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In this work, we established a fluorescence resonance energy transfer (FRET) system between ZnSe:Mn/ZnS quantum dots and Hypocrellin A (HA, a photosensitizer used for photodynamic therapy of cancer) in aqueous solution, excited by four-photon. Here, the QDs are the donors and the HA are the acceptors. The four-photon-excited fluorescence resonance energy transfer spectrum was obtained under 1300nm femtosecond laser pluses. The experimental results indicated that the highest efficiency of FRET can reach up to 61.3%. Furthermore, the viability test in cancer cells was further demonstrated for biological applications of FRET system. When FRET occurs the cell killing rate of the cancer cells will reach to 84.8% with the 1mM concentration of HA. Our work demonstrates that while the four-photon excited FRET system is promising in both optics and biological applications, is also needs further investigation.
© 2016 Optical Society of America
1. Introduction
Recently, the FRET technique exhibits great potential as a new type of analysis methods due to its high sensitivity [1,2]. Fluorescence resonance energy transfer (FRET) is a process whereby excited state energy is transferred nonradiatively from an excited donor molecule to an acceptor molecule. A series of conditions should to be met for occurring of FRET: (1) The emission spectrum of donor should have competent overlap with the absorption spectrum of acceptor; (2) The donor and acceptor have to get close enough; (3) The emission and absorption dipole moment of donor and acceptor must not be perpendicular. The extent of FRET occurrence is usually expressed as FRET efficiency: the proportion of the photons absorbed by the donor whose energy is transferred to the acceptor [3,4].
However, the wavelength of the materials used in FRET always in visible region, they should be excited in short wavelengths. This drawback greatly limits the applications of FRET, because the short wavelength light cannot penetrate deeply into living tissues under common one-photon excitation. As an effective technique, longer wavelength was needed in FRET system [5–8]. Alternatively, four-photon excitation using near-infrared wavelength light has been suggested as a better way for improvement in applications, since the light in NIR region has the best penetration depth into living tissues [9]. In four-photon excitation conditions, longer near infrared wavelengths can be used, which are more suitable for applications in optical, biological and medical areas, especially in the area of photodynamic therapy [10].
Nanotechnology can satisfactorily address this concern the development of four-photon nanoparticle provides both structural and functional properties for four-photon excitation. Among the various nanoparticles being actively investigated for biomedical (FRET) applications, QDs have attracted increasing attention as donor of FRET, since their outstanding optical properties comparing to the traditional organic fluorescent molecules, It has been shown that QDs exhibit one and two-photon absorption cross sections in the visible and NIR regions much larger than the bulk due to the quantum confinement effect [11–18].
Similar with the two-photon excitation, Four-photon excitation means the molecular fluoresce accompanied by absorption of three photons. Four-photon excitation fluorescence has a better effect than two-photon excitation. It can detect the deeper level of samples because a longer wavelength excitation and a lower scattering [19–21].
Hypocrellin A (HA), one natural pigment extracted from fungus Hypocrella, a parasitic fungus of Siramudinaria, has been used in China for many years in a variety of traditional medicinal treatments, especially for skin diseases. Hypocrellin A has also been shown to accumulate selectively in tumor cells and can photodynamically kill them [22–24].
Herein, we used ZnSe:Mn/ZnS QDs and HA to construct the FRET systems, excited by four-photon for the first time. Steady-state photoluminescence, fluorescence lifetime measurements, and four-photon excited fluorescence property are completed to analyze the four-photon excited fluorescence. The FRET efficiency are calculated to demonstrate the quality of FRET systems, and extend to biological applications.
2. Experimental
Sodium hydroselenide (NaHSe) was prepared by the reaction of selenium powder and sodium borohydride (NaBH4, ≥ 98.0%) in water at 80°C. A transparent solution was obtained after the complete reduction of selenium. A zinc precursor solution was prepared by mixing 0.5 mmol Zinc chloride (ZnCl2, 98%), Manganese chloride(MnCl2, 98%) and mercaptopropionic acid (MPA, ≥ 99%) in water. The pH of the zinc precursor solution was adjusted to 11.6. The freshly prepared NaHSe solution was injected into the zinc solution under continuous stirring and the temperature was increased gradually to 100 °C to obtain ZnSe QDs. The weight ratio of ZnCl2 and MnCl2 is 1:0.02. 20ml ZnS shell stock solution, which contained ZnCl2 and MPA with a molar ratio of 1.0:1.9 in pH 11.6 solution was rapidly injected into 100 mL freshly prepared Mn:ZnSe solution at boiling temperature, followed by refluxing for 1 h.
Hypocrellin A (HA, ≥ 99%), the acceptor, was purchased from Nanjing herb biological science and Technology Co. Due to the low solubility of HA in water, 1.0 mM HA solution was prepared in DMSO solution and was protected from exposure to the light.
The FRET system was constructed in this way: 0.5ml QDs (1mg/mL) solution and 0.2ml HA solution (1mM) was added into cuvettes 1.2cm*1.2cm in turn. And the solution was kept at room temperature for 30 min before being used.
Transmission electron microscopy (TEM) images were obtained using G220 S-TWIN transmission electron microscope (FEI Tecnai, Hillsboro, USA). The steady-state photoluminescence spectrum was acquired through a Cary Eclipse Fluorescence Spectrometer (Agilent Technologies Inc, California, USA) with 1mm quartz cuvettes at room temperature. The excitation wavelength was kept at 350 nm to avoid the interference caused by the absorption of the acceptor (HA). The excitation and emission slots were both set at 5mm. The absorption measurements of solutions were studied using a Cary 5000 UV-Vis spectrophotometer (Agilent Technologies Inc, California, USA). The fluorescence lifetime of ZnSe:Mn/ZnS QDs-HA systems were measured separately using a FLS980 Fluorescence Spectrometer (Edinburgh Instruments, Livingston, UK).
To study the four-photon excited luminescence, the Fluorescence spectral measurements were performed using QE65000 fiber optic spectrometer (Ocean Optics, Shanghai, China). The samples were excited at the wavelength of 350nm and 1300nm separately with femtosecond laser pulses, produced by Ti: sapphire femtosecond laser (Coherent Inc, CA, USA) with pulse widths of 50fs, a repetition frequency of 1kHz.
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium reduction assay was used to measure the cytotoxicity of ZnSe:Mn/ZnS QDs on cells. The MCF-7 cells seeded in 96-well plates were incubated with ZnSe:Mn/ZnS QDs solutions with different concentrations for 24h and 48h. Afterwards, 20uL of MTT solution (5 mg mL-1) was added to each well. After another 4h of incubation, the solution in the wells was removed and the purple precipitate was dispersed in 150 mL of dimethyl sulfoxide (DMSO, Sigma). The absorbance of the solution in the wells was measured using a microplate reader (TECAN, Austria) at a wavelength of 495 nm. The cell viability was calculated by normalizing the absorbance of the sample wells to that of the control wells.
MTT assay was also used to measure the effect of QDs-HA systems on cells after irradiation by femtosecond laser. MCF-7 cells were seeded in 96-well microtiter plates for used. HA (PH = 11.6) and QDs-HA in different concentrations were added into the medium of treatment wells (except for the control wells) and incubated for 4h. Then, Femtosecond laser with pulse widths of 50fs, a repetition frequency of 1kHz, and fixed power of 100mW was used to irradiate the wells for 120 seconds. The cells were incubated for further 24h after irradiation. MTT cell viability assay were used to evaluate the results.
3. Results and discussion
3.1 Characterization and linear optical property
TEM images of ZnSe:Mn/ZnS QDs are shown in Fig. 1(a). Particles appear spherical in shape and did not induce aggregation. The average diameters of ZnSe:Mn/ZnS QDs are 12nm. The absorption peak of ZnSe:Mn/ZnS QDs was around 325nm. Figure 1(b) shows the normalized absorption and fluorescence spectral curves of ZnSe:Mn/ZnS QDs and HA, which indicates HA have three absorption bands, located in 473nm, 540nm, 581nm respectively, and the fluorescence peak of QDs are located at 575nm, respectively. There is a significant overlay between fluorescence peak of QDs and the absorption peak of HA, which suitable for the occurrence of FRET.
Fig. 1 (a) TEM images of ZnSe:Mn/ZnS QDs samples with average sizes of 12nm. (b) Normalized QDs fluorescence spectrum and the absorption and fluorescence spectra of HA.
Figure 3(a) shows the emission spectra of pure ZnSe:Mn/ZnS QDs, ZnSe:Mn/ZnS QDs-HA and pure HA. Comparing to ZnSe:Mn/ZnS QDs, the emission intensity of FRET system decreased to 34%, that FRET had occurred. In addition, there is an obvious red shift (about 75nm) of HA fluorescence spectra. The reason is HA will be dehydrated into hypocrellin B(HB) in alkaline conditions. Similar to HA, HB is also isolated from a parasitic fungus Hypocrella. It has similar absorption peak and photosensitivity with HA. The HA fluorescence spectra in different PH values were obtained to verify this phenomenon (Fig. 2(a)). The red shift extent of the spectral increased gradually with the increasing of PH value. The absorption spectrum almost has no changes when the pH value was 9,10,11,12. So, we set the following two pH values (PH = 7, PH = 11.6) as the samples. The results are shown in Fig. 2(b). HA has absorption peaks at 473nm, 540nm and 581nm, and HB has absorption peaks at 467nm, 543nm and 582nm. The absorption peak will have a minute shift with the changing of the PH values, but it will not affect the efficacy of the photosensitizer (HA or HB) during the experiments. The pH value of the ZnSe:Mn/ZnS QDs is 11.6, so the HA fluorescence peak of FRET system is around 700nm. For the sake of simplicity, we still call the acceptor HA in the following experiments.
Fig. 2 (a)Fluorescence spectra of HA solutions in different PH values (b)Absorption spectra of HA solutions in two different PH values.
Figure 3(b) shows the fluorescence spectra for different concentrations of HA in the FRET system; the fluorescence intensity of QDs increased gradually with increasing HA concentrations. Because the number of HA molecules around the ZnSe:Mn/ZnS QDs increased, the energy-transfer efficiency of FRET systems were higher than before. When there are multiple acceptors simultaneously around one QDs donor, The Förster energy-transfer efficiency can be expressed as [25]
where is the average number of acceptor molecules interacting with donor molecules. We can clearly see that the energy-transfer efficiency increases with . The fluorescence spectra results of FRET systems correspond with the conclusions deduced from Eq. (1), which means the energy transfer efficiency is proportional to concentration of the acceptor in FRET systems.Fig. 3 (a) Normalized absorption and fluorescence spectrum of HA and the fluorescence spectra of the QDs. (b) Fluorescence spectra of ZnSe:Mn/ZnS QDs-HA systems with different concentrations.
The fluorescence lifetime of FRET systems with the different concentration of HA are measured using a FLS980 Fluorescence Spectrometer which selects the fluorescence peak wavelengths of donor (QDs, 575nm) and acceptor (HA, 700nm) as the sampling points [26]. The results of the FRET systems under the same experimental conditions were shown in Fig. 4, Table 1 and Table 2. For understanding the results more visualizedly, the lifetime curves were analyzed using the software of FLS980 Fluorescence Spectrometer. The lifetime data was fitted by the biexponential function:
where τ1 and τ2 are the time constants, respectively, A1 and A2 are the normalized amplitudes of the components, respectively. The average lifetime was calculated by:Fig. 4 Fluorescence lifetime curves of QDs-HA solutions with various A/D concentration ratios at (a)a wavelength of 570 nm (the fluorescence peak wavelength of QDs), (b) a wavelength of 700nm (the fluorescence peak wavelength of HA).
Table 1. Fitted fluorescence lifetime of QDs with various A/D ratios in FRET systems. The fitting formula is y = A1*exp(-t/τ1) + A2*exp(-t/τ2), in which A1 + A2 = 1.
Table 2. Fitted fluorescence lifetime of HA with various A/D ratios in FRET systems.
The lifetime of HA is shown to be prolonged with increasing concentration of HA, while the lifetime of the QDs reduce, showing the energy transfer from the donor (QDs) to the acceptor (HA). Also, the presence of FRET is initially confirmed here. We also calculated the efficiency of FRET using the following equation [27]:
whereandare the fluorescence lifetime of the donor with and without the presence of the acceptor, respectively. The FRET efficiency is calculated for the QDs and HA mixed solutions with various HA concentration ratios using Eq. (4), and the results are shown in Table 3. It is shown that the lifetime of QDs drops as the HA concentration increases, while the FRET efficiency increases to 61.3%. We can see from Table 3, the energy-transfer efficiency decreased when the concentration of HA reach to 2mM, indicating that the concentration(1mM) was close to the limits of the FRET system. The energy-transfer efficiency of QDs& 1mMHA sample was the highest, so the HA concentration of 1mM was chosen as ideal in the remaining experiments in this study.Table 3. Fitted fluorescence lifetime and FRET efficiency of FRET systems
3.2Four-photon induced fluorescence spectral property of FRET system
Two-photon absorption is that the molecules absorb two incident photons to complete the level transition process. The same principle was also apply to a molecule absorb four photons simultaneously to complete the level transition between three real process, It was known as four-photon absorption.
The occurrence of four-photon absorption process must meet the following conditions of energy conservation [28]
The physical meaning can be expressed like this, The sum of photon energy absorbed by molecular should be equal to the level spacing between initial energy level and termination level. The attenuation of a bunch of quasi-monochromatic light in the nonlinear optical medium can be expressed as [28],
where I (z) is the intensity changes of light when it propagate along the z axis; are one, two, three, four-photon absorption coefficient, respectively. if the media is linear transparent in terms of the frequency of the incident light, there is. In this case, if we only consider the production of four-photon absorption process, the formula can be written as [28],The physical meaning of the formula can be expressed like this, the probability of four-photon absorption is proportional to the fourth power of the light intensity in a position of propagation process [29,30]. We can get the number of absorb photons in the four-photon-excited process, through measuring the relationship between fluorescence intensity excited by laser and intensity of incident light.
According to the research experience, four-photon excitation wavelength selection may follow following rules [28]:
Based on the empirical formula and spectral feature shown in Fig. 5, 1300nm wavelength were chosen for effective four-photon excitation of QDs-HA systems.
First, we measured the fluorescence intensity of QDs as a function of intensities of the input ~1300nm laser pulses to verify the four-photon excitation identities, respectively. The experimental data are shown in Fig. 6, the data excited at ~1300nm can be fitted by a straight line with a slope of 4.12. These results basically indicated the four-photon excitation features at these wavelengths, it consistent with the principles of the formula(7).
Fig. 6 Measured fluorescence intensity as a function of the input laser intensity at 1300nm wavelengths, and the best fitting straight lines.
The four-photon absorption induced fluorescence spectra for two FRET system were excited by ~1300nm and ~50fs laser pulses from a Femtosecond laser system (Libra–USP-HE from FEI, Inc.) operating at a repetition rate of 1 kHz. The results, recorded by a spectrometer (Ocean QE65000), are shown in Fig. 7 by the dash curves. We also measure one-photon induced fluorescence spectra by using ~350nm for the sample solution, the results are also shown in Fig. 7 by the solid-line curves. From Fig. 7 we can see that the one- and four-photon induced spectral curves for each given systems all have three peaks, and the position of these paks are nearly the same, showing that the systems obtained great effects under four-photon excited, the results are beneficial for further biological application. There is a little red-shift about 9nm of the peak emission position for ZnSe:Mn/ZnS QDs. This little red-shift is due to re-absorption of the fluorescence signal propagating in a high concentration solution sample, which can be ignored in a low concentration sample.
4. Evaluation on cells by femtosecond laser and QDs-HA systems
Prior to embarking on in vitro and in vivo studies, the cytotoxicity of ZnSe:Mn/ZnS QDs were evaluated by MTT cell viability assay, using the MCF-7 cell line. From our findings, we have observed that the cell viability of MCF-7 cells was maintained above 70% with concentration as high as 300ug/mL, at 24 and 48h post-treatment (Fig. 8). The cytotoxicity data suggests that the prepared ZnSe:Mn/ZnS QDs have negligible in vitro toxicity and demonstrate their usefulness for long term in vitro and in vivo imaging studies.
In order to confirm the occurrence of FRET between ZnSe:Mn/ZnS QDs and HA, femtosecond laser was used to irradiate cells, the excitation wavelength were selected at 1300nm. The 1300nm wavelength can induce FRET in the QDs-HA systems because of the four-photon excitation effect of NIR femtosecond laser of ZnSe:Mn/ZnS QDs, but had no influences on HA itself. These create a fair condition for the comparison of the free HA and QDs-HA systems (Fig. 9(a)).
Fig. 9 (a) The FRET damaging on MCF-7 cells treated with different FRET systems. (b) The FRET damaging on MCF-7 cells with and without irradiation. The concentration of samples 1-6 were 1µM, 5µM, 50µM, 0.5mM, 1mM, and 1.5mM, respectively.
Under the irradiation of 1300nm femtosecond laser, no significant damaging could be found for most HA samples. Only when the concentration was 2mM, the cell viability come to 50%, improving this concentration is a little high for cells. These high concentration needs to be used according to the severity of disease. This result can be understood as 1300nm are not the suitable excitation wavelength of HA. In this conditions, although HA molecules can enter into the cells, the damaging effect of HA alone is slight.
Completely contrary conditions could be observed in QDs-HA samples, the effect of QDs-HA are obvious, as most cells have been killed after irradiation of 1300nm femtosecond laser, the reasons could be explained like, ZnSe:Mn/ZnS QDs can be excited by NIR femtosecond laser, due to its excellent nonliner optics properties (a high multi-photon absorption cross section). Once the FRET system was excited by laser, the energy will transfer from donor (QDs) to the acceptor (HA), this energy inspired the efficacy of HA to kill the cells. We can see from Fig. 9(a), the cells viability decreased with the increasing acceptor (HA) concentration. When the concentration is 1mM, the cells viability is lowest (15.2%) because of the highest FRET efficiency (61.3%) in the ZnSe:Mn/ZnS QDs-1mM HA FRET system.
In Fig. 9(b), cells were incubated with QDs-HA for 4h, the red line represent the cell viability without irradiation. The blue line represent the cells irradiated by 1300nm femtosecond laser after incubating with QDs-HA conjugates for 4h, significant damaging could be found for MCF-7 cells. The cell viability reached to 15.2%. These results suggest that the QDs-HA conjugates in the cellular environment can further perform FRET in cells.
With the low power femtosecond (1300nm) laser beam, effective damaging of cancer cells by ZnSe:Mn/ZnS QDs-HA system was achieved, demonstrating that the new ZnSe:Mn/ZnS QDs-HA system have great potential in both optics and biological applications.
5. Conclusion
We achieved FRET system with ZnSe:Mn/ZnS QDs and HA solutions. Four-photon excitation properties of the FRET systems were studied by using ~50fs laser pulses at wavelengths of 1300nm. The experimental and theoretical analysis indicated that the FRET efficiency increases as acceptor concentration increases. When the acceptor concentration reaches to 1mM. The cell killing rate of cancer cells can reach to 84.8% . This study shows that there is a bright future for these materials in bio-probing, bio-imaging, and other biological and optoelectronic applications.
Funding
National Natural Science Foundation of China (NSFC NO.11204020); Nanophotonics and Biophotonics Key Laboratory of Jilin Province, China. (20140622009JC and 14GH005)
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