A near IR photosensitizer based on self-assembled CdSe quantum dot-aza-BODIPY conjugate coated with poly(ethylene glycol) and folic acid for concurrent fluorescence imaging and photodynamic therapy

Abstract

An effective photosensitizer for the fluorescence imaging and photodynamic therapy of tumour cells has been developed on the basis of a self-assembled CdSe quantum dot-thiophene-substituted aza-BODIPY conjugate coated with poly(ethylene glycol) and folic acid via Förster resonance energy transfer (FRET).

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The development of biocompatible nanosystems for cancer therapeutics and diagnostics (theranostics) is highly desirable for preventing over- or under-treatment during cancer treatment.¹ Aza-boron-dipyrromethene (aza-BODIPY) dyes, in which the meso carbon atom of the classic BODIPY molecule is replaced with a nitrogen atom,^2a display superior photophysical properties for applications in near IR fluorescence imaging and photodynamic therapy.² It has been well recognized that the combination of strong fluorescence for diagnostics and the efficient generation of singlet oxygen for photodynamic therapy in one sensitizer used to be in conflict, because efficient excited-state (S₁ → T₁) intersystem crossing normally quenches the fluorescence.^2a Quantum dots (QDs) have broad excitation spectra, which allow the excitation of QDs at wavelengths far removed (>100 nm) from their respective emission bands (large Stokes shift).³ Moreover, the narrow, size-tunable and symmetric emission spectra of QDs have made them excellent energy donors for Förster resonance energy transfer (FRET) materials.⁴ Hence, carefully designed QDs-aza-BODIPY conjugates can be self-assembled to undergo FRET to achieve the concurrent functions of imaging and photodynamic therapy, because QDs enhance the transfer of energy from the excited state of aza-BODIPY to the ground state of molecular O₂.

One of the most widely employed approaches for connecting organic dyes to QDs is the formation of amide bonds using a coupling agent.⁵ However, the relatively long center-to-center distance lowers the efficiency of energy transfer (ET_eff).⁶ In addition, the hydrophobicity of QDs-organic conjugates limits their applicability in biological systems. An alternative approach that is conveniently employed is phase transfer.⁷ However, the numerous surface functional sites of QDs prefer to crosslink with hydrophilic groups instead of the dye, which also limits the efficiency of FRET. Moreover, the low stability of hydrophilic QDs often gives rise to intermediate aggregates in neutral/acidic buffers and blood serum.^3a By constructing a FRET system comprising quantum dots and aza-BODIPY, clear potential advantages of the QD-aza-BODIPY conjugate over aza-BODIPY alone include extending the range of excitation wavelengths of aza-BODIPY to achieve better separation of excitation and emission wavelengths in fluorescence, the ability to target molecules of interest, and high singlet oxygen efficiency.

We describe here a self-assembled conjugate containing 3,5-dithiophene-substituted aza-BODIPY bound to the surface of CdSe QDs encapsulated by biocompatible FA-DSPE-PEG₂₀₀₀, resulting in high FRET efficiency (Scheme 1a), which may be used as a multifunctional photosensitizer for the targeting of live cells and simultaneous fluorescence imaging and photodynamic therapy via excitation in the near IR region.

Scheme 1 (a) Schematic diagram showing a particle composed of single CdSe QDs and aza-BODIPY (aza-a) self-assembled with FA-DSPE-PEG. (b) TEM image of a QD-aza-a@PEG conjugate. The inset shows the expanded core–shell structure.

Firstly, 3,5-dithiophene-substituted aza-BODIPY (aza-a) was prepared according to the literature^8,9 (the details are provided in the ESI†). The molecular structure was characterized by ¹H NMR, MALDI-TOF-MS and absorption spectroscopy (Fig. S1, ESI†). The CdSe QD-aza-a@PEG conjugate was prepared via the self-assembly of aza-a, CdSe QDs, DSPE-MPEG₂₀₀₀ and DSPE-PEG₂₀₀₀-FA (in a molar ratio of 8.5 : 1.5) with a total mass ratio of 15% using a single-step sonication method¹⁰ (the experimental details are provided in the ESI†). The morphology of the QD-aza-a@PEG conjugate was determined by HRTEM imaging and dynamic light scattering (DLS) analysis (Fig. S2–S4, ESI†). A thin, uniform layer of DSPE-PEG₂₀₀₀-coated QD-aza-a cores was observed in the corresponding HRTEM image, which showed their homogeneous size of about 15 nm and successful surface modification with no apparent agglomeration (Scheme 1b), in which the measured hydrodynamic diameter was 90 nm, and they did not exhibit significant change even after they had been kept in an aqueous solution for 6 months.

The minimum requirement for effective energy transfer in a FRET pair is that the emission spectrum of the donor molecule must overlap adequately with the absorption spectrum of the acceptor molecule.¹¹ CdSe QDs were selected as an energy transfer donor because their emission band centered at 655 nm overlaps effectively with the absorption band of aza-a (Fig. 1a). The spectral overlap integral (J) can be determined by the equation:

where F_D(λ) and ε_A(λ) are the fluorescence intensity of the donor (CdSe QDs) and the molar extinction coefficient of the acceptor (aza-a), respectively, at a wavelength of λ.

Fig. 1 (a) Superposition of the normalized absorption spectrum of aza-a (red) ([aza-a] = 1.7 × 10⁻⁴ M) and the fluorescence spectrum of CdSe QDs (black) ([QD] = 1 × 10⁻⁵ M) in aerated toluene at 298 K. (b) UV-visible absorption spectra of CdSe QD-aza-a@PEG conjugate ([QD] = 1 × 10⁻⁵ M; [aza-a] = 1.7 × 10⁻⁴ M), PEGylated QDs ([QD] = 1 × 10⁻⁵ M) and PEGylated aza-a ([aza-a] = 1 × 10⁻⁵ M) at room temperature.

The absorption spectrum of the conjugate comprised the sum of the absorption spectra of aza-a and the QDs (Fig. 1b and Table 1). The absorption and emission bands of the conjugate were slightly red-shifted (ca. 3–4 nm) in comparison with those of free aza-a. This might be attributed to a difference in the dielectric constant of the fluorophore in solution and near the surface of the QDs.¹²

Table 1 Absorption and fluorescence data: fluorescence lifetime (τ), fluorescence quantum yield (Φ), donor–acceptor distance (r), energy transfer efficiency (ET_eff) and rate (K_ET) calculated by eqn (S1) and (S2)

Compound	λex	λem	τ (ns)	Φ^c	r (Å)	ETeff	KET (10^8 s^−1)
a Lowest-energy absorption peak of QDs.b No fluorescence was observed upon irradiation at 420 nm. The Förster radius R0 was calculated to be 32.8 Å. Tetraphenylporphyrin was used as the standard (ΦF = 0.11).c λex = 420 nm.d λex = 635 nm.
QD-aza-a@PEG conjugate
QDs	616^a	655	2.52	0.02^c, 0.01^d	21.3	86.9%	0.34
Aza-a	728	753	2.15	0.4^c, 0.65^d
PEGylated QDs and aza-a
QDs	619^a	655	19.19	0.8^c, 0.53^d
Aza-a	724	750	—^b	0.03^c, 0.12^d

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As shown in Fig. 2a, upon excitation at 420 nm, where only the QDs display a substantial amount of absorption, the QD-aza-a@PEG conjugate exhibited dual emissions from both QDs (centered at 655 nm) and aza-a (centered at 750 nm). In comparison with the emission spectra of PEGylated aza-a and QDs excited under the same conditions, the emission from the QDs decreased significantly (>90% loss in comparison with that of QDs with no aza-a), with a concomitant increase in the emission from aza-a, which indicates efficient FRET from the QDs to aza-a. Therefore, this confirmed that the enhancement in intensity at 750 nm that was observed for the QD-aza-a@PEG conjugate was a result of energy transfer from the QDs to the attached aza-a.¹³ Because UV light is harmful to biological samples, the QD-aza-a@PEG conjugate was excited at 635 nm (Fig. 2b) and the emission intensity at 750 nm increased significantly (threefold) in comparison with that of PEGylated aza-a at the same concentration. This result indicates that efficient FRET from QDs to aza-a can be achieved using a red visible wavelength suitable for application in biological systems.

Fig. 2 Photoluminescence spectra of the CdSe QD-aza-a@PEG conjugate (blue), PEGylated QDs (black) and PEGylated aza-a (red) at the same concentration in water: (a) λ_ex = 420 nm and (b) λ_ex = 635 nm. ([QDs] = 1 × 10⁻⁶ M, [aza-a] = 1.7 × 10⁻⁵ M).

The values of the fluorescence emission maxima (λ_em), quantum yields (Φ_F) and lifetimes (τ) are summarized in Table 1. The fluorescence quantum yields were determined using the free base of tetraphenylporphyrin as a standard (Φ_F = 0.11).¹⁴ When a comparison is made of the fluorescence quantum yields, the emission from the QDs in the QD-aza-a@PEG conjugate decreased significantly, with a concomitant increase in that of aza-a in comparison with those of PEGylated aza-a and QDs. Furthermore, the photoluminescence of the QDs in the conjugate exhibited single-exponential decay with a lifetime value of 2.52 ns, which was obviously shorter than the lifetime (19.19 ns) for free QDs (Fig. S5, ESI†). Moreover, fitting of the lifetime data demonstrated that the build up lifetime of aza-a (2.15 ns) was a result of the transfer of energy from the QDs to aza-a, but not by direct excitation of the photosensitizer itself. On the basis of the above fluorescence quantum yield and lifetime data, the obvious explanation is that the QDs behaved as an energy transfer donor and aza-a as an acceptor during the FRET process.

The transfer efficiency can also be determined from the lifetimes of the donor measured in the absence (τ_D) and presence (τ_DA) of the acceptor using the equation .^14c,d The efficiency of FRET was calculated to be 86.9%. The high ET efficiency demonstrates that the QD-aza-a@PEG conjugate provides an effective way to construct an energy transfer system. Moreover, the rate of energy transfer (i.e., K_ET) was calculated using the equation K_ET = (1/τ_DA) − (1/τ_D).^14c,d The values of K_ET are also summarized in Table 1. In brief, qualitative analysis of data for the efficiency (ET_eff) and rate (K_ET) of ET as standard criteria for the Förster energy transfer mechanism is highly dependent on the center-to-center donor–acceptor separation and the spectral overlap of the donor emission and the acceptor absorption. With the self-assembly conjugation strategy, two apparent advantages substantially improved the efficiency of FRET: (i) thiophene-substituted aza-a has an affinity for the surface of CdSe QDs and therefore the donor–acceptor distance (r) of 21.3 Å in the resulting conjugate is shorter than the linkage through covalent amide bonds;⁵ and (ii) the self-assembly approach can increase the number of aza-a units bound to each QD to 17. Therefore, the efficiency of FRET can be tuned by choosing a size-controlled QD and an acceptor with an appropriate spectral overlap and binding mode to control the distance and the amount of dye bound to each QD surface.

In order to investigate the photosensitizing capability of the QD-aza-a@PEG conjugate, the production of singlet oxygen was determined using a steady-state method with 1,3-diphenyl-isobenzofuran (DPBF) as the ¹O₂ scavenger. Fig. 3a shows the extensive bleaching of DPBF as a function of time (reduction in the amplitude of the spectral features at 411 nm) when incubated with the QD-aza-a@PEG conjugate and irradiated with a 635 nm laser beam (20 mW cm⁻²) at intervals of 3 min in an air-saturated aqueous solution. The QD-aza-a@PEG conjugate induced a 65% reduction in the absorbance of DPBF after 30 min, whereas this was only 5% in the presence of PEGylated aza-a. The irradiation of PEGylated QDs had almost no noticeable effect (Fig. S6†). Control experiments with DPBF alone under the same excitation conditions showed no bleaching. These results indicate that by employing QDs as a carrier for aza-a its singlet oxygen yield was significantly improved and it thus behaved as an efficient NIR photosensitizer to act as a PDT therapeutic agent via a FRET-mediated process.

Fig. 3 (a) Plot of changes in the intensity of DPBF at 411 nm against time in the presence of the QD-aza-a@PEG conjugate (red triangles), aza-a at the same concentration (green dots) and DPBF alone (black squares) irradiated at 635 nm. ([QDs] = 1 × 10⁻⁶ M, [aza-a] = 1.7 × 10⁻⁵ M). (b) MTT assay of HeLa cells incubated with the QD-aza-a@PEG conjugate at different concentrations irradiated at 635 nm (red solid line) and in the dark (black dotted line).

The cytotoxicity of the QD-aza-a@PEG conjugate to HeLa human cervical carcinoma cells was examined in the presence and absence of irradiation at 635 nm using an MTT assay. Fig. 3b shows that the cell viability was 93% in the dark in the presence of 0.25 μM QD-aza-a@PEG conjugate. Moreover, with a concentration of up to 0.5 μM, 90% of the cells maintained proliferation. Therefore, the low toxicity in the dark further demonstrated high biocompatibility and stability in vitro owing to the QD-aza-a conjugate being encapsulated by PEG-phospholipids. The photocytotoxicity to cancer cells was examined by irradiating with laser light at 635 nm (20 mW cm⁻²) for 3 min. Fig. 3b shows that when the concentration of the QD-aza-a@PEG conjugate was increased to 0.5 μM, the cell viability dramatically decreased and a cell destruction rate of approximately 80% was observed. Therefore, the QD-aza-a@PEG conjugate displayed high photocytotoxicity under near IR irradiation and could act as a potential PDT agent for cancer therapy. In the presence of both the QD-aza-a@PEG conjugate and irradiation, HeLa cells exhibited high death rates. To study the mechanism of cell death during PDT mediated by the QD-aza-a@PEG conjugate, β-carotene, vitamin C, vitamin E and mannitol were used as scavengers of ¹O₂, hydroxyl radicals (˙OH), hydrogen peroxide (H₂O₂) and superoxide anions (O₂⁻), respectively. Fig. S7a and b† show that cell death was effectively inhibited in the presence of β-carotene or vitamin C during PDT. The β-carotene group exhibited slightly improved efficiency, with a cell viability of 84% in comparison to that of 81% in the vitamin C group. Moreover, the protection afforded was lower in the vitamin E and mannitol groups (Fig. S7c and d†). On the basis of these data, regarding the possible mechanism of cell death, it could be reasonably assumed that PDT mediated by the QD-aza-a@PEG conjugate damaged cancer cells by ROS that were generated, which was mainly due to ¹O₂ as well as ˙OH and partially due to other reactive species such as H₂O₂ and O₂⁻.

As shown in Fig. 4a, bright red fluorescence could be observed for HeLa cells after incubation with the QD-aza-a@PEG conjugate. No obvious morphological change in the cells was observed, which implied high biocompatibility. In contrast, the HeLa cells treated with the PEGylated QD-aza-a conjugate exhibited negligible fluorescence (Fig. 4a) owing to the lack of targeting of FA. Therefore, the QD-aza-a@PEG conjugate could enter the cancer cells via an endocytosis pathway mediated by FA receptor.¹⁵ To further study the targeting of cells, HeLa cells as model cancer cells and immortalized HaCaT human epidermal cells as model normal cells were treated with the QD-aza-a@PEG conjugate and investigated by confocal fluorescence imaging (Fig. 4b); only the HeLa cells displayed strong red fluorescence, but the fluorescence from the HaCaT cells was negligible. This observation leads us to conclude that the intracellular internalization of the QD-aza-a@PEG conjugate was limited to cancer cells, which was attributed to the specific binding of FA to FA receptor.^15c Thus, the QD-aza-a@PEG conjugate could target cancer cells and did not injure normal cells, which ensured high therapeutic efficacy and minimal side effects. The photocytotoxicity of the QD-aza-a@PEG conjugate was also investigated by confocal fluorescence imaging using annexin V-FITC as a probe for identifying apoptotic cells.¹⁶ Strong green fluorescence from annexin V-FITC was observed on the membrane after HeLa cells were incubated with the QD-aza-a@PEG conjugate and irradiated with a laser light at 635 nm for 3 min (Fig. 4c). Furthermore, in a bright-field image, the morphology of the HeLa cells was incomplete and the outer membrane was ruptured. In contrast, HeLa cells without irradiation in the dark group exhibited no fluorescence (Fig. S8c†), which indicated that the QD-aza-a@PEG conjugate possessed high photocytotoxicity to cancer cells with no toxicity in the dark. Thus, the QD-aza-a@PEG conjugate could target the cancer cells with high therapeutic efficacy and did not injure the normal cells, with minimal side effects. These results suggested that the QD-aza-a@PEG conjugate could be employed for concurrent cancer cell diagnostics and photodynamic therapy.

Fig. 4 Confocal fluorescence and bright field images of (a) HeLa cells incubated with QD-aza-a@PEG conjugate and PEGylated QD-aza-a without FA irradiated with a 635 nm laser light. (b) HeLa and HaCaT cells incubated with QD-aza-a@PEG conjugate. (c) Annexin V-FITC stained HeLa cells with different treatments to study the cell apoptosis induced by QD-aza-a@PEG conjugate mediated PDT, HeLa cells were irradiated with a 635 nm laser for 3 min at a power density of 20 mW cm⁻². ([QDs] = 5 × 10⁻⁷ M, [aza-a] = 8.5 × 10⁻⁶ M).

In summary, we have developed a biocompatible near IR photosensitizer for targeting tumors with concurrent imaging and photodynamic therapy on the basis of a self-assembled CdSe QD-aza-a@PEG conjugate. The strong near IR emission of the aza-BODIPY moiety at 750 nm upon excitation of the QD donor via efficient FRET makes it useful for bioimaging. Moreover, the generation of singlet oxygen for photodynamic therapy can be achieved using a laser light at 635 nm. The ability to make this conjugate water-soluble and target it to specific tumor cells is promising for applications in cellular labelling, deep-tissue imaging, assay labelling, diagnosis and photodynamic therapy.

Acknowledgements

We gratefully acknowledge the financial support provided by the Major State Basic Research Development Program of China (No. 2013CB922101) and the National Natural Science Foundation of China (No. 21371090).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Methods and synthesis, HRTEM imaging, and additional optical spectra. See DOI: 10.1039/c6ra23113f

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