubbling for 30 min. The mixture was refluxed at 100 ℃ for 90 min to

19

support the growth of the nanocrystals. Additional MPA (0.4 mmol) and Zn (OAc) 2·2H2O (0.1

20

mmol) were added to the crude ZnSe QDs reaction solution (0.02 mmol) and the pH of the

21

solution was adjusted to 9.5. The reaction mixture was heated under N2 atmosphere and stirred for

22

60 min. Subsequently, the mixture was refluxed at 100℃ for 60 min to keep constant for the

23

growth/annealing process of the ZnSe-ZnS QDs. The reaction was terminated by allowing the

24

reaction mixture to cool down to room temperature. The obtained samples were purified by

25

centrifugation and decantation with the addition of 1.5 times volume anhydrous ethanol, followed

26

by resuspension in a minimal amount of ultrapure water, and the purified QDs were dried

4

1

overnight at 60℃ temperature in vacuum. The excess ligand and un-reacted precursors were

2

removed by extensive purification prior to analysis.

3

2.3 Fluorescence spectra

4

The absorption spectra were recorded on an UV-1800 spectrophotometer (Shimadzu, Japan)

5

and the fluorescence analysis was performed on a RF-5301 spectrofluorimeter (Shimadzu, Japan)

6

under 37°C. The fluorescence spectra were recorded in the wavelength range of 300-500 nm upon

7

excitation at 295 nm when BSA samples were titrated with QDs or anticancer drugs. The

8

excitation and emission slit widths were 3nm, and scan speed were kept constant with each data

9

set. Quartz cells (1.0 cm optical path) as the container were used for all measurements. In each

10

titration, the fluorescence spectrum was collected with the concentrations of BSA at 1.0 × 10−5

11

mol L-1. The fluorescence quenching was studied by changing the quencher (anticancer drugs or

12

QDs) concentration. Each experiment was repeated at lest three times and found to be

13

reproducible within experimental errors.

14 15

3. Results and discussion

16

3.1 Fluorescence studies

17

BSA has three intrinsic fluorophore: tryptophan, tyrosine and phenylalanine. Because the

18

quantum yield of phenylalanine is very low and the fluorescence of tyrosine is almost totally

19

quenched, the intrinsic fluorescence of BSA is almost entirely due to tryptophan [17]. When the

20

fluorescence emission spectra of BSA are measured with a series of concentrations of quencher by

21

fixing the excitation wavelength at 295 nm, the fluorescence emission peak of BSA at 348 nm

22

gives the information of tryptophan residues. ZnSe-ZnS QDs are nanocrystals with unique

23

photophysical properties including broad excitation spectra, stable fluorescence intensity, high

24

quantum yields and the narrow full width at half-maximum (FWHM) [18]. So they were chosen in

25

order to maximize the spectral overlap of the donor–acceptor emission and absorption spectra (red

26

line) while still maintaining good spectral resolution of the donor and acceptor emission. The

27

absorption and emission spectra obtained from ZnSe-ZnS QDs and BSA are shown in Fig.1. The

28

maximal fluorescence emission peak of the BSA is at 348 nm (black line), while that of the QDs is

29

at 387nm (red line). So there is appreciable overlap between the emission spectrum of the BSA

30

(donor) and the absorption spectrum of the QDs (acceptor). 5

1

Fig.1 The absorption and emission spectra of ZnSe-ZnS QDs (red line) and the emission spectrum

2

of BSA (black line).

3 4

On the basis of the relationship between quenching of excited states and quencher

5

concentration, the fluorescence intensity data were then analyzed according to Stern–Volmer

6

relation (Eq.1) to get a better insight into the type of quenching [19]:

7

F0 / F = 1 + Kq τ0 [Q] = 1 + KSV [Q]



(1)

8

Where F0 is the fluorescence intensity of fluorophore in absence of the quencher, and F is the

9

fluorescence intensity in the presence of the quencher. Kq is the quenching rate constant of the

10

bimolecular, τ0 is the average lifetime of the fluorophore without quencher, KSV is the quenching

11

constant of Stern-Volmer equation, and [Q] is the concentration of quencher.

12

In many instances the fluorophore can be quenched both by collision and by complex

13

formation with the same quencher. In this case, the Stern-Volmer plot exhibits an upward

14

curvature, concave toward the y-axis at high [Q], and F0 / F are related to [Q] by the modified

15

form of the Stern-Volmer equation:

16 17 18 19

F 0 / F = (1+K D [Q]) (1+KS [Q])

(2)

KD and KS are the dynamic and static quenching constants, respectively. The above modified equation is a quadratic function of the [Q], which explains the curve toward the y-axis. The binding constant is calculated according to the double-logarithm equation:

20

log10 (F 0 − F) / F = log10 Ka + n log10 [Q]

21

Where Ka is the binding constant and n is the number of binding sites per molecule.

(3)

22

The fluorescence energy transfer efficiency (E), commonly determined by the fluorescence

23

intensity of donor in the absence or presence of acceptor, is determined from steady-state

24

fluorescence data as the equation (4):

25

E = 1 − F / F0

(4)

26

To obtain the binding parameters of BSA with ZnSe-ZnS QDs, the related fluorescence

27

signals of the ZnSe-ZnS QDs and BSA were determined in the reaction mixture. The change of

28

fluorescence spectra of BSA–QDs solutions with gradually increasing quantity of QDs (0 mmol

29

L-1 ; 0.0025 mmol L-1; 0.005 mmol L-1; 0.0075 mmol L-1; 0.0100 mmol L-1; 0.0125 mmol L-1;

30

0.0150 mmol L-1; 0.0175 mmol L-1; 0.0200 mmol L-1) to a fixed amount of BSA (1.0 × 10-5 mol 6

1

L -1) was studied. As expected, a significant enhancement of QDs fluorescence intensity at about

2

380 nm and the corresponding quenching of the emission of BSA at about 348 nm were observed

3

(Fig.2). The FRET efficiency of BSA–QDs conjugation can reach to 24.87% (Eq.4). The data

4

suggests that relatively short distances are needed to obtain adequate energy transfer [20].

5

Therefore, biomolecules can be coupled onto semiconductor nanoparticles without any spacer. In

6

addition, there is an obvious red-shift of the maximum emission of ZnSe-ZnS QDs from 380 to

7

384 nm. The red-shift of the fluorescence spectrum is explained by the partial delocalization of

8

charge excitons into the formed shell region [21]. It indicated that the excited-state dipole–dipole

9

coupling between QDs and BSA results in an excitation energy transfer from BSA to QDs, i.e.,

10

fluorescence resonance energy transfer. The emission change here implied that the BSA would

11

adopt a more incompact conformation state on the surface of QDs and resulted in the exposure of

12

the hydrophobic cavities [22]. Adsorption of protein molecules on nanoparticles surface changed

13

their surface functionality, and influenced their behavior in biological systems [23]. For the

14

nanoparticles, formation of nanoparticle-protein conjugates provides stability over broad range of

15

pH and ionic strengths.

16

Fig.2 Fluorescence emission spectra from BSA and different concentration of QDs solution

17 18

3.2. Binding mode of ZnSe-ZnS QDs with BSA

19

The forces that govern the interaction between small molecules and biological

20

macromolecules or nanoparticles include covalent bond, hydrophobic force, hydrogen bond

21

formation, Vander Waals force and electrostatic interaction and so on [24]. The thermodynamic

22

parameters of binding reaction are the main evidence for confirming acting forces. Therefore, the

23

thermodynamic parameters dependent on temperatures were analyzed in order to characterize the

24

acting forces between QDs and BSA. For this purpose, the temperature-dependence of the binding

25

constant was studied. The temperatures chosen were 298K, 303K, 310K and 313K. According to

26

the Vant Hoff equation (Eqs.5 and 6) [25] the thermodynamic parameters were obtained from a

27

linear Vant Hoff plot via Fig. 3 and tabulated in Table 1.

28

lnK = - △H/RT + △S/R

(5)

29

Where K is analogous to the quenching constant Ksv at the corresponding temperature, R is

30

molar gas constant and T is the temperature. △H and △S are the standard enthalpy and entropy 7

1

change for the reaction, respectively. The values of lnK are plotted against 1000/ T according to

2

Eq.5 at different temperatures.

3 4 5

The free-energy changes (∆G) at different temperatures are calculated for the above reaction by the following relationship: △G=△H - T△S

(6)

6

The ∆H and ∆S for the binding reaction between QDs and BSA respectively were found to be

7

−27.37 kJ mol−1 and 6.31 J mol−1 K−1, which indicated that the binding process was an exothermic

8

reaction. The negative values of ∆G suggest that the binding process of QDs to BSA is

9

spontaneous. The negative value of enthalpy (∆H) and positive entropy (∆S) values indicated that

10

electrostatic interaction played a major role in the binding reaction between BSA and QDs. It was

11

also proved by literatures results [26] that the electrostatic interactions occurred between the

12

negatively charged QDs and the cationic ‘‘hot spot’’ around the active site of the protein.

13

Table 1 Thermodynamic parameters of BSA–QDs system.

14

Fig.3. Van’t Hoff plot for the binding of ZnSe-ZnS QDs with HAS

15 16

The binding constant and the number of binding sites between ZnSe-ZnS QDs and BSA were

17

determined as 3.63 × 107 L mol-1 and 1.75 according to the double-logarithm equation (Eq. 3).

18

Xiao and co-workers [27] determined the magnitudes of binding constants between three colors

19

QDs and BSA were almost in the range of 105-108 L mol-1, which was consistent with the affinity

20

of ZnSe-ZnS QDs for BSA in the current study. The magnitudes of binding constants drugs for

21

serum albumins were almost in the range of 104 -106 L mol-1 [28]. If the carrier is highly bound to

22

plasma proteins, it is facilely to associate this complex to deliver free drug molecule, which would

23

enhance the bioavailability efficacy of a drug. These differences in binding constants also

24

illustrated that different nanomaterials show different function when bound to plasma proteins [29].

25

Smaller nanoparticles favor native-like protein structure more strongly, resulting in higher intrinsic

26

enzyme activity. But the larger nanoparticles provide larger surface area of contact for adsorbed

27

proteins and thus results in stronger interactions between proteins and nanoparticles.

28

3.3 Interaction of BSA with anticancer drugs

29

The interaction of serum albumins with drugs has been studied widely by many research

30

groups [30, 31]. Upon titration of anticancer drugs to the BSA solution, the fluorescence intensity 8

1

attenuated gradually with increasing concentration of anticancer drugs. Approximately 29.64% to

2

81.80% of BSA fluorescence quenching was observed when the concentration of anticancer drugs

3

reached 0.045 mmol L-1. The extent of the fluorescence attenuation was in an order:

4

MTX>DOX>VCR>HCPT. In addition, MTX, DOX, VCR and HCPT resulted in an obvious blue

5

shift of the maximum λem of BSA from 348 to 344 nm. However, there was no shift of the

6

emission wavelength of BSA with the addition of SAHA, DOC and BUCN. The obvious

7

blue-shift of the maximum λem of BSA is indicative of changes in the immediate environment of

8

the tryptophan residues, typically, the polarity of tryptophan residues and the hydrophobicity of

9

the hydrophobic cavity of BSA [32]. The hydrophobic groups are in the interior of the tertiary

10

structure and the polar groups are on the surface of native protein. The emission of BSA may be

11

blue-shifted if the indole group of tryptophan residue is buried within native protein, and its

12

emission may be red-shifted when the protein is unfolded [33].

13

Fig. 4 (left) shows the Stern –Volmer plots for the BSA fluorescence quenching by anticancer

14

drugs. The Stern –Volmer curve for most drugs were linear. However, the Stern –Volmer plots

15

largely deviated from linearity toward the y-axis at high MTX concentrations, which indicated that

16

both dynamic and static quenching were involved for MTX on BSA fluorescence. The magnitudes

17

of binding constants (Fig.4 right) were in the range of 104–107 L mol-1, in agreement with common

18

affinities of drugs for serum albumins (104–106 L mol-1) in vivo. The binding constants and the

19

number of binding sites between anticancer drugs and BSA according to the double-logarithm

20

equation are shown in Table 2. The number of binding sites per BSA molecule (n) was determined

21

as 1.01 ± 0.29, which indicated that one binding site formed between the anticancer drugs and

22

BSA.

23

MTX>DOX>VCR>HCPT>BCNU>DOC>SAHA.

24

Fig.4 Stern-Volmer plots (left) and double-logarithm curves (right) for BSA fluorescence

25

quenching by anticancer drugs.

26

Table 2 Binding constants (log10 Ka) and number of binding sites per BSA molecule (n) of

27

interaction in the absence and presence of ZnSe-ZnS QDs.

The

extent

of

the

fluorescence

attenuation

was

in

an

order:

28 29 30

3.4 Interaction of ZnSe-ZnS QDs with anticancer drugs The interaction of ZnSe-ZnS QDs with drugs has been studied by trickling the anticancer 9

1

drugs to the QDs solution, the fluorescence intensity attenuated gradually with increasing

2

concentration of anticancer drugs. Approximately 5.32% to 29.64% of the QDs fluorescence

3

quenching was observed when the concentration of anticancer drugs reached 0.045 mmol L-1. The

4

degree of fluorescence quenching was far lower than that interaction between BSA and anticancer

5

drugs. Moreover, there was no obvious shift of the emission wavelength of QDs with the addition

6

of any drugs. The Stern –Volmer curve (Fig. 5 left) for all drugs were linear which indicated that

7

static quenching were involved for anticancer drugs on QDs fluorescence. The magnitudes of

8

binding constants (Fig.5 right) were in the range of 103–105 L mol-1. The number of binding sites

9

per QDs (n) was determined as 1.07 ± 0.20. The extent of the fluorescence attenuation was in an

10

order: BCNU>DOX>MTX>DOC>HCTP>VCR>SAHA.

11

The phenomenon was indicative of no change in the interior structure and the polar groups on

12

the surface of ZnSe-ZnS QDs. These results indicated that the quenching effect of drugs on QDs

13

fluorescence depended on the structures of drugs [34]. As Table 3 showed，the more polar groups

14

of drugs, for instance -COOH、-OH and -NH2, the more binding sites and constants and the higher

15

affinity between QDs and drugs. It may be due to the electrostatic attraction between ZnSe-ZnS

16

QDs and drugs. Particularly, there is –Cl instead of above polar groups mentioned with the BCNU

17

molecular so that drug molecular has positive charge after the –Cl deviates. Though the degree of

18

fluorescence quenching was no obvious, the binding sites and constants were the highest in this

19

research.

20

In addition, the magnitudes of binding constants（103 – 105 L mol-1 ） for drugs and QDs was

21

far lower than that for drugs and BSA (104 – 107 L mol-1), which illustrated the affinity of drugs

22

and QDs

23

disassociate this complex to give a free drug molecule, which would weaken the bioactivity of a

24

drug. Because the affinities of drugs for serum albumins (104 – 106 L mol-1) in vivo was slightly

25

higher than that for drugs and QDs, nano-delivery can easily released drugs molecular after

26

playing the carrier role of drug deliver system (DDS) [35]. Then free drug molecular binding with

27

serum protein in the body and expressed drugs effect.

28

Table 3 Binding constants (log10 Ka) and number of binding sites per QDs molecule (n) of

29

interaction with anticancer drugs.

30

Fig.5 Stern-Volmer plots and double-logarithm curves for QDs fluorescence quenching by

was weaker. In generally, if nano-delivery was highly bound to drugs, it was difficult to

10

1

anticancer drugs

2 3

3.5 Fluorescence quenching of BSA induced by anticancer drugs in the presence of QDs

4

When anticancer drugs were continuously added to the BSA solution (1.0 × 10-5 mol L-1 )

5

containing 30 µmol L-1 of ZnSe-ZnS QDs, further attenuation in the fluorescence of BSA was

6

observed. The obvious red-shifts of the maximum emission of BSA with the addition of anticancer

7

drugs were also observed in the presence of QDs. However, the extents of shifts induced by drugs

8

in the presence of QDs were much smaller than that in the absence of QDs. The quenching effects

9

of BSA fluorescence induced by DOX, HCPT, SAHA, DOC and BUCN were enhanced in the

10

presence of QDs but MTX or VCR. The quenching effects of BSA fluorescence induced by

11

anticancer drugs in the presence of QDs were significantly stronger than those in the absence of

12

QDs.

13

Figure 6 (left) showed the Stern-Volmer plots for BSA fluorescence quenching by anticancer

14

drugs in the presence of ZnSe-ZnS QDs. As shown in Figure 6 (left), the Stern-Volmer plots

15

largely deviated from linearity toward the y-axis at high concentrations of MTX and DOX.

16

According to Soares et al [36], both dynamic and static quenching involved in these drugs

17

quenching BSA fluorescence the presence of ZnSe-ZnS QDs. In many cases this upward curvature

18

showed that the fluorophore was quenched by both mechanisms with the same quenchers.

19

However, in other cases the upward curvature illustrated that the presence of a sphere of action.

20

Quenching occurs due to the quencher being adjacent to the fluorophore at the moment of

21

excitation [37]. This type of apparent static quenching is usually interpreted in terms of the model

22

“sphere of action”.

23

Figure 6 (right) showed the double-logarithm curves of anticancer drugs quenching BSA

24

fluorescence in the presence of QDs, and Table 2 presented the corresponding calculated results.

25

The magnitudes of binding constants in the presence of QDs were in the range of 103–106 L mol-1,

26

in agreement with common affinities of drugs for serum albumins (104–106 L mol-1) in vivo. The

27

number of binding sites per BSA molecule (n) was determined as 1.03 ± 0.17, and the obtained

28

values of n (0.8740–1.3617) was correspond to the binding sites with high affinity.

29

Fig.6 Stern-Volmer plots and double-logarithm curves for BSA fluorescence quenching by

30

anticancer drugs in the presence of ZnSe-ZnS QDs. 11

1 2

3.6 Effect of ZnSe-ZnS QDs on the affinities of anticancer drugs for BSA

3

The cooperative binding of small-molecule ligand to multimeric proteins has played a special

4

role in the exploring of physiological affinity and delivery of drugs [38]. Proteins have the

5

fundamental ability to selectively bind to other molecules. For most of proteins, the binding of the

6

first ligand to the protein can influence the affinity for the second ligand, which is called

7

cooperative-effect binding [39]. As shown in Figure 7(left), ZnSe-ZnS QDs obviously increased

8

the affinities of SAHA, DOC and BUCN for BSA. The QDs significantly improved the binding

9

constant of DOC for BSA by 36.72%. The affinities of DOX and HCPT for BSA were hardly

10

changed in the presence of QDs. However, the affinity of MTX and VCR for BSA was slightly

11

reduced.

12

Recently Berezhkovskiy [40] illustrated that the increase in the quantity of sites lead to the

13

increase of ligand bound to them. So the number of binding sites increasing with increasing

14

binding constant could be considered as one theory to evaluate these models. The relationship

15

between the logKa and n between anticancer drugs and BSA in the absence and presence of

16

ZnSe-ZnS QDs were shown in Fig.7 (right). The values of log10 Ka were proportional to n. This

17

result confirmed that Eq.3 used here was suitable to study the interaction among anticancer drugs,

18

QDs and BSA.

19

Fig.7 The affinities of anticancer drugs for BSA in the presence of ZnSe-ZnS QDs (left) and the

20

relationship between the affinities (log10Ka) and the number of binding sites (n) between drugs and

21

BSA (right).

22 23

Here, ZnSe-ZnS QDs showed a positive cooperativity, which enhanced the affinity of SAHA,

24

DOC, BUCN, DOX and HCPT for BSA. According to theory of Soares et al, ligand of drug

25

molecules binds quickly with ZnSe-ZnS QDs owning to the big surface area of QDs. The drug

26

molecular approach BSA through FRET interaction between BSA and QDs, then present the static

27

quenching sphere of action. Quenching further occurs due to the drug as quencher being adjacent

28

to the area at the moment of excitation. The enhancement of binding sites and binding constant

29

indicated increase of affinity between drugs and BSA then enhanced the absorbance and delivery

30

of drugs in vivo. If a drug molecular strongly bond with BSA, the addition of QDs could compete 12

1

the affinity of drugs and reduce binding interaction between BSA and drugs. So the ZnSe-ZnS

2

QDs showed a negative cooperativity of MTX and VCR for BSA, which duo to the competition

3

between QDs and BSA. The schematic is represented in Fig.8.

4

Fig.8 The schematic representation of ZnSe-ZnS QDs for influence of binding affinities of

5

anticancer drugs to BSA based on FRET theory.

6 7

4. Conclusion

8

Based on FRET theory, seven anticancer drugs were studied for their affinities for bovine

9

serum albumin (BSA) in the presence and absence of ZnSe-ZnS QDs as representative. The higher

10

binding constant (3.63 × 107 L mol-1) and number of binding sites (1.75) between ZnSe-ZnS QDs

11

and BSA demonstrated that the QDs carrier could facilely associate to plasma proteins and

12

enhance the bioavailability of drugs [41]. The magnitudes of binding constants (103–106 L mol-1 )

13

in the presence of QDs were in agreement with common affinities of drugs for serum albumins

14

(104–106 L mol-1) in vivo. ZnSe-ZnS QDs in blood could improve the transporting ability of BSA

15

for SAHA, DOC, BUCN, DOX and HCPT, but showed a negative cooperativity of MTX and

16

VCR for BSA. From this research, the interaction between nanoparticles and drugs will be taken

17

into account when we construct a nanoscale drug delivery system (DDS).

18 19

Acknowledgments

20

The authors gratefully acknowledge the financial support from the National Natural Science

21

Foundation of China (No. 81173023) and Fundamental Research Funds for the Central

22

Universities (No. JKY2011097).

23 24

References

25 26 27 28 29 30 31 32

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[8] J. Qian, C. Wang, X. Pan, S. Liu, Anal Chim Acta, 763 (2013) 43-49. [9] X. Cao, Y. Ye, S. Liu, Anal Biochem, 417 (2011) 1-16. [10] J. Liu, W. Zhao, R.-L. Fan, W.-H. Wang, Z.-Q. Tian, J. Peng, D.-W. Pang, Z.-L. Zhang, Talanta, 78 (2009) 700-704. [11] Q. Ma, X.-G. Su, X.-Y. Wang, Y. Wan, C.-L. Wang, B. Yang, Q.-H. Jin, Talanta, 67 (2005) 1029-1034. [12] D. Brambilla, B. Le Droumaguet, J. Nicolas, S.H. Hashemi, L.-P. Wu, S.M. Moghimi, P. Couvreur, K. Andrieux, Nanomed-Nanotechnol, 7 (2011) 521-540. [13] P.B. Kandagal, J. Seetharamappa, S.M.T. Shaikh, D.H. Manjunatha, J Photochem Photobiol A, 185 (2007) 239-244. [14] P. Daneshgar, A.A. Moosavi-Movahedi, P. Norouzi, M.R. Ganjali, A. Madadkar-Sobhani, A.A. Saboury, Int J Biol Macromol, 45 (2009) 129-134. [15] S.K. Sahoo, V. Labhasetwar, Drug Discovery Today, 8 (2003) 1112-1120. [16] C. Shu, B. Huang, X. Chen, Y. Wang, X. Li, L. Ding, W. Zhong, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 104 (2013) 143-149. [17] S. Singh, R. Kaur, J. Chahal, P. Devi, D.V.S. Jain, M.L. Singla, J Lumin, 141 (2013) 53-59. [18] Y.-Q. Li, J.-H. Wang, H.-L. Zhang, J. Yang, L.-Y. Guan, H. Chen, Q.-M. Luo, Y.-D. Zhao, Biosens Bioelectron, 25 (2010) 1283-1289. [19] M.A. Perillo, A. Arce, J Pharmacol Toxicol, 35 (1996) 69-76. [20] Y. Zhang, Q. Zeng, Y. Sun, X. Liu, L. Tu, X. Kong, W.J. Buma, H. Zhang, Biosens Bioelectron, 26 (2010) 149-154. [21] J.-W. Yoo, N. Doshi, S. Mitragotri, Adv Drug Deliver Rev, 63 (2011) 1247-1256. [22] M. Li, Y. Ge, Q. Chen, S. Xu, N. Wang, X. Zhang, Talanta, 72 (2007) 89-94. [23] Y. Liang, Y. Yu, Y. Cao, X. Hu, J. Wu, W. Wang, D.E. Finlow, Spectrochim Acta A, 75 (2010) 1617-1623. [24] Z. Peng, Y. Bang-Ce, J Agr Food Chem, 54 (2006) 6978-6983. [25] L. Ding, P. Zhou, H. Zhan, X. Zhao, C. Chen, Z. He, Chemosphere, 92 (2013) 892-897. [26] J. Xiao, L. Chen, F. Yang, C. Liu, Y. Bai, J Hazard Mater, 182 (2010) 696-703. [27] J. Xiao, M. Wu, G. Kai, F. Wang, H. Cao, X. Yu, Nanomed-Nanotech, 7 (2011) 850-858. [28] J. Xiao, H. Cao, Y. Wang, J. Zhao, X. Wei, J Agr Food Chem, 57 (2009) 6642-6648. [29] Q. Xiao, S. Huang, J. Ma, W. Su, P. Li, J. Cui, Y. Liu, J Photochem Photobiol A, 249 (2012) 53-60. [30] H. Härmä, T. Soukka, A. Shavel, N. Gaponik, H. Weller, Anal Chim Acta, 604 (2007) 177-183. [31] N. Shahabadi, M. Maghsudi, S. Rouhani, Food Chem, 135 (2012) 1836-1841. [32] N. Janakiraman, A. Mohan, A. Kannan, G. Pennathur, Spectrochim Acta A, 95 (2012) 478-482. [33] M. Sarkar, S.S. Paul, K.K. Mukherjea, J Lumin, 142 (2013) 220-230. [34] E. Petryayeva, U.J. Krull, Anal Chim Acta, 706 (2011) 8-24. [35] J.-H. Wang, T.-C. Liu, Y.-C. Cao, X.-F. Hua, H.-Q. Wang, H.-L. Zhang, X.-Q. Li, Y.-D. Zhao, Colloid Surface A, 302 (2007) 168-173. [36] M.N. Soares S, Freitas V, J Agric Food Chem, 55 (2007) 6726-6735. [37] L. Trapiella-Alfonso, J.M. Costa-Fernandez, R. Pereiro, A. Sanz-Medel, Talanta, 106 (2013) 243-248. [38] W.R. Algar, A.J. Tavares, U.J. Krull, Anal Chim Acta, 673 (2010) 1-25. [39] B. Haley, E. Frenkel, Urol Oncol, 26 (2008) 57-64.

14

1 2 3

[40] B. LM, J Pharm Sci 96 (2007) 249-257. [41] P. Amornphimoltham, A. Masedunskas, R. Weigert, Adv Drug Deliver Rev, 63 (2011) 119-128.

4 5

15

BSA FL QDs FL QDs UV

400

2.5 2.0

300

1.5 200

1.0 0.5

100

Fluorescence intensity

Absorbance intensity

3.0

0.0 -0.5 300

350

400

0 450

Wavelength(nm)

1 2

Fig.1 The absorption and emission spectra of ZnSe-ZnS QDs (red line) and the emission spectrum

3

of BSA (black line).

4

16

1

Fluorescence intensity

600

400

BSA

QDs

200

0 320 340 360 380 400 420 440 460

2 3 4

Wavelength (nm)

Fig.2 Fluorescence emission spectra from BSA and different concentration of QDs solutions

17

12.0

lnK = 3292.4/T + 0.7588 2 R = 0.9904

11.8

ln K

11.6 11.4 11.2 11.0 3.20

1 2

3.25

3.30

3.35

3.40

-1 1000 / T (K )

Fig.3 Van’t Hoff plot for the binding of ZnSe-ZnS QDs with BSA

3 4

18

BCNU DOC SAHA HCPT DOX VCR MTX

5

F0 / F

4

0.5

3 2 1

1

BCNU DOC SAHA HCPT DOX VCR MTX

1.0

log10 (F0 - F) / F

6

0.0 -0.5 -1.0 -1.5 -2.0 -5.4

-6 Drug concentration (10 mol/L)

-5.2

-5.0

-4.8

-4.6

-4.4

-4.2

log10 [ Q ]

2

Fig.4 Stern-Volmer plots and double-logarithm curves for BSA fluorescence quenching by

3

anticancer drugs.

4 5

19

BCNU DOC SAHA HCPT DOX VCR MTX

1.8

F0 / F

1.6 1.4

-0.5

1.2 1.0 0.8

-1.0 -1.5 -2.0 -2.5

0

1

BCNU DOC SAHA HCPT DOX VCR MTX

0.0

log (F0 - F) / F

2.0

10

20

30

40

-6 -1 Drug concentration(10 mol L )

50

-5.4

-5.2

-5.0

-4.8

-4.6

-4.4

-4.2

log10 [ Q ]

2

Fig.5. Stern-Volmer plots and double-logarithm curves for QDs fluorescence quenching by

3

anticancer drugs

4 5

20

BCNU DOC SAHA HCPT DOX VCR MTX

F0 / F

4

0.5

3

2

0.0 -0.5 -1.0 -1.5

1 0

1

BCNU DOC SAHA HCPT DOX VCR MTX

1.0

log (F0 - F) / F

5

10

20

30

40

50

-5.4

-6 Drug concentration (10 mol/L)

-5.2

-5.0

-4.8

-4.6

-4.4

-4.2

log10 [ Q ]

2

Fig.6 Stern-Volmer plots and double-logarithm curves for BSA fluorescence quenching by

3

anticancer drugs in the presence of ZnSe-ZnS QDs.

4 5

21

40 SAHA

8

DOC

y = 5.76x - 1.584 2 R = 0.9901

7 BCNU

20 10

DOX

6

log10 Ka

Affinities (%)

30

HCPT

0 -10

5 4 3

MTX VCR -20

2 0.6

0.8

1.0

1.2

1.4

1.6

n

1 2

Fig.7 The affinities of anticancer drugs for BSA in the presence of ZnSe-ZnS QDs (left) and the

3

relationship between the affinities (log10Ka) and the number of binding sites (n) between drugs and

4

BSA (right).

5 6

22

1

2 3

Fig.8 The schematic representation of ZnSe-ZnS QDs for influence of binding affinities of

4

anticancer drugs to BSA based on FRET theory.

5 6 7 8 9 10

23

1 T

.

△HӨ/KJ mol-1

.

.

△SӨ/J mol-1 K-1

298K

-29.26

303K

-29.29

310K

-27.37

6.309

313K 2

.

△GӨ/KJ mol-1

-29.33 -29.35

Table 1 Thermodynamic parameters of BSA–QDs system

3 4 5

24

R

0.9904

1 2

Table 2 Binding constants (log10 Ka) and number of binding sites per BSA molecule (n) of

3

interaction in the absence and presence of ZnSe-ZnS QDs.

Drug-BSA

Drug-QDs/BSA

log10Ka

n

R

log10Ka

n

R

MTX

7.2743

1.5301

0.9906

6.3962

1.3617

0.9947

DOX

5.0231

1.1931

0.9976

5.2714

1.1269

0.9903

VCR

4.7913

1.1390

0.9969

4.2820

0.9911

0.9981

HCPT

3.8224

0.9821

0.9956

4.0999

0.9228

0.9935

BCNU

3.1961

0.8070

0.9994

3.7688

1.0260

0.9949

DOC

2.6408

0.7222

0.9964

3.6106

0.9065

0.9981

SAHA

2.5508

0.7072

0.9941

3.4056

0.8740

0.9980

4 5 6 7

25

1 2

Table 3 Binding constants (log10 Ka) and number of binding sites per QDs molecule (n) of log10Ka

3

n

R

-COOH

-NH2

-OH

BCNU

5.0074

1.4120

0.9930

0

0

0

DOX

4.8097

1.1643

0.9983

0

1

5

MTX

4.6079

1.0753

0.9929

2

2

0

DOC

4.1794

1.0339

0.9972

0

0

4

HCPT

4.0065

0.9737

0.9974

0

0

2

VCR

3.4519

0.9431

0.9916

0

0

2

SAHA

2.1774

0.7963

0.9959

0

0

1

interaction with anticancer drugs.

4 5 6

26

1

Highlights

2

►The interaction between ZnSe-ZnS QDs and BSA was systematically investigated. ►ZnSe-ZnS

3

QDs could enhance the binding affinity of some drugs for BSA based on FRET. ►The

4

double-logarithm strategy detecting the binding affinities of drugs to BSA was proposed. ►Drugs

5

quenched the fluorescence intensity of BSA or QDs effectively relate to polar groups of drugs.

6

►The mechanism will be available on whether or not the nanoparticles affect drug transport.

7 8 9 10 11

27

1

Graphical abstract

2

3 4

The schematic representation of ZnSe-ZnS QDs for influence of binding affinities of

5

anticancer drugs to BSA based on FRET theory. Here, ZnSe-ZnS QDs could enhance the affinity of

6

SAHA, DOC, BUCN, DOX and HCPT for BSA, but showed competitive effect of MTX and VCR for

7

BSA. So the interaction between nanoparticles and drugs will be available on whether or not the

8

nanoparticles affect drug transport in blood.

9 10 11 12 13

28