Förster resonance energy transfer properties of a new type of near-infrared excitation PDT photosensitizer: CuInS₂/ZnS quantum dots-5-aminolevulinic acid conjugates

Abstract

Recently, near-infrared (NIR) excitation has been suggested for PDT improvement and therapy of cancer. In this study, 5-aminolevulinic acid (ALA), a photosensitizer used for photodynamic therapy (PDT) of cancer, was coordinated to CuInS₂/ZnS (CIS/ZnS) QDs to form CIS/ZnS–ALA conjugates, by chemical bonding. An efficient transfer of energy from the donor (QDs) to the acceptor (ALA) was demonstrated through Förster resonance energy transfer (FRET), and the FRET efficiency of QDs–ALA system can reach as high as 58.49% under near-infrared excitation. The optical properties (fluorescence spectra, Fourier transform-infrared spectra and fluorescence lifetimes) were also determined for the conjugates. In addition, the PDT killing in vitro was further achieved under the 800 nm and 1300 nm femtosecond laser pulses. These results demonstrate that the CIS/ZnS–ALA conjugates are promising for PDT and needed to be investigated further.

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1 Introduction

Photodynamic therapy (PDT) is a localized light-activated treatment to destroy tumor tissues, and it is applied to treat not only skin and eye diseases but also to eliminate other cancer cells^1,2. The photosensitivity and the singlet oxygen accumulation ability of cancer cells are better than those of normal cells, so irradiation of the target region by a light source kills the cancer cells preferentially.^3,4 This fact makes PDT a candidate for a targeted therapeutic strategy in cancer treatment.^5,6

Attention to ALA-based photodynamic therapy (PDT) is increasing in the biomedicine field. ALA is a naturally occurring substance, which is normally synthesized from succinyl CoA and glycin.⁷ This synthesis process is inhibited in the presence of heme. ALA does not have the characteristics of a photosensitizer, but it has the function of a photosensitizer after undergoing a metabolic process transformation to the photosensitizer protoporphyrin IX (PpIX) in vivo. This photosensitizer has low toxicity to cells, and the total body clearance (24–48 h) is rapid.⁸ If 5-ALA is applied exogenously, it can be absorbed selectively by cancer cells. The negative feed-back mechanism is surpassed and excessive protoporphyrin IX accumulates in the cells. However, PpIX can only be excited by light in the 300–650 nm region. This disadvantage greatly limits its PDT applications, because the short wavelength light cannot penetrate deeply into living tissues under one-photon excitations. Therefore, longer excitation wavelength (near-infrared wavelength) light has been suggested for PDT improvement, and the so-called multi-photon excitation PDT needs to be seriously studied.

Under these conditions, the use of quantum dots conjugated to photosensitizers such as ALA is a very promising approach for PDT; this new type of functional nanoparticle give us the ability to perform both imaging and treatment. Quantum dots (QDs) possess excellent photoluminescence properties that make them potential candidates for biological applications (e.g. tissue imaging, cancer treatment).^9,10 However, most of the high-quality QDs were composed of heavy metal elements (such as Cd, Pb and Hg) in previous studies.¹¹ These heavy metal elements raise tremendous concerns about the toxicity of these QDs in the biomedical community.¹² Development of QDs composed of fewer toxic components is a potential strategy to deal with the heavy metal-related toxic effects in the area of biomedical research.

Moreover, in recent years, many researchers have devoted a lot of efforts to synthesizing near-infrared (NIR) fluorescent QDs with long lifetime photoluminescence, excellent nonlinear optical properties, and good biocompatibility.^13,14 The most interesting spectral regions are 750–850 nm and 1000–1400 nm, the so-called first and second optical window of the tissue. A good example of promising QDs are the ternary CIS QDs and core/shell CIS/ZnS QDs, which have already demonstrated spectral characteristics matching those required for red and NIR region applications.^15–18 These types of CIS/ZnS QDs exhibit two-photon absorption cross sections (TPACS) in the visible and NIR regions that are much larger than the bulk due to the quantum confinement effect. In the previous decades, the CIS/ZnS QDs were widely used in bioimaging, because of their unique optical and biological properties (low toxicity, long fluorescence lifetimes and nonlinear properties), but they are rarely applied in photodynamic therapy.^19,20

These types of NIR QDs can be used to activate a photosensitizer through the process called Förster resonance energy transfer (FRET). FRET is a process whereby excited state energy is transferred nonradiatively from an excited donor molecule to an acceptor molecule.^21–23 The extent of FRET occurrence is usually expressed as FRET efficiency, i.e. the proportion of the photons absorbed by the donor that is transferred to the acceptor.²⁴ Due to the excellent nonlinear properties of QDs, the covalent linking of the QDs and ALA allow us to use the excitation wavelengths wherein the ALA alone couldn't achieved. The QDs–ALA conjugates have the optical advantages of quantum dots and the treatment efficacy of ALA, and they can overcome the weaknesses of QDs or ALA alone in cancer treatment.

In this study, we first put forward a new multi-photon excitation PDT photosensitizer, i.e. CIS/ZnS QDs–ALA conjugates. CIS/ZnS QDs with different emission wavelengths were synthesized. Then, the CIS/ZnS QDs were coordinated with ALA to form CIS/ZnS QDs–ALA conjugates. These types of conjugates overcome the limitation of the excitation wavelength of ALA. The multi-photon excitation advantage of CIS/ZnS QDs determines the ability of the conjugates to be excited by a longer wavelength laser to achieve the therapeutic effect. The excitation wavelength can range from 635 nm to 1300 nm in our work, compared to the 700 nm excitation wavelength of Wang's study.²⁵ Longer wavelength of the laser used means deeper penetration depth and less damage to the organism, and all this can greatly improve the therapeutic effect in biomedicine.

2 Experimental details

2.1. Materials

Copper(II) chloride (CuCl₂, 99%), indium chloride hydrate (InCl₃·4H₂O, 99.9%), zinc chloride (ZnCl₂, 98%), sodium sulfide (Na₂S, 99%), 3-mercaptopropionic acid (MPA, >99%) and 5-aminolevulinic acid (ALA, >97%) were purchased from Sigma Aldrich. N-Hydroxysuccinimide (NHS, 98%) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, >98%) was obtained from Tokyo Chemical Industry (TCI). All chemicals were used as received. Deionized water (DI water) was used throughout the experiment.

2.2. Equipment

Ultraviolet-visible (UV-vis) spectra were obtained using a Cary 5000 UV-vis spectrophotometer (Agilent Technologies Inc, California, USA). The emission spectra were obtained on a Cary Eclipse Fluorescence Spectrometer (Agilent Technologies Inc, California, USA). Transmission electron microscopy (TEM) images were obtained using a G² 20 S-TWIN transmission electron microscope (FEI Tecnai, Hillsboro, USA). Infrared spectra were obtained on a Nicolet iS50 FT-IR spectrometer (Thermo Scientific, Massachusetts, USA). Fluorescence lifetimes were measured using a FLS980 Fluorescence Spectrometer (Edinburgh Instruments, Livingston, UK). A Ti:sapphire femtosecond laser (Coherent Inc, CA, USA) was used in PDT measurements. Fluorescence images of cells were observed with a DMI3000B Microscope (Leica, Wetzlar, Germany).

2.3. Synthesis of QDs–ALA conjugates

2.3.1. Preparation of core/shell CIS/ZnS QDs. The synthesis method for forming a ZnS shell on CIS QDs was adapted from Zhang et al.²⁶ CIS QDs solution was prepared in advance. Separately, 0.04 M of ZnCl₂ and 50 μL of MPA were dissolved in 5 mL of DI water. The pH was adjusted to 10 by dropwise addition of NaOH solution. The reaction mixture was heated to 60 °C and then injected into the hot CIS QDs solution. The reaction temperature of the mixture was held at 98 °C for 30 min to form the core/shell CIS/ZnS QDs.

2.3.2. Synthesis of QDs–ALA conjugates. ALA was covalently linked to CIS/ZnS QDs as follows. CIS/ZnS QDs (2.5 mM) was dissolved in 2 mL DI water. Then, 100 μL of 1.2 mM EDC was added to activate the carboxylic group (–COOH) of the QDs. The mixture was allowed to stir for 5 min at room temperature. After this time, 100 μL of 3.4 mM NHS was added to the activated QDs solution, and it was stirred for 30 min at room temperature. Lastly, 1 mL of 1 mM ALA was added to the mixture solution and stirring continued for a further 4 h to allow conjugation of the CIS/ZnS QDs to ALA to occur. The compounds were precipitated out of solution with ethanol and centrifuged to remove unlinked QDs or ALA. The conjugates are represented as QDs(1)–ALA, QDs(2)–ALA, QDs(3)–ALA for the functional conjugates of different emission wavelengths.

2.4. Detection of fluorescence in MCF-7 cells due to QDs–ALA treatment

SKMEL-30 cells were seeded into a petri dish and incubated overnight to allow cell attachment. QDs–ALA conjugates were added to the medium of treatment wells and incubated for 4 h to ensure that the conjugates entered the cells. Then, the cells were trypsinized and washed twice with PBS. Freezing and thawing was used for cell disruption, letting the FRET system flow out of cells, and the treated liquid was collected into new dark tubes for the detection of fluorescence spectra and lifetimes.

2.5. Cytotoxicity assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium reduction assay was used to measure the cytotoxicity of QDs–ALA conjugates on cells. The MCF-7 cells seeded in 96-well plates were incubated with CIS/ZnS–ALA solutions with different concentrations for 24 h. Subsequently, 20 μL of MTT solution (5 mg mL⁻¹) was added to each well. After another 4 h 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.

2.6. PDT measurements of QDs–ALA conjugates in cells by near-infrared excitation

MCF-7 cells were seeded in 96-well microtiter plates for use. Then, 1 mM ALA, 2 mM ALA, CIS/ZnS–1 mM ALA conjugates and CIS/ZnS–2 mM ALA conjugates were added to the medium of treatment wells (except for the control wells) and incubated for 4 h. Then, a femtosecond laser with pulse widths of 50 fs, a repetition frequency of 1 kHz, and fixed power of 100 mW was used to irradiate the wells for 120 seconds. The cells were incubated further for 24 h after irradiation. MTT cell viability assay and fluorescence imaging of the cells were used to evaluate the results.

3 Results and discussion

3.1. Characterization of QDs

In this study, QDs with three different wavelengths (635 nm, 660 nm and 730 nm) were conjugated with ALA.²⁷ The evolution of the absorption and emission spectra of CIS/ZnS QDs during the synthesis at various molar ratios of Zn is shown in Fig. 1(a). In the case of CIS/ZnS QDs, the content of Zn plays an important role in determining the emission peak of the QDs. When Zn was introduced at the content ratio of 10% (CIS/ZnS(3)) and 20% (CIS/ZnS(2)), the absorption and emission peaks of the QDs shifted to shorter wavelength. A more significant blue-shift of fluorescence spectra from 730 nm to 635 nm can be observed when the Zn content increased to 40% (CIS/ZnS(1)). The blue-shift of the absorption and emission spectra indicates widening of the bandgap as a result of the Zn incorporation. Moreover, an enhancement of emission intensity was observed when the CIS QDs were incorporated with 10%, 20% and 40% of Zn. The possible explanation for the above phenomenon is that the incorporation of Zn reduced the structural defects, stabilized the crystal structure and inhibited the nonradiative recombination.

Fig. 1 (a) Emission spectra of CIS/ZnS QDs with different Zn content. (b) The absorption spectrum of protoporphyrin IX.

Once ALA enters the cells, the external addition of ALA results in enhanced synthesis of protoporphyrin IX (PpIX) by the cellular heme biosynthetic pathway. Therefore, the absorption spectrum of PpIX was also given in Fig. 1(b). It demonstrated four weaker absorption bands at 506 nm, 542 nm, 575 nm and 630 nm.

3.2. Characterization of QDs–ALA conjugates

In the conjugation, the addition of EDC and NHS to the QDs solution resulted in the formation of a highly reactive intermediate (NHS-carboxylate). This activated ester then reacted with the free amino group of ALA, giving the conjugates of ALA with CIS/ZnS QDs as illustrated. In this study, we intended to use the CIS/ZnS–ALA conjugates to achieve multi-photon excitation PDT, because the CIS/ZnS dots have very high two-photon absorption cross section. The design of this study is shown in Scheme 1.

Scheme 1 Schematic of the covalent linkage of the CIS/ZnS QDs to ALA.

TEM images of CIS/ZnS QDs, ALA and ALA-conjugated CIS/ZnS QDs are shown in Fig. 2. All particles appear spherical in shape and surface functionalization did not induce aggregation. The average diameters of ALA, CIS/ZnS QDs, and ALA-conjugated CIS/ZnS QDs are 12 nm, 7 nm and 20 nm, respectively.

Fig. 2 TEM images of: (1) ALA, (2) CIS/ZnS QDs and (3) CIS/ZnS QDs–ALA conjugate.

ALA molecules were conjugated to the quantum dots using EDC/NHS coupling agents that activate the carboxylic groups. FT-IR was employed to further characterize the complexes and also to confirm the formation of CIS/ZnS–ALA nanoconjugates. In Fig. 3(a), C=O vibration of ALA is observed at 1683 cm⁻¹ and the –NH₂ band is found at 3438 cm⁻¹. With the CIS/ZnS QDs (Fig. 3(b)), we observe a COO– stretching band at 1655 cm⁻¹ and the corresponding –OH band at 3300 cm⁻¹.²⁸ The amide bond can be characterized by the complex bands of –CO–NH–, the –CO–NH– mode that contains contributions from the –C–O– stretching vibration and the –NH– bending vibration, which originate from bonding between the carboxy groups of the MPA capped QDs and the amide groups of ALA.²⁹ In Fig. 3(c), formation of characteristic amide bands at 1578 cm⁻¹ confirms the success of the linkage. This band was absent in the spectrum for the QDs alone. Moreover, the disappearance of the COO– stretching band at 1655 cm⁻¹ and –NH₂ band at 3438 cm⁻¹ obtains further confirmation of this process.

Fig. 3 FT-IR spectra of: (a) ALA, (b) CIS/ZnS QDs and (c) CIS/ZnS QDs–ALA conjugate.

Fig. 4 displays the fluorescence lifetime spectrum of CIS/ZnS(1) quantum dots, CIS/ZnS(1)–ALA conjugates and a CIS/ZnS–ALA mixture excited at 377 nm. The concentration of CIS/ZnS quantum dots is the same in all three samples. Once the CIS/ZnS(1)–ALA conjugates are synthesized successfully, the energy is transferred from the donor to the acceptor. The FRET process can be clearly reflected in the fluorescence lifetime studies, as shown in Fig. 4. The fluorescence lifetime of CIS/ZnS–ALA mixture is nearly the same as that of the CIS/ZnS quantum dots, which shows that FRET cannot occur in the mixture solution. However, in the CIS/ZnS(1)–ALA conjugates, the fluorescence lifetime is much shorter than that of the pure CIS/ZnS(1) quantum dots. It is well known that the fluorescence resonance energy transfer can shorten the fluorescence lifetime and quench the fluorescence of QDs. Similar changes were observed for CIS/ZnS(2)–ALA conjugates and CIS/ZnS(3)–ALA conjugates (fluorescence lifetime spectra of the other two conjugates CIS/ZnS(2)–ALA and CIS/ZnS(3)–ALA are not shown).

Fig. 4 Fluorescence lifetimes for CIS/ZnS, CIS/ZnS–ALA conjugated sample, and a mixture of CIS/ZnS and ALA.

3.3. Förster resonance energy transfer (FRET) studies

Because it is an absorption wavelength of the QDs with no influence on ALA, the excitation wavelength of the QDs–ALA conjugates was set at 450 nm. FRET between the photosensitizer (PpIX, from conversion of ALA) and the various QDs was verified by a decrease in the emission intensity of the donor (CIS/ZnS QDs). The different extents of decrease in the emission intensity of the CIS/ZnS QDs in the conjugates can be observed in Fig. 5. Black lines are the QDs alone and red lines represent the emission spectra of CIS/ZnS–ALA conjugates. The fluorescence intensity of QDs–ALA conjugates gradually decreased with the reduction of the fluorescence wavelength of QDs, proving that there was added energy transfer from the QDs to photosensitizer, namely, the extent of fluorescence resonance energy transfer increased.

Fig. 5 Fluorescence emission spectra of (a) CIS/ZnS(1) QDs and CIS/ZnS(1)–ALA conjugates, (b) CIS/ZnS(2) QDs and CIS/ZnS(2)–ALA conjugates and (c) CIS/ZnS(3) QDs and CIS/ZnS(3)–ALA conjugates (black lines are the QDs alone, red lines are CIS/ZnS–ALA conjugates).

FRET efficiency was estimated from the change of lifetimes in the QDs–ALA conjugates and the values were summarized in Table 1. The lifetimes were obtained from the fitted fluorescence lifetime curves (red lines in Fig. 4). The fitting formula is³⁰

y = A₁ × exp(−t/τ₁) + A₂ × exp(−t/τ₂) (1)

where τ₁ and τ₂ are the fluorescence lifetimes of QDs–ALA conjugates and A₁ + A₂ = 1. The efficiency of FRET could be calculated based on the following equation:³¹where τ_DA and τ_D are the fluorescence lifetime of the donor with and without the presence of the acceptor, respectively. The FRET efficiency was calculated for the QDs–ALA conjugates with different emission peaks of QDs based on eqn (2), and the results were shown in Table 1.

Table 1 Förster resonance energy transfer parameters of the QDs–ALA conjugates

Samples	Fluorescence peak	Average lifetime	FRET efficiency (%)
CIS/ZnS(1)	635 nm	155.741
CIS/ZnS(1)–ALA	635 nm	82.025	47.33
CIS/ZnS(2)	660 nm	159.057
CIS/ZnS(2)–ALA	654 nm	109.208	31.34
CIS/ZnS(3)	730 nm	164.354
CIS/ZnS(3)–ALA	723 nm	151.485	7.83

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The FRET efficiency obtained in this study is significantly different in different QDs–ALA conjugates. The efficiency can reach 47.33% in CIS/ZnS(1)–ALA conjugates, whereas only 7.83% in CIS/ZnS(3)–ALA conjugates. Obviously, as expected, we chose CIS/ZnS(1)–ALA conjugates as the most suitable candidate in the following experiments, because its higher efficiency and better treatment effect could be useful in further applications.

We used CIS/ZnS(1) QDs and ALA in different concentrations to synthesize different CIS/ZnS–ALA conjugates, and fluorescence spectra of different CIS/ZnS–ALA conjugates were shown in Fig. 6. With the increasing concentrations of ALA, the fluorescence intensity of the CIS/ZnS–ALA conjugates decreased gradually. Because the number of ALA molecules around the QDs increased in the conjugate solution, the energy transfer efficiency of the QDs–ALA conjugates became higher.

Fig. 6 Fluorescence spectra of CIS/ZnS–ALA conjugates in different concentrations.

We obtained fluorescence lifetime spectra of the CIS/ZnS–ALA conjugates in different concentrations, because the FRET process can be clearly demonstrated in the fluorescence lifetime studies. Using the same method used to generate Fig. 4, we obtained the lifetimes of the CIS/ZnS–ALA conjugates from the fitted fluorescence lifetime curves. The results were summarized in Table 2. With the increasing concentrations of ALA, the fluorescence average lifetime of CIS/ZnS–ALA conjugates decreased and the FRET efficiency increased gradually. We can observe from Table 2 that the FRET efficiency became nearly 50% when the concentration of CIS/ZnS–ALA conjugates reached 1 mM; when the concentration reached about 5 mM, the efficiency became 60%, and the treatment effect become more obviously. Therefore, we can select the appropriate concentration in the proper range (ALA: 1–5 mM) to synthesize CIS/ZnS–ALA conjugates according to the severity of the disease (Fig. 7).

Table 2 The decay constants of the fitted fluorescence lifetimes curves and FRET efficiency of CIS/ZnS–ALA conjugates

Samples	A1	τ1 (ns)	A2	τ2 (ns)	Average lifetime	FRET efficiency (%)
CIS/ZnS	0.0264	10.709	0.9736	159.674	155.741
CIS/ZnS–0.5 mM ALA	0.2675	8.186	0.7325	133.544	100.011	35.78
CIS/ZnS–1 mM ALA	0.2942	7.804	0.7058	112.962	82.025	47.33
CIS/ZnS–2 mM ALA	0.2807	6.977	0.7193	107.963	79.616	48.88
CIS/ZnS–3 mM ALA	0.3032	6.261	0.6968	101.856	72.872	53.21
CIS/ZnS–5 mM ALA	0.2607	6.135	0.7393	85.291	64.655	58.49

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Fig. 7 Fluorescence lifetime spectra of CIS/ZnS–ALA conjugates in different concentrations.

3.4. Cell viability study of CIS/ZnS–ALA conjugates

Prior to embarking on in vitro and in vivo studies, the cytotoxicity of the CIS/ZnS–ALA conjugates was evaluated by MTT cell viability assay, using the MCF-7 cell line. From our findings, we observed that the cell viability of MCF-7 cells was maintained above 70% with concentrations as high as 300 μg mL⁻¹, at 24 and 48 h post-treatment (Fig. 8). The cytotoxicity data suggests that the prepared CIS/ZnS–ALA conjugates have negligible in vitro toxicity and demonstrate their usefulness for long term in vitro and in vivo imaging studies.

Fig. 8 Cell viability of MCF-7 cells treated with CIS/ZnS–ALA for 24 h and 48 h.

3.5. Evaluation of photodynamic therapy on cells by femtosecond laser and CIS/ZnS–ALA conjugates

A femtosecond laser was used to irradiate cells for the purpose of photodynamic therapy; the excitation wavelengths were selected as 800 nm and 1300 nm. The 800 nm and 1300 nm wavelengths can induce FRET in the CIS/ZnS–ALA conjugates because of the multi-photon excitation effect of the NIR femtosecond laser on CIS/ZnS QDs,³² but they had no influence on ALA itself. This creates fair conditions for the comparison of the free ALA and CIS/ZnS–ALA conjugates (Fig. 9).

Fig. 9 The PDT damaging of MCF-7 cells, measured by an MTT assay.

Under the irradiation of the 800 nm and 1300 nm femtosecond laser, no significant damage could be found for 1 mM ALA and 2 mM ALA samples. This result can be understood as NIR wavelength are not the suitable excitation wavelength for ALA. In these conditions, although ALA molecules can enter the cells, the non-existent two-photon absorption cross section of ALA make the PDT ineffective, and the damaging effect of ALA alone is slight.

Completely different results could be observed in CIS/ZnS–1 mM ALA conjugates and CIS/ZnS–2 mM ALA conjugates samples; the PDT effect of CIS/ZnS–ALA conjugates is obvious, as most cells are killed after irradiation by the 800 nm and 1300 nm femtosecond laser. The results could be explained by the following. CIS/ZnS can be excited by the NIR femtosecond laser, due to its excellent nonlinear optics properties (a high two-photon absorption cross section). Once the QDs are excited, the energy is transferred from the donor to the acceptor, and this energy enables the ALA to effectively kill the cells. With this low power density femtosecond laser beam, effective damaging of cancer cells by CIS/ZnS–ALA conjugates was achieved, demonstrating that the new CIS/ZnS–ALA conjugates have great potential for PDT.

According to the results, representative micrographs showing cell morphology were presented (the results of irradiation of 1300 nm femtosecond laser was set as example). In Fig. 10(a), MCF-7 cells were irradiated by a 1300 nm femtosecond laser and then incubated in growth medium for 24 h. Fig. 10(b) shows that the cell morphology was destroyed after CIS/ZnS–ALA treatment. It was deduced from the results that CIS/ZnS–ALA conjugates are suitable for photodynamic therapy applications.

Fig. 10 (a) MCF-7 cells after 24 h incubation in growth medium (b) MCF-7 cells after 24 h of 1 mM 5-ALA treatment.

The cellular uptakes of these conjugates were studied with MCF-7 cells by fluorescence imaging. The fluorescence images of the CIS/ZnS–ALA conjugates in cells are shown in Fig. 11. In Fig. 11(b), cells were incubated with CIS/ZnS–ALA for 4 h, but without treatment. The cell morphology remains intact and the cells were labeled successfully with quantum dots. In Fig. 11(c), cells were irradiated by a 1300 nm femtosecond laser after incubating with CIS/ZnS–ALA conjugates for 4 h, and significant damage could be found of MCF-7 cells. These results suggest that the CIS/ZnS–ALA conjugates in the cellular environment can further perform FRET in cells.

Fig. 11 Fluorescence images of conjugates in MCF-7 cells. (a) Control untreated MCF-7 cells irradiated by 1300 nm. (b) Cells incubated with CIS/ZnS–ALA conjugates for 4 h without irradiating. (c) Cells incubated with conjugates for 4 h and irradiated by 1300 nm.

4 Conclusion

Designing and developing a new “photosensitizer” with high TPACS is a route to promoting multi-photon excitation PDT, because multi-photon excitation PDT with NIR lasers is believed to be an excellent way for PDT developments. However, traditional photosensitizers are not efficient for multi-photon excitation PDT. Therefore, establishing new conjugates is a systematical work; not only TPACS but other properties are concerned, including the stability, biocompatibility and toxicity, and they should be thoroughly studied and made suitable for practical PDT treatments. In this study, we designed a new type of CIS/ZnS–ALA conjugate. CuInS₂/ZnS (CIS/ZnS) quantum dots with different emission peaks were conjugated to 5-aminolevulinic acid (ALA), and the conjugates are represented as CIS/ZnS(1)–ALA, CIS/ZnS(2)–ALA and CIS/ZnS(3)–ALA. There was a decrease in the fluorescence lifetimes of CIS/ZnS quantum dots in the presence of ALA due to the Förster resonance energy transfer (FRET). We calculated the FRET efficiency of the three types of CIS/ZnS–ALA conjugates and found that CIS/ZnS(1)–ALA was the most suitable conjugate for further application because of its highest FRET efficiency, which reached 58.49%. The FRET mediated PDT of CIS/ZnS–ALA conjugates under NIR femtosecond laser can be successfully carried out to achieve destruction of cancer cells. Our results suggest that the conjugation of multi-photon excitation QDs with ALA could be a simple and effective modality to improve practical PDT applications. The cells viability results of the conjugates after irradiation by 800 nm and 1300 nm femtosecond lasers were less than 40%, suggesting that these conjugates are suitable candidates for PDT as functional anticancer agents for cancer treatment.

Acknowledgements

The authors would like to acknowledge the support of the National Natural Science Foundation of China (NSFC No. 11204020), Nanophotonics and Biophotonics Key Laboratory of Jilin Province. P. R. China (20140622009JC) and (14GH005).

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