Kinetic study of the self-assembly behavior of Mn-TCPP assemblies
Self-assembly pathway of Mn-TCPP assemblies
In aqueous solution, Mn2+ and TCPP were mixed in aqueous solution under vigorous stirring followed by slow addition of OH- at 328 K to initiate self-assembly (Figure 1a). After reaction for ca. 1 h, Mn-TCPP colloid was formed, which was verified by Tyndall effect under laser irradiation (Figure S1a). The size and morphology of the formed Mn-TCPP assemblies were characterized by transmission electron microscope (TEM) and dynamic light scattering (DLS). As shown in Figure S1b, nanoparticles with ca.10 nm were clearly seen in the TEM images. DLS profile (Figure S1b) showed that the hydrodynamic diameter (Dh) of the Mn-TCPP assemblies was 20 nm with PDI 0.196 which indicated there were some degrees of aggregation. Atomic force microscope (AFM) images confirmed the height of the nanosheets was about 5 nm (Figure S2a) which suggested nanosheets were formed. The nanosheet structures were further verified by scanning electron microscope (SEM) images shown in Figure S2b. Moreover, energy dispersive spectrometer (EDS) and X-ray photoelectron spectroscopy (XPS) profiles (Figure S3 and Figure S4) confirmed the constituent of the Mn-TCPP nanosheets consisted of Mn and N element. The components of the as-prepared Mn-TCPP nanosheets were also confirmed by TG analysis (Figure S5). TG analysis demonstrated that the Mn content in the Mn-TCPP nanosheets was about 14 wt%.
Interestingly, during the formation of the Mn-TCPP colloid, we found the solution color changed from yellow to pink before the colloid transformed to final brown as shown in the insert image of Figure 1c. UV/Vis spectrum was employed to measure the three different states. As shown in Figure 1c, the maximum absorption peak was 435 nm (solution color: yellow) shifted to 415 nm (solution color: pink) with gradient addition of OH-. With further addition of OH-, the solution color changed from yellow to brown accompanied redshift of the maximum absorption peak from 415 nm to 465 nm. Here we could see that during the self-assembly process of Mn-TCPP nanosheets, there were three different energy states formed: initial state, intermediate state and thermodynamically favored state. This interesting phenomenon encouraged us to attempt to study the self-assembly pathway of Mn-TCPP assemblies. UV/Vis spectra were further used to monitoring changes in the absorption spectrum from an initial state to an intermediate state, then to thermodynamically favored state. The absorption intensity changes at 465 nm as a function of OH- amount exhibited an obvious non-isodesmic model where there was a lag seen in the initial process (Figure 1d).29 This kinetic profile was characteristic of a cooperative process.30 In addition, the self-assembly process was temperature-dependent which was verified by temperature-dependent kinetic profiles (Figure S6). Here we could see that the self-assembly pathway could be divided into three different phases. We referred to the pathway from the initial state to the intermediate state as phase I and the pathway from the intermediate state to the thermodynamically favored state was named as phase II. The thermodynamic stable state was referred as phase III.
In the phase I, addition of OH- quickly induced the decrease of the peak (435 nm) while the peak at 415 nm increased sharply as shown in Figure 2a. The corresponding plot of I415/I435 as a function of OH- amount was shown in Figure 2b. The plot could be fitted to a first-order kinetic model. This I415/I435 change could be ascribed to the formation of TCPP-COO-, which was confirmed by the control experiment where addition of OH- into the TCPP solution without Mn2+. In the control experiment, a similar trend in I415 and I435 was observed (Figure 2c). However, by comparing the two plots of I415/I435 versus OH- amount, we could see that there was a big gap shadowed in Figure 2b. This gap indicated that OH- could be consumed in another form in the Mn/TCPP solution. This view was supported by the formation of Mn(OH)2 (Figure S7a) when Mn2+ and TCPP were mixed in a higher molar ratio (TCPP : Mn2+) of 1:10 followed by addition of OH-. This experiment suggested that TCPP and Mn2+ both consumed OH- in a competitive manner. Moreover, with the further addition of OH-, no absorption peak at 465 nm was observed in the control experiment (Figure 2c), which suggested that Mn2+ was crucial in the formation of thermodynamic stable Mn-TCPP aggregates. The formation of -COO- in TCPP could provoke the coordination between Mn2+ and TCPP. This was verified by the shift of -C=O stretching peak in the IR spectrum (Figure S7b). The formed intermediate Mn-TCPP aggregates were characterized by TEM, EDS, XPS and XRD. As shown in Figure 2d and Figure S8a, the Mn-TCPP aggregates revealed one-dimensional assemblies. The height profile in the AFM image (Figure S8b) indicated that the height of Mn-TCPP assemblies was about 2 nm, which suggested the one-dimensional Mn-TCPP assemblies were tape structures. The EDS and XPS profiles (Figure S9 and Figure S10) confirmed the formed intermediate Mn-TCPP tape structures constituted Mn and N elements. And TG analysis (Figure S11) demonstrated that the content of Mn in the intermediate Mn-TCPP structures was as high as 55% which was similar to the results obtained from ICP-AAS (Mn content : 54 wt%). This confirmed that the high content of Mn in the intermediate Mn-TCPP aggregates. XRD profile verified that the formed intermediate Mn-TCPP tape structures were amorphous structures (Figure S12). Moreover, from UV-Vis spectra, we could see that the maximum absorption wavelength was at 415 nm, which indicated the TCPP was in a monomer state in the formed intermediate Mn-TCPP aggregates.31 According to the above data, we could see that the intermediate Mn-TCPP tape structures could consist of large amounts of Mn(OH)2 and few Mn-TCPP complexes where the Mn-TCPP complexes separately anchored in the amorphous Mn(OH)2 complexes.
From the above experiments, we could summary the possible self-assembly pathway in the phase I. Addition of OH- could induce the formation of -COO- in TCPP and large amounts of Mn(OH)2 in a competitive manner. Subsequently, Mn-TCPP complexes formed by coordination between Mn2+ and TCPP (COO-) anchored into the amorphous Mn(OH)2 aggregates in a single monomer state.
However, the formed intermediate Mn-TCPP aggregates were kinetically formed state and not stable, which could further transform to thermodynamically favored state. The transformation from kinetically formed state to thermodynamically favored state was investigated by UV/Vis spectra. As shown in Figure 3a, continue addition of OH- could decrease the absorption intensity at 415 nm (I415) with I465 increasing. The change of I415/I465 versus additional OH- amount was seen in Figure S13. I415/I465 decreased as a function of OH- amount in an exponential decay mode. Moreover, the decay rate was temperature-dependent which was verified by comparing plots under 308 K and 328 K (Figure S13). Interestingly, even without addition of OH-, the transformation from kinetic favored Mn-TCPP assemblies to thermodynamically favored Mn-TCPP assemblies with time was observed seen in Figure 3b. As time going, the I415/I465 decreased sharply and quickly achieved a thermodynamically favored state in 0.5 h at 328 K. The transformation rate was temperature-dependent seen in Figure 3b. decreasing temperature could significantly decline the transformation rate. For example, when the temperature decreased to 318 K, it needed ca. 1 h to achieve the thermodynamic equilibrium state shown in Figure 3b. In addition, the transformation rate was also dependent on solution conditions. We centrifuged the intermediate Mn-TCPP assemblies and monitored the I415/I435 change with time in the deionized water instead of the original solution (Figure 3c). In this case, the transformation from intermediate Mn-TCPP assemblies to thermodynamic equilibrium state needed much longer time (more than 6 h) at 328 K. Figure 3c revealed that in deionized water, increasing temperature could accelerate the transformation of intermediate Mn-TCPP aggregates as well. By comparing the transformation rates of intermediate Mn-TCPP aggregates in original solutions and deionized water, we hypothesized that the transformation from Mn-TCPP intermediates to the thermodynamically favored state could be related to TCPP monomers. To verify this view, we added TCPP/OH- into the intermediate TCPP-Mn assemblies in water. From the plot of absorption intensity at 465 nm versus time (Figure S14), we could clearly see that adding TCPP/OH- could accelerate the transformation process, which indicated that TCPP monomer involved in the transformation process. We also did the dialysis experiment of intermediate Mn-TCPP assemblies against water with a dialysis bag (molecular cut-off value: 3500). The solution outside of the dialysis bag was monitored by UV. After 14 h, the absorption peak at 415 nm was detected which was corresponding to the phase I process (Figure 3d). After 3 days, the absorption intensity at 465 nm began to increase which suggested the phase II occurred. From these experiments, we could see that in the phase II, the intermediate Mn-TCPP tape structures first disassembled into monomer TCPP and Mn2+ followed by reassembling into thermodynamically favored Mn-TCPP nanosheets.
Photophysical properties of Mn-TCPP assemblies
The photophysical properties of Mn-TCPP assemblies in different states were investigated. The absorption profiles (Figure 1c) of Mn-TCPP assemblies in different states have been discussed above. And the various absorption behaviors of Mn-TCPP assemblies were resulted from the aggregation types of TCPP ligands. Here Figure 4a compared fluorescence of the Mn-TCPP assemblies in different states. From Figure 4a, we could find that the fluorescence of the thermodynamic stable Mn-TCPP nanosheets was completely quenched, while the intermediate Mn-TCPP aggregates still exhibited relatively strong fluorescence.
Meanwhile, as shown in nanosecond time-resolved transient absorption spectra (NTAS) (Figure 4b), a significant feature of transient absorption was observed at 460 nm under 355 nm laser excitation, which suggested that there was a triplet excited signal for intermediate Mn-TCPP tape structures. This could be attributed to the monomer state of porphyrin in the intermediate structures. However, under the same experimental conditions, no triplet excited signal was detected for thermodynamic stable Mn-TCPP nanosheets (Figure S15). No FL emission and triplet state in the thermodynamic stable Mn-TCPP nanosheets could be attributed to the J-aggregate induced quenching effect of the porphyrin moieties.18,32These different photophysical properties between intermediate Mn-TCPP assemblies and thermodynamic stable Mn-TCPP assemblies demonstrated that the metal-organic nanomaterials could exhibit self-assembly pathway-dependent properties.
In vitro PDT/PTT experiment
The different photophysical properties endowed these two Mn-TCPP aggregates with different applications. In terms of the intermediate state, the triplet signal detected by NATS demonstrated that the intermediate Mn-TCPP assemblies could possess light-induced 1O2 generation capability as PDT photosensitizers. And the thermodynamic favored aggregates with no FL emission could convert light energy into heat for PTT. Based on this hypothesis, we first evaluate the photoinduced singlet oxygen generation ability of the intermediate Mn-TCPP aggregates with 1,3-diphenylisobenzofuran (DPBF) as a probe. The DPBF was widely used as a 1O2 sensor due to its decreasing absorbance intensity at approximately 410 nm when exposure to O2 (Figure 5a). Upon light irradiation (660 nm, 12 mW cm–2) within 20 min, negligible changes were noted in the DPBF control group. However, a continuous decrease of the DPBF absorption at 410 nm was both observed in the two Mn-TCPP aggregates samples, which suggested their 1O2 generation ability. Moreover, for the intermediate Mn-TCPP aggregates, a significant decrease was clearly noted, suggesting the highly efficient light-induced 1O2 generation ability. This experiment demonstrated that the intermediate Mn-TCPP aggregates possessed excellent 1O2 generation ability for PDT.
To verify the photothermal property of the Mn-TCPP aggregates, we recorded the temperature changes of the two Mn-TCPP aggregates samples with an infrared (IR) thermal mapping apparatus. And the PBS solution was served as a control. As shown in Figure 5b, under laser irradiation at a low laser power density of 200 mW cm−2 within 10 minutes, the temperature in the thermodynamic favored aggregates increased from 24 oC to 41 oC maximumly, which exhibited good photothermal conversion ability. And the photothermal conversion behavior of the thermodynamic favored aggregates were further explored in various laser-irradiation time and concentrations (Figure S16a and Figure S16b), suggesting that the photothermal behavior was concentration-and laser-irradiation time dependent. At last, the photostability of the thermodynamic stable aggregates were explored, As shown in Figure S16c, when exposed to a laser for 24 min, negligible change in the maximum temperature was noted in the thermodynamic stable Mn-TCPP aggregates sample. This suggested that the thermodynamic stable Mn-TCPP aggregates exhibited excellent photothermal stability. Here the good photothermal conversion capability of the thermodynamic stable Mn-TCPP aggregates could be caused by strong π–π stacking proposed by Zheng et al.32 Thus, the thermodynamic stable Mn-TCPP aggregates could be promising photothermal agents (PTAs) for PTT.
The effects of PDT or PTT on the cell viability were further investigated towards HeLa cells. We first assessed the intracellular 1O2 generation by the intermediate Mn-TCPP aggregates after internalization. The dichlorofluorescein diacetate (DCFH-DA) was selected as a 1O2 sensitizer, which could convert DCFH-DA into highly fluorescent dichlorofluorescein (DCF) and then detected using monitored using confocal laser scanning microscopy (CLSM). As shown in Figure 5c, upon light irradiation, green fluorescence was observed in the cells cultured with intermediate Mn-TCPP aggregates, suggesting good 1O2 generation of intermediate Mn-TCPP aggregates in cells. Moreover, from a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay (Figure 5d and Figure 5e), owing to the contribution of 1O2 or photothermal ability of different Mn-TCPP aggregates, an obvious phototoxicity towards HeLa cells was observed. The 50% growth inhibitory concentration (IC50) values against HeLa cells under light irradiation were measured to be ca. 50 µg/mL for intermediate Mn-TCPP aggregates and ca. 0.73 mg/mL for thermodynamic stable Mn-TCPP aggregates.This vitro toxicity studies demonstrated that the formed Mn-TCPP aggregates controlled by self-assembly pathway exhibited good antitumor efficiency via PDT or PTT. This suggested that the Mn-TCPP aggregates showed self-assembly pathway-dependent anticancer therapy.