Introduction

Photodynamic therapy (PDT) is a useful method involving PS and specific light source for the treatment of various cancers, and it has drawn considerable attention due to its high selectivity, few side effects, low invasion and drug resistance [1,2,3]. However, the hydrophobicity, self-aggregation and low quantum yield of usual photosensitizers (PSs) were adverse to apply in aqueous physiological environment [4, 5]. Furthermore, a certain amount of specific light source is critical to activate PS to generate reactive oxygen species (ROS) [6]. While the penetration depth of light required for PS activation in clinic usually remains a major problem for wide application of PDT [7, 8]. Several external lights such as X-ray and near-infrared light have been explored to enhance the efficiency of PDT in combination with two-photon excitation and upconversion nanoparticles [9, 10]. All the same, there is still a challenge to reach most intracorporal tumors. Thus, designing a PS that can be activated inside the cells will increase immensely the role of PDT in deep lesions.

Firefly bioluminescence (BLS) is undoubtedly the most well known and studies BLS system. The luciferase of firefly is found in nature and can be obtained from the market, it has been widely used for enzymatic measurement of ATP [11], and its gene can be applied as a reporter for gene expression experiments [12]. The first attempt to test the potential of firefly BLS-mediated PDT was made in 2003 by Theodossiou [13]. In this work, the cancer cells were transfected with a modified luciferase gene and substrate and the PDT system led to a 90% toxicity rate to the cancer cells. However, this report was based on the gene delivery, which was still only at clinical trials level. Schipper and co-workers following measured the low intensity of firefly BLS could not generate sufficient light to induced PDT in vitro [14]. The difference between two reports can be attributed to the d-luciferin (DL) concentration. Whereas Theodossiou et al. used concentrations of 500 µM, Schipper and co-workers only used concentrations as high as 20 µM. There remain some doubts as to whether firefly BLS generates sufficient light to induce photodynamic toxicity in cancer cells. Herein, we select middle DL concentrations of 100 µM to further explore in this paper.

CDs, first discovered during the purification of single-walled carbon nanotubes in 2004 [15], have been proved the availability in the fields of cell imaging [16], chemical analysis [17], optical device [18], gene delivery [19], and photo-catalysis [20]. We [21], and others [22], had demonstrated the CDs could be used as nano-carriers for PS and enhanced the PDT efficiency through FRET. Meanwhile, CDs are sensitive to the photons, which is facilitated to absorb the light of BLS to avoid wasting. So here firefly luciferase (FLUC)-induced BLS was explored as inner illumination light to extend the use of PDT in deeper seated cancers. The water-solubility and excitation-independent PL properties of CDs were carefully prepared to construct a bridge between BLS and PIX, and then to form a united PDT system (Scheme 1).

Scheme 1
scheme 1

Schematic illustration of BLS-initiated PDT supported by high-luminescent and upconverted carbon dots

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Experimental section

Chemicals and materials

Citric acid, magnesium sulfate (MgSO4), and ethylenediamine (EDA) were purchased from Alfa Aesar. 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), PIX, and 1,3-diphenylbenzofuran (DPBF) were obtained from Sigma. FLUC, d-luciferin (DL), and adenosine triphosphate (ATP) were supplied by Fluka. All the other reagents were of analytical grade and used without further purification.

Characterization

UV–Vis absorption was measured on a TU-1810 UV–Vis Spectrophotometer (Pgeneral, China). Photoluminescence emission measurements were performed using FLS920 fluorometer (Edinburgh Instruments, Britain). The morphology and microstructure of the CDs were examined by high-resolution transmission electron microscopy (HRTEM) on a Philips Tecnai G2 F20 microscope (Philips, the Netherlands) with an accelerating voltage of 200 kV. The samples for HRTEM were made by dropping an aqueous solution onto a 300-mesh copper grid coated with a lacy carbon film. The FTIR spectra of the samples were measured on a Nicolet 380 spectrometer (Thermo, America). Zeta potential was tested by NICOMP 380ZLS (PSS, America) in PBS.

Synthesis of CDs

Typically, 2.1 g citric acid was dissolved in 50 ml distilled water followed by an addition of 2.68 ml EDA. The mixed solution was stirred vigorously to form a colorless, transparent, and homogeneous solution. Then, a normal hydrothermal method was applied at 200 °C for 5 h around. Subsequently, the obtained products were cooled down to room temperature. At last, CDs can be acquired after further filtration, dialysis, and lyophilization.

Synthesis of CDs-PIX conjugate

1 mg PIX dissolved in 1 ml DMF was added to 1 mg EDC and 0.6 mg NHS. The solution was stirred at 25 °C for 30 min. Then, 1 mg CDs dissolved in 5 ml Tris buffer was added, and the solution was stirred for 24 h. The as-prepared CDs-PIX conjugate was washed with deionized water and ethanol alternatively to remove the unreacted CDs and PIX and then further dialyzed to replace the solvent.

SO detection in vitro

The generation of SO by the CDs-PIX conjugate was detected chemically using the spectrophotometric method, in which DPBF was used as the chemical detector. In a typical experiment, 100 µl 20 µM CDs-PIX was placed in quartz cuvette and followed by adding 100 µl 5 mM DPBF dissolved in DMF. The solution was aerated for 10 min. Then, FLUC and its substrates were added to produce BLS as exciting light. The CDs-PIX and BLS alone were set up as two control experiments. All the solutions were detected in the fluorescence spectrometer, and the visible absorbance was recorded at intervals.

Measurement of ROS production

Intracellular ROS level in SMMC-7721 cells treated was detected by staining treated with the fluorescent dye 2′7′-dichlorodihydrofluorescein diacetate (H2DCFDA). Cells were seeded in five plates as follows: two control groups (negative control: PBS; positive control: 40 mM H2O2) and three experimental groups (CDs-PIX, FLUC/DL/Mg2+/ATP, CDs-PIX/FLUC/DL/Mg2+/ATP). The positive control cells were treated with 40 mM H2O2 at 37 °C for 30 min, while the other groups were cultured at 37 °C for 24 h. Then, the cells in all groups were washed with PBS and incubated with 10 µM H2DCFDA for 50 min at 37 °C. Subsequently, 200 µl DAPI solution (original solution diluted to one thousand times when used) was added and incubated for 10 min. The samples were examined under a Leica confocal laser scanning microscope (Mannheim, Germany) using an excitation of 488 nm for H2DCFDA and an excitation of 405 nm for DAPI. Intracellular ROS generation caught by H2DCFDA was also quantitatively measured by flow cytometer.

Results and discussion

HRTEM and PL emission spectra were conducted to characterize the size and optical properties of the as-prepared CDs. The HRTEM images, as shown in Fig. 1a, displayed that CDs were well distributed without apparent agglomeration and the average particle diameter was 2 nm around. The quantum yield of as-prepared CDs reached 79.7% using quinine sulfate (54%) as a standard (Fig. S1). To further explore the optical properties of as-prepared CDs, we carried out a detailed PL study (Fig. 1b, c). With excitation from 320 to 480 nm, the maximum emission peak position shifted to long wavelength gradually but in a non-uniform mode. While PL intensity of maximum emission peak increased from 320 to 360 nm initially and reached a vertex at 360 nm, then it decreased quickly. This classical bathochromic shift phenomenon of CDs (downconverted PL property) has been reported previously [23, 24]. However, when CDs were excited by even long-wavelength light (from 520 to 620 nm), an obvious upconverted PL property was revealed (shown in Fig. 1c) by using a long-pass filter in front of the sample. The PL intensity of these upconverted emissions decreased firstly and reached a nadir at 580 nm; then, it went up gradually. It’s interesting to note that the locations of these upconverted emission peaks were very close to that of former downconverted PL emissions. But the upconverted PL emissions showed an unknown blue-shift phenomenon along with increased excitations, which was opposite to that of downconverted PL emissions. The upconverted PL property of CDs should possibly be attributed to the multiphoton active process similar to previous reports [25, 26].

Figure 1
figure 1

a HRTEM image of CDs (scale bar: 50 nm). b Downconversion PL emission spectra of CDs (λex = 320–480 nm). c Upconversion PL emission spectra of CDs (λex = 520–620 nm). d BLS emission spectra produced by firefly luciferase under different pH values

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FLUC, derived from Photinus pyralis, is a coelenterazine-type luciferase. When FLUC was exposed to its substrates, energy would be released and the BLS spectra were recorded, as shown in Fig. 1d. The emission spectra of BLS were sensitive to pH values. The maximum emission peaks were observed at 558 nm under pH 8.0 (Tris buffer), and 615 nm under pH 6.8 (PBS). As to pH 7.4 (PBS), it showed a very broad peak, which was just like the mixture of BLS obtained under pH 8.0 and pH 6.8. In any case, FLUC could produce effective luminescence in the range from 520 to 640 nm, which was satisfactory for the excitation of as-prepared CDs.

To estimate the stability of CDs-PIX conjugate, the size and surface potential in physiological environment were measured. As shown in Fig. S2, the sizes of CDs-PIX were not uniform anymore and distributed in the range of 1.93–4.16 nm in comparison with CDs (Fig. 1a), and the average size was 2.55 ± 0.58, which was higher than the average size of CDs. Zeta potential (Fig. S3) of CDs and CDs-PIX was recorded in PBS (pH 7.4). The values of CDs were − 13.3 ± 1.30, due to the existence of oxygen functional groups on the surface of CDs. After conjugation, the zeta potential of CDs-PIX conjugate turned into − 26.7 ± 2.81, which demonstrated the CDs-PIX was more stable than the CDs. All samples bear negative charges which may be attributed to the high solubility and dispersity of particles. The inset in Fig. S3 also proved the CDs-PIX was stable in various media such as PBS (pH 7.4), saline, bovine, and rabbit serum, which met the requirements to apply in physiological environment.

The conjugation state of CDs-PIX was examined by means of FTIR spectra, UV–Vis absorption spectra, and fluorescence detection. As shown in Fig. 2a, FTIR spectra revealed that CDs had many characteristic absorption, and the strong peaks at 1564 cm−1, 1108 cm−1, and 1660 cm−1 were attributed to N–H, C–N, and –CO–NH–, respectively. It could be concluded that the as-prepared CDs were abundant in –NH2 and other organic groups, which ensured the excellent solubility of CDs in aqueous solution and reaction feasibility with PIX. The peak at 1702 cm−1 was attributed to the C=O stretching of carboxyl in PIX. After the conjugation reaction, there were no peaks at 1702 cm−1 or 1660 cm−1 in the spectrum of CDs-PIX, but a new sharp peak belonging to amide linkage of –CO–NH– was found at 1650 cm−1, proving the successful chemical binding between CDs and PIX.

Figure 2
figure 2

a FTIR spectra of CDs, PIX, and CDs-PIX. b The UV–Vis spectra of CDs, PIX (0.12 mM), and CDs-PIX (0.12 mM). c PL emission spectra of CDs-PIX (λex = 550–590 nm; insets: magnification of CDs part). d Emission spectra of BLS and CDs-PIX conjugate activated by the BLS under pH 6.8 (insets: magnification of CDs part)

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The UV–Vis absorption spectra (Fig. 2b) showed that PIX had a broad absorption which spanned nearly whole visible range with maximum absorption peak at 370 nm around. The CDs without PIX showed virtually no absorption in the range of 550–700 nm. After conjugation with PIX, it could be found that the absorption of CDs-PIX was similar to that of PIX, indicating that the functional chromophores of PIX were kept well even after the conjugation reaction. The absorption of CDs-PIX was much higher than that of PIX from 300 to 500 nm under the same concentration, which demonstrated the CDs-PIX conjugate was prepared successfully. Furthermore, the absorption of CDs-PIX, especially in the range from 520 to 640 nm, was just the effective range of FLUC-induced BLS (Fig. S4). It was obviously that the emission peak of BLS was not quite suitable for the sensitization of PIX. However, taking the upconverted PL properties of as-prepared CDs into consideration, once excited by the BLS, CDs would exhibit effective emission with wavelengths from 400 to 500 nm, which could provide an excellent overlap of the absorbance spectrum of PIX and satisfy the pre-requisite for CDs-PIX conjugate to realize an efficient FRET process (Fig. S4).

The fluorescence properties of CDs-PIX were explored by PL emission spectra, as shown in Fig. 2c. There was a nearly fixed emission peak at 620 nm around belonging to the PIX content in CDs-PIX conjugate. With excitation from 550 to 590 nm, the PL intensity of these maximum emission peaks attributed to PIX increased initially and reached a vertex at 560 nm around and then it decreased gradually. As to CDs part (insets of Fig. 2c), the emission peaks decreased all the way in the whole excitation scope, which was unlike the tendency of CDs alone shown in Fig. 1c. It was inferred that the lost energy of CDs part in CDs-PIX conjugate was contributed to PIX part through the FRET process. An intuitive measurement was conducted by contrasting PL emission spectra of PIX and CDs-PIX with same concentration. As shown in Fig. S5, the PIX was excited weakly at 560 nm, while the CDs-PIX showed two emission peaks, which were quite in accordance with the characteristic peaks’ positions of CDs and PIX, respectively, but differed in the PL intensity. The PL intensity of emission peak attributing to PIX part in CDs-PIX conjugate was about ninefold than that of PIX alone. The enhancement of PL intensity was due to the following reasons: (1) a result of energy transfer from CDs, which was a typical FRET phenomenon according to our conjecture; (2) CDs improved the solubility of PIX in aqueous solution; and (3) under the assistance of bridge function of CDs, the PIX could be excited in direct and indirect two paths. Since CDs-PIX was successfully activated by external light source with same wavelengths according to BLS produced by FLUC, here the BLS was also tested to activate CDs-PIX directly (Fig. 2d). Though the emission peak of PIX in CDs-PIX conjugate was covered by sustained BLS at the same location, the upconverted emission belonging to CDs part could be found with careful examination (insets of Fig. 2d), indicating the successful activation of CDs-PIX conjugate by BLS. Combining the results of Fig. S5, we inferred the PIX in CDs-PIX conjugate could be directly excited by BLS and indirectly excited through CDs upconversion functions.

Singlet oxygen (SO) is a prominent member of ROS in PDT. The SO generation from BLS-activated CDs-PIX was detected (Fig. S6). Here, 1,3-diphenylbenzofuran (DPBF) was used as the chemical detector, which absorbed strongly at 410 nm, and the resulting loss of absorbance at 410 nm was indicative of the SO produced [27]. Results indicated BLS was powerful enough to activate CDs-PIX and produced plenty of SO, leading to a 60% reduction in DPBF absorbance after 40 min compared to only 2% for CDs-PIX alone and 25% for BLS control groups. Intracellular ROS generation in SMMC-7721 cells was evaluated by H2DCFDA staining with confocal microscopy and flow cytometry [28]. As shown in Fig. 3, the green fluorescence signal standing for ROS generation was detected in SMMC-7721 cells treated with either 40 mM H2O2 (positive control: 34.77%) or CDs-PIX/FLUC/DL/Mg2+/ATP combination (34.30%) but was not significantly detected by confocal microscopy or flow cytometry in the negative control (PBS), CDs-PIX conjugate, or FLUC/DL/Mg2+/ATP combination groups (13.80, 10.09 or 21.96%, respectively). It should be noted that the BLS group also reflects certain ROS generation that is consistent with SO detection, indicating the BLS had some cytotoxicity to the cells which might be attributed to high concentration of DL and need to be further explored.

Figure 3
figure 3

a Quantification of ROS of CDs-PIX plus sensitized FLUC-mediated PDT in SMMC-7721 cells with H2DCFDA staining measured by flow cytometry. (Concentration: H2O2, 40 mM; CDs-PIX, 20 µM; FLUC/DL/Mg2+/ATP, 17 µM/100 µM/0.1 M/1 mM; CDs-PIX/FLUC/DL/Mg2+/ATP, 20 µM/17 µM/100 µM/0.1 M/1 mM) b ROS production of CDs-PIX plus sensitized FLUC-mediated PDT in SMMC-7721 cells observed by confocal microscopy. Green: ROS indicator H2DCFDA; blue, DAPI (nuclei); scale bar, 10 µm

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The cytotoxicity was also measured via a MTT assay (Fig. S7). CDs-PIX plus sensitized FLUC showed clear cytotoxicity in contrast to the other materials under the same concentration (20 µM), and approximately 60% cells were killed. Thus, it was proved that an external light source was not necessary for PDT and BLS-initiated endogenous FRET process could activate a PS (such as PIX) and therefore killing some target cells with the assistance of CDs.

Conclusions

In summary, we have explored a BLS-initiated PDT strategy to activate a designed photosensitizer CDs-PIX conjugate without an external light source. The high-luminescent and excitation-independent CDs are used as a bridge to deliver PIX and regulate the BLS in the FRET process to meet the qualification for PIX sensitization; thus, the PIX can be activated by the BLS in direct and indirect two paths. The preliminary evaluation on SMMC-7721 cells has yet proved that intracellular ROS is produced with enhanced PDT effect for tumor cell killing. To the best of our knowledge, this is the first report for using inner light to activate CDs-modified PDT system. Although the therapeutic effect remains to be improved, this work opens a new therapy modality for tumor and holds a potential for biomedical applications.