Porphyrin-based metal coordination polymers with self-assembly pathway-dependent properties for photodynamic and photothermal therapy

Porphyrin-based metal coordination polymers (MCPs) have attracted numerous attention due to their great promise application in phototherapy including photodynamic therapy (PDT) and photothermal therapy (PTT). However, the detailed self-assembly process of porphyrin-based MCPs still remains poorly understood. This work provides a detailed study of the self-assembly process of MCPs constructed by Mn2+ and TCPP (TCPP: 4,4′,4′′,4′′′-(Porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid)) in aqueous solution. Unlike traditional nucleation and growth mechanism, we discover that there is a metastable metal-organic intermediate which is kinetically favored in the self-assembly process. And the metastable metal-organic intermediate nanotape structures could convert into thermodynamically favored nanosheets through disassembling into monomers followed by reassembling process. Moreover, the two structurally different assemblies exhibit distinct photophysical performances. The intermediate Mn-TCPP aggregates show good light-induced singlet oxygen 1O2 generation for PDT while the thermodynamic favored stable Mn-TCPP aggregates exhibit good photothermal conversion ability as photothermal agents (PTAs). This study could facilitate controlling self-assembly pathway to fabricate complex MCPs with desirable applications.


Introduction
Metal coordination polymers (MCPs) are constructed from coordination metal ions and organic linkers followed by supramolecular assembly with hydrogen bonding, π-π stacking or hydrophobic interactions. [1][2][3][4][5] Owing to their facile synthetic procedure, high drug loading capability and low long-term toxicity, the MCPs have attracted numerous interest in biomedical application. [6][7][8][9][10] In the past decades, large numbers of metal-organic nanomaterials for anticancer therapy were fabricated with different sizes and morphologies. 4,11−12 Among the reported MCPs, porphyrin-based MCPs are widely studied. [13][14][15][16] Porphyrins are well-known photosensitizers for photodynamic therapy due to their highly e cient singlet oxygen 1 O 2 generation under light irradiation. [16][17] Moreover, Zheng et, al reported that porphyrins could possess photothermal conversion capability when porphyrins aggregated with high packing density. [18][19] So, porphyrins could also be utilized as photothermal agents (PTAs) for photothermal therapy (PTT). These dual functionalities endow porphyrin great promise in phototherapy. Moreover, in the MCPs, porphyrins directly participate in constructing the structure of MCPs, which makes MCPs have relatively high photosensitizer loading e ciency. 14,20 At last, the metal ions such as Mn 2+ , Gd 3+ offer magnetic resonance imaging (MRI) which endows the formed MCPs with bioimaging applications. [21][22] All these merits make the porphyrin-based MCPs provide great therapeutic e ciency in precise anticancer therapy.
Although much attention has been paid to the fabrication of porphyrin-based MCPs with various morphologies and functions, the deep understanding of the self-assembly mechanism which controlled the nal nanostructures remained elusive. [23][24][25] The kinetic detailed study was rarely reported but crucial in view of controlling the self-assembly pathway to fabricate the metal-organic nanoparticles with desired applications. 23-24,26−27 And the corresponding formed nanostructures usually exhibited unique properties such as diversity, adaptivity and complexity. 28 In this paper, we studied the self-assembly process of Mn-TCPP (TCPP: 4,4′,4′′,4′′′-(Porphine-5,10,15,20tetrayl)tetrakis(benzoic acid)) nanoparticles (NPs) (Scheme 1). Interestingly, we discovered that during the self-assembly process, kinetically formed metastable amorphous Mn-TCPP intermediates (tape structures) were rst formed before converting into thermodynamically favored form. The kinetically formed metastable Mn-TCPP aggregates exhibited different morphologies and photophysical properties from the thermodynamic stable states. We also found that the metastable Mn-TCPP aggregates converted into thermodynamic stable assemblies (nanosheets) via disassembling into monomers followed by the reassembling process. Moreover, the different photophysical properties endowed the Mn-TCPP aggregates different phototherapy applications. The kinetically formed metastable Mn-TCPP aggregates showed e cient photoinduced singlet oxygen generation ( 1 O 2 )for PDT application.
Meanwhile, the thermodynamic stable Mn-TCPP aggregates exhibited photothermal conversion ability as photothermal agents (PTAs). This research provided a new insight of the formation of porphyrin-based MCPs for phototherapy including PDT and PTT.

Results And Discussion
Kinetic study of the self-assembly behavior of Mn-TCPP assemblies Self-assembly pathway of Mn-TCPP assemblies In aqueous solution, Mn 2+ and TCPP were mixed in aqueous solution under vigorous stirring followed by slow addition of OHat 328 K to initiate self-assembly ( Figure 1a). After reaction for ca. 1 h, Mn-TCPP colloid was formed, which was veri ed 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 pro le ( Figure S1b) showed that the hydrodynamic diameter (D h ) 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 con rmed the height of the nanosheets was about 5 nm ( Figure S2a) which suggested nanosheets were formed. The nanosheet structures were further veri ed by scanning electron microscope (SEM) images shown in Figure S2b. Moreover, energy dispersive spectrometer (EDS) and X-ray photoelectron spectroscopy (XPS) pro les ( Figure S3 and Figure   S4) con rmed the constituent of the Mn-TCPP nanosheets consisted of Mn and N element. The components of the as-prepared Mn-TCPP nanosheets were also con rmed 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 nal 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 OHamount exhibited an obvious non-isodesmic model where there was a lag seen in the initial process ( Figure 1d). 29 This kinetic pro le was characteristic of a cooperative process. 30 In addition, the self-assembly process was temperature-dependent which was veri ed by temperaturedependent kinetic pro les ( 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 OHquickly induced the decrease of the peak (435 nm) while the peak at 415 nm increased sharply as shown in Figure  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 OHcould decrease the absorption intensity at 415 nm (I 415 ) with I 465 increasing.
The change of I 415 /I 465 versus additional OHamount was seen in Figure S13. I 415 /I 465 decreased as a function of OHamount in an exponential decay mode. Moreover, the decay rate was temperaturedependent which was veri ed 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 I 415 /I 465 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 signi cantly 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-  Figure S14), we could clearly see that adding TCPP/OHcould 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 rst disassembled into monomer TCPP and Mn 2+ 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 pro les (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  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 o C to 41 o C 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.