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Interaction between upconversion nanoparticles (UCNPs) and natural photosensitizers (Ps) has not been investigated so far. In order to make new near infrared photosensitizers, the conjugates (Ps-UCNPs) were synthesized by NaYF4 as a substrate and hydrothermal reaction of lanthanide ions Yb3+ with Er3+ or Tm3+, and then interaction with pheophorbide A (Pha) and resveratrol (Res) respectively. Based on the intensity of the fluorescence emission by Ps-UCNPs, the optimal reaction conditions are 16 mM Yb3+/0.4 mM Er3+ at 120°C for 20 h for Pha-UCNPs, and 80 mM Yb3+/0.1 mM Tm3+ at 180°C for 20 h for Res-UCNPs. The nanoparticles have a hexagonal phase or cubic phase with an average diameter size of 220 nm, and selectively emit the stronger red (670 nm) or violet (380 nm) fluorescence. Pha-UCNPs show the strong effects, and the maximum yield of singlet oxygen was seven times more than UCNPs and pheophorbide A alone under 980 nm illumination. It is attributed to the efficient resonance energy transfer from UCNPs to pheophorbide A. Pha-UCNPs is an effective NIR photosensitizer with potential for deep tissue disease photodynamic therapy.
© 2017 Optical Society of America
1. Introduction
Photodynamic therapy (PDT), a treatment modality for tumors based on the photochemical reaction, involved in a combination of light, photosensitizers and single oxygen [1, 2]. PDT is a minimally invasive therapy and possesses good specificity and selectivity on a particular diseased tissue by the light of specific wavelengths such as ultraviolet or visible light [3]. However, the treatment efficiency of PDT is seriously challenged since the limited tissue penetration depths (less than 1 cm) of the excitation wavelength of photosensitizers in the range of ultraviolet or visible light [4]. Notably, the upconversion nanoparticles (UCNPs) provide a potential strategy to overcome the drawbacks of PDT [5]. Near infrared (NIR) light easily pass ‘optical tissue penetration window’ to deep tissues, and can be converted to ultraviolet or visible light through a process termed ‘upconversion’ [6–8]. That is to say, the conjugation of photosensitizers with upconversion nanoparticles will make it possible to convert NIR to ultraviolet or visible light and excite photosensitizers to achieve deep tissues PDT, but the interaction between UCNPs and photosenitizers has not been reported so far.
The upconversion nanoparticles with the host materials of NaYF4 doped by lanthanide ions Yb3+ with Er3+or Tm3+ have been widely applied in biological labeling, bioimaging and photodynamic therapy because of their advantages including low toxicity, weak background fluorescence, high sensitivity, deep organs penetration [9–12], but the upconversion efficiency from infrared to visible light or ultraviolet could be remarkably changed in various synthetic conditions such as concentration of absorber, emitter, reaction time and reaction temperature [13–16].
Water insolubility is another trouble for the application of most of UCNPs, but polymer-coated UCNPs can enhance the water solubility [17]. We design the PVP-coated UCNPs conjugated with photosensitizers in order to improve the NIR sensitivity and solubility of the nanoparticles.
PDT induces the death of diseased tissues or cells due to reactive oxide species (ROS) generated by photosensitizers [18]. ROS, metabolic products of aerobic respiration including singlet oxygen (1O2), hydrogen peroxide (H2O2), hydroxyl radical (•OH), superoxide anion (O2•) and lipid peroxide radical (ROO•) etc [19], can create oxidation reaction with a variety of biological molecules such as DNA, RNA, amino acids, proteins and so on [20]. ROS are not only useful to kill cancers, but also expected to kill bacteria and parasites to treat infectious diseases [21].
In this research, two kinds of UCNPs conjugated photosensitizers were synthesized and compared to find the best conditions to produce singlet oxygen under NIR. Pheophorbide A and resveratrol are natural compounds and respectively excited by red light, blue and violet for activation [22–24]. The photon energy transition and transfer from UCNPs to photosensitizers are discussed to reveal the mechanism.
2. Materials and methods
2.1 Chemicals
Yttrium oxide (Y2O3, 99.9%), ytterbium oxide (Yb2O3, 99.9%), erbium oxide (Er2O3, 99.9%), thulium oxide (Tm2O3, 99.9%), 9,10-diphenyl anthracene (DPA, 99.5%) were supplied by Aladdin Chemistry Co. Ltd. (Shanghai, China). Y(NO3)3 and Yb(NO3)3 stock solutions (80 mM), Er(NO3)3 and Tm(NO3)3 stock solution (20 mM) were prepared by dissolving corresponding oxides in nitric acid. Sodium fluoride (NaF) was of analytical grade and bought from Tianjin Chemical Reagents Development Center (Tianjin, China) and 400 mM stock solution was prepared in deionized water. Polyvinylpyrrolidone (PVP-K30) was purchased from Shanghai Bioscience Technology Co. Ltd. (Shanghai, China) and dissolved in deionized water to prepare the stock solution (4 w%). Pheophorbide A (Pha) was extracted from the silkworm excrement in our previous research [25]. Resveratrol (Res, 98%) was bought from Guangzhou Qiyun Biotechnic Company (Guangzhou, China).
2.2 Synthesis of PVP coated UCNPs
The PVP coated UCNPs (PVP-UCNPs) were synthesized by hydrothermal reaction following literature protocol with slight modifications [26]. In a typical synthesis of NaYF4:Yb3+/Er3+ nanoparticles, 15.6 mL of Y(NO3)3, 4 mL of Yb(NO3)3, 0.8 mL of Er(NO3)3 solution, 20 mL PVP solution were mixed with stirring at 50°C for 0.5 h to make solution A. After then, 20 mL of NaF solution was added into solution A drop by drop under vigorous stirring at 50°C for 1 h. The mixture was transferred to a 100 mL Teflon-lined autoclave and heated subsequently to 180°C for 20 h. After the autoclave was cooled to room-temperature naturally, the precipitate was collected, washed with ethanol and deionized water alternatively several times, and then dried at 80°C. As for the NaYF4: Yb3+/Tm3+ nanoparticles, only Er(NO3)3 was used for substitution of Tm(NO3)3 in above procedure.
2.3 Synthesis of photosensitizers conjugated with UCNPs
The conjugation of UCNPs with photosensitizers (Ps-UCNPs) was by means of molecular interaction and was carried out with the following procedure. One hundred and fifty milligrams of PVP-UCNPs, 100 mg pheophorbide A and resveratrol were respectively dissolved in dichloromethane and stirred at room temperature for 5 h. The products were obtained by evaporating dichloromethane and washing with ethanol. Co-doping concentration, reactive temperature and time were optimized on the base of fluorescence intensity of Ps-UCNPs.
2.4 Characterization of Ps-UCNPs
Upconversion fluorescence spectra were recorded on Hitachi F-4500 fluorescence spectrophotometer (Hitachi Instrument Co, Ltd, Japan) under the excitation of a 980 nm diode laser. The size of the nanoparticle was observed by Nano 2S MDT-2 Malvern Particle Size Analyzer (Malvern Instrument Co, Ltd, United Kingdom). X-ray powder diffraction patterns were measured on a Bruker D2 Phaser (Bruker Company, Germany) with Cu-Ka radiation (40 kv, 30 mA, λ = 1.5406 Å). The morphology of the nanoparticles was observed under a JEM-2100F high resolution scanning electron microscope (SEM) (JEOL Company, Tokyo, Japan) at 200 kV.
2.5 Measurement of singlet oxygen
Singlet oxygen production of Ps-UCNPs was measured by 9,10-Diphenylanthracene (DPA) bleaching method [27, 28]. Briefly, 5 mL of 2 × 10−5 mol/L DPA ethanol solution and 10 mL of 0.5 mg/mL Ps-UCNPs ethanol solution were mixed and continuously irradiated by 980 nm laser (0.5 W/cm2) for 90 min, and the absorbance of DPA at 355 nm was detected as the initial value, and the change of absorbance (∆A) between the initial value and the measured value after mixture with Ps-UCNPs is expressed as the production of singlet oxygen.
2.6 Statistical analysis
Data were expressed as mean ± standard deviation (), and analyzed with SPSS13.0 software (IBM, Armonk, NY, USA). Significant tests among the groups were based on one-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) test.
3. Results and discussion
3.1 Spectra matching of UCNPs and photosensitizers
In order to activate photosensitizers by upconversion photons which are produced by UCNPs, it should be a match between the emission spectra of UCNPs and the absorption of photosensitizers [29–31]. The upconversion spectra of UCNPs and the absorption spectra of photosensitizers were measured in order to elucidate the possibility of energy transfer. There are two kinds of typical upconversion spectra respectively emitted by NaYF4: Yb/Er and NaYF4: Yb/Tm nanoparticles as illustrated in Fig. 1. The emission peak of the NaYF4: Yb/Er nanoparticles overlaps obviously with the absorption spectra of pheophorbide A at 419 nm and 650 nm (Fig. 1(a)), and the maximum absorption of resveratrol covers the emission peak of the NaYF4: Yb/Tm nanoparticles at 293 nm (Fig. 1(b)). The results elucidate an occurrence of energy transfer between UCNPs and the photosensitizers at difference wavelength.
Fig. 1 Matching of emission spectra of UCNPs with absorption spectra of pheophorbide A (Pha, a) and resveratrol (Res, b).
3.2 Optimization of factors on synthesis of Ps-UCNPs
The excited photosensitizers can emit special fluorescence spectra, which can be used to evaluate real energy transfer from UCNPs to the photosensitizers. The pheophorbide A emits fluorescence at 670 nm while excitation at 419 nm, and resveratrol emits fluorescence at 380 nm while excitation at 293 nm (Fig. 2). Because the maximum peaks are respectively 670 nm and 380 nm which are different from the emission peaks (650 nm and 360 nm) of the NaYF4: Yb/Er and NaYF4: Yb/Tm nanoparticles, the intensity of fluorescence at 670 nm and 380 nm can be used as indexes to optimize the synthetic conditions of Ps-UCNPs.
Fig. 2 Fluorescence emission spectra of pheophorbide A and resveratrol excited at 419 nm and 293 nm respectively.
The upconversion spectra could be regulated by selecting the type of rare earth ions, doping proportion and changing reaction conditions [32–34], therefore, the co-doping concentration of emitter and absorber, reactive temperature and time were investigated to synthesize Ps-UCNPs with strong emission at certain wavelength.
3.2.1 Pha-UCNPs
The fluorescence emission spectra of NaYF4: Yb3+/Er3+ nanoparticles conjugated with pheophorbide A were measured at different concentration of Yb3+ and Er3+, reactive temperature and time, the results are shown in Fig. 3. 16 mM, 32 mM and 80 mM Yb3+ as the absorber of photon energy had the maximum intensity of the fluorescence emission at 670 nm, 480 nm and 545 nm respectively (Fig. 3(a)), suggesting that Yb3+ can change the ratio of different emission wavelength. 16 mM Yb3+ is the best concentration for Pha-UCNPs because of the strongest intensity at 670 nm which is the emission of pheophorbide A, indicating the best energy transfer from UCNPs to pheophorbide A. The lower concentration (8 mM) of Yb3+ is not enough to make effective upconversion and energy transfer, but the higher concentrations of Yb3+ have lower transfer efficiency, and the possible mechanism is that more photons absorbed by Yb3+ from near-infrared light combine to form shorter wavelength of photons, whose resonance energy is mismatching for the organic vibration groups of pheophorbide A.
Fig. 3 Emission fluorescence spectra of Pha-UCNPs with different concentration of Yb3+ (a) and Er3+ (b), reactive temperature (c) and time (d).
Er3+ as the emitter of upconversion light directly influences the energy transfer efficiency. The emission fluorescence at 670 nm reached maximum intensity at 0.4 mM Er3+ concentration, and then decreased with Er3+ concentration further increase (Fig. 3(b)). It suggests that more Er3+ ions are excited with the concentration increase before 0.4 mM, but when the concentration is more than 0.4 mM, since every Yb3+ ion could absorb a certain amount of energy in the fixed power of 980 nm laser, the energy carried by every Er3+ ion decreases with the increase of Er3+concentration, leading to weaker energy level of transition and less excitation of pheophorbide A.
Reaction temperature had obvious effects on the emission spectra of Pha-UCNPs (Fig. 3(c)). The fluorescence intensity increased from 90°C to 120°C and then decreased from 120°C to 180°C, and the temperature of 120°C had the highest emission intensity at 670 nm. It is possible that 120°C could decrease the internal defects of nanoparticles and improves crystallinity related with emission efficiency.
Pha-UCNPs had the highest emission in 20 h of reaction time (Fig. 3(d)). Emission intensity continuously increased with reaction time until 24 h. Because the nanoparticle size is gradually enlarged with the reaction time, and it will change the luminous intensity of different emission spectra due to the relative concentration decrease of the doping ions on the surface with the increase of particle size, and larger size inhibits surface effect of the material and enhances the fluorescence emitting [35], but it also increases the molecular distance between UCNPs and photosensitizer leading to weakness of energy transfer.
3.2.2 Res-UCNPs
NaYF4: Yb3+/Tm3+ nanoparticles were conjugated with resveratrol to make Res-UCNPs. Its fluorescence emission spectra were compared at different concentration of Yb3+ and Tm3+, reactive temperature and time, the results are shown in Fig. 4. The fluorescence intensity at 380 nm gradually went up with the absorber Yb3+ concentration from 20 mM to 80 mM but decreased thereafter (Fig. 4(a)). It is due to the increase of photon number absorbed by the higher concentration of Yb3+ ions. It indicates that Yb3+ ions can be main block of the nanoparticles to enhance the upconversion emission of high-order photons, and make ultraviolet intensity stronger. But higher concentration (100 mM) of Yb3+ relatively decreases Tm3+ level and the interaction between Yb3+ and Tm3+.
Fig. 4 Fluorescence emission spectra of Res-UCNPs with different concentration of Yb3+ (a) and Tm3+ (b) at different temperature (c) and reactive time (d).
The effect of Tm3+ concentration on emission spectra of Res-UCNPs was opposed to Yb3+ (Fig. 4(b)). The fluorescence intensity decreased with Tm3+ concentration from 0.1 mM to 1 mM. It indicates that 0.1 mM Tm3+ is the best concentration to transit the energy. The lower concentration (0.05 mM) of Tm3+ is not enough to make effective upconversion, but when Tm3+ continuously increase from 0.1 mM, the shortened distance and the strengthened interaction force among Tm3+ ions eventually leads to concentration-quenching and cross-relaxation effects with few energy transfer to resveratrol.
From 90°C to 150°C the fluorescence intensity decreased, but increased with the reaction temperature from 150°C to 180°C until 210°C (Fig. 4(c)). The former increase of temperature may lead to the internal defects of nanoparticles, and latter increase of temperature may change the crystallinity causing transfer of more powerful energy, but continuous increase of temperature can change crystal structure leading to weakness of energy transfer.
The reactive time had the same affecting tendency as the temperature. The fluorescence intensity decreased with reaction time from 4 h to 16 h, but increased thereafter until 24 h (Fig. 4(d)). The former reactive time increase enlarges the size of nanoparticles leading to decrease of relative concentration of Yb3+, and longer time reaction may change crystal structure of the nanoparticles leading to the increase (20 h) or the decrease (24 h) of different pattern of energy transfer to excite resveratrol.
3.3 Crystal structure of the Ps-UCNPs
The crystal phase and defect mode could also affect the luminous intensity of different emission spectra as well as the size [36]. The crystal structures of Pha-UCNPs and Res-UCNPs were determined by x-ray diffraction (XRD) as shown in Fig. 5. It showed that the hexagonal phase of β-NaYF4 (JCPDS card 27-1427 as the standard) was a dominant phase in Pha-UCNPs and the cubic phase of α-NaYF4 (JCPDS card 39-0724 as the standard) was a dominant phase in Res-UCNPs. Although the cubic phase existed in the nanoparticles was less efficient for converting NIR light to visible compared to the hexagonal phase, its ultraviolet upconversion fluorescence was still strong. The higher intensity of diffraction peaks in Res-UCNPs than Pha-UCNPs suggests better crystallinity, which effectively decreases the surface defects of the crystals and enhances luminous property. The diffraction peaks were the highest at 20 h reaction time indicating that the crystal structure at that time is proper for both Pha-UCNPs and Res-UCNPs.
Fig. 5 X-ray powder diffraction pattern (XRD) of Ps-UCNPs: (a) Pha-UCNPs, (b) Res-UCNPs, (c) β-NaYF4, (d) α-NaYF4.
3.4 Size and morphology of the nanoparticles
The size distribution measured by Melvin particle size analyzer showed that the average sizes of NaYF4: Yb3+/Er3+ and NaYF4: Yb3+/Er3+ were 68 nm and 54 nm respectively with size distribution in the range of 40-70 nm. While conjugation with pheophorbide A or resveratrol, the average size was up to 220 nm with distribution in the range of 180-240 nm. Morphology of the nanoparticles was observed and photographed with SEM (Fig. 6), and the image shows that the UCNPs have irregular shapes with good dispersity, but change to sphere shape after conjugation with photosensitizers. The size gradually increased from 180 nm to 240 nm with reactive time from 4 h to 24 h, but had different effects on energy transfer from UCNPs to photosensitizers.
3.5 Single oxygen production of Ps-UCNPs
Single oxygen was generated by energy transfer from the excited photosensitizers to molecular oxygen and determined by DPA, a singlet oxygen quencher. The change of absorbance (∆A) between DPA and its mixture with Ps-UCNPs is expressed as the production of singlet oxygen in the experiments. As for pheophorbide A (Fig. 7(a)), the singlet oxygen production continuously increased with the irradiation time in 980 nm, but for resveratrol, it increased during initial 3 min and dropped rapidly after that time because of quenching effect. By contrast, Pha-UCNPs had a higher and more stable singlet oxygen production and a certain potential application value. The maximum was about seven times more than UCNPs and pheophorbide A alone, and reached stable after 30 min illumination (Fig. 7(b)).
Fig. 7 Comparison of singlet oxygen production among Ps-UCNPs (a) and Pha-UCNPs under 980 nm illumination at different time (b). Data were presented as mean ± standard deviation in 3 repetitions. There was significant difference (p<0.01) between Pha-UCNPs and Res-UCNPs, and between Pha-UCNPs and UCNPs or pheophorbide A.
3.6 Mechanism of energy transfer
The upconversion emission spectra and photographs of the NaYF4: Yb3+/Er3+ or NaYF4: Yb3+/ Tm3+ nanoparticles, excited by a 980 nm NIR laser, are given in Fig. 8. The green and red emission bands were clearly observed at about 419, 529, 545 and 650 nm which are attributed to the 2H9/2→4I15/2, 2H11/2→4I15/2, 4S3/2→4I15/2 and 4F9/2→4I15/2 transitions of Er3+ ion separately (Fig. 8(Aa)). The ultraviolet and visible light emission bands were clearly observed at about 293, 350, 360, 454, 481 and 650 nm which are attributed to the 1I6→3H6, 1I6→3F4, 1D2→3H6, 1D2→3F4, 1G4→3H6 and 1G4→3F4 transitions of Tm3+ ion separately (Fig. 8(Ab)). The strongest emission peak of nanoparticles doped with Er3+ located at 545 nm radiating green fluorescence, and the nanoparticles doped with Tm3+ had two stronger emission peaks at 481 nm and 360 nm radiating blue and violet respectively.
Fig. 8 Upconversion emission spectra and photographs ofNaYF4:Yb3+/Er3+ (a) and NaYF4:Yb3+/Tm3+ (b) with the 980 nm laser as excitation light source in the left (A). Schematic energy level diagram showing the upconversion mechanism of the Yb3+, Er3+/Tm3+ co-doped nanoparticles in the right (B).
The mechanism of upconversion emission for the Yb3+/Er3+ or Yb3+/Tm3+ co-doped nanocrystals has ever been investigated [37–39]. As illustrated in Fig. 8(B), the absorber Yb3+ ions absorb near-infrared light, and transfer the resonance energy to the emitter Er3+ or Tm3+ ions which can emit shorter wavelength such as ultraviolet or visible light [40, 41]. Although the emitter Er3+ or Tm3+ can be activated directly, co-doping of the absorber with Yb3+ ions in the nanoparticles usually generate stronger upconversion fluorescence due to the broad and strong absorption of Yb3+ at 980 nm. After conjugation with pheophorbide A and resveratrol, 419 nm light emitted by the nanoparticles doped with Er3+ excites pheophorbide A, and 293 nm light emitted by the nanoparticles doped with Tm3+ excites resveratrol by energy transfer to emit distinguished fluorescence at 670 nm and 380 nm respectively, and produce singlet oxygen leading to pharmacological effects.
4. Conclusion
Ps-UCNPs are synthesized by NaYF4 as the substrate and hydrothermal reaction of lanthanide ions Yb3+ with Er3+ or Tm3+, and then conjugation with pheophorbide A or resveratrol. Based on intensity of fluorescence emission by Ps-UCNPs, the optimal reaction conditions are 16 mM Yb3+/0.4 mM Er3+ at 120°C for 20 h for Pha-UNCNPs, and 80 mM Yb3+/0.1 mM Tm3+ at 180°C for 20 h for Res-UCNPs. The nanoparticles have a hexagonal phase or cubic phase with an average diameter size of 220 nm, and selectively emit the stronger red or violet fluorescence.
Pha-UCNPs have the high and stable singlet oxygen production under 980 nm illumination, which is attributed to the efficient resonance energy transfer from UCNPs to pheophorbide A. It is an effective NIR photosensitizer hopeful for the deep tissue diseases photodynamic therapy.
Funding
Guangzhou and Guangdong Scientific Plan Project (No. 1563000123, No. 2016B090918086).
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