ONâ Photodynamic Therapy by a Hybrid

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NIR-activated “OFF/ON” Photodynamic Therapy by a Hybrid Nanoplatform with Upper Critical Solution Temperature Block Copolymers and Gold Nanorods Baoxuan Huang, Jia Tian, Dawei Jiang, Yun Gao, and Weian Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00963 • Publication Date (Web): 06 Sep 2019 Downloaded from pubs.acs.org on September 7, 2019

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NIR-activated “OFF/ON” Photodynamic Therapy by a Hybrid Nanoplatform with Upper Critical Solution Temperature Block Copolymers and Gold Nanorods

Baoxuan Huang, Jia Tian*, Dawei Jiang, Yun Gao and Weian Zhang*

Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, People’s Republic of China.

ACS Paragon Plus Environment

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ABSTRACT: Photodynamic therapy (PDT) is a promising treatment modality for cancer treatment owing to its minimally invasive nature and negligible drug resistance. However, the disadvantage of conventional photosensitizers including universal aggregation-caused quenching (ACQ) effect or non-selective activation is still a major hurdle for PDT clinical application. Herein, a new strategy for flexible manipulating photosensitizers in effective quenching and quick recovery of photoactivation is presented by introducing porphyrin units into an upper critical solution temperature (UCST) block copolymer decorated gold nanorods (AuNR-P(AAmco-AN-co-TPP)-b-PEG). The UCST block copolymer can achieve self-quenching effect to make the porphyrin photosensitizers in “Off” state by π-π stacking and hydrogen bonding interactions at physiological temperature, which greatly minimize non-selective phototoxicity of photosensitizers to meet the requirement of phototherapy protected from sunlight. After the immigration of AuNR-P(AAm-co-AN-co-TPP)-b-PEG nanoparticles into the tumor tissue and the internalization by cancer cells, the UCST polymer chains can be extended under the local heating of AuNRs by NIR light irradiation, and then porphyrin photosensitizers are turned “On” to dramatically boost the PDT efficiency. Therefore, the process of PDT could be well manipulated in “Off/On” state by the hybrid nanoplatform with UCST block copolymers and AuNRs, which will open new horizons for clinical treatments of PDT.


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INTRODUCTION Photodynamic therapy (PDT), as a noninvasive therapeutic modality, has gained great attention in cancer treatments.

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PDT mainly involves specific redox processes where photosensitizers

upon light irradiation at an appropriate wavelength can transfer the photon energy to generate reactive oxygen species (ROS), notably singlet oxygen (1O2), for irreversibly damaging of malignant cells and tissues.8-13 Thus, the photosensitizers play a crucial and fundamental role in PDT.14 However, the disadvantages of conventional photosensitizers such as non-selective activation are still a major hurdle for PDT clinical application, which often causes the therapyrelated toxicity and side effects on surrounding normal tissues.15-20 Moreover, the aggregationcaused quenching (ACQ) effect of photosensitizers also greatly decreases the PDT efficiency.21 During the past decades, much effort focused on PDT has been contributed to overcoming the ACQ effect. For example, to overcome the ACQ effect, many excellent works to enhance the PDT efficiency have been done,22-24 including the dehydration of photosensitizers on the backbone of hydroxypropyl cellulose,25 introduction of high steric cage-shaped polyhedral oligomeric silsesquioxane (POSS) into photosensitizer-containing alternating copolymers,26 and construction of photosensitizers-based metal-organic frameworks (MOFs).1 Although some achievements have been obtained to reduce the aggregation of photosensitizers and improve the efficiency of PDT, these obstacles are mostly considered individually. There is rarely systemic attention on the states of photosensitizers in the whole processes of PDT, especially, the state of photosensitizers during blood circulation was rarely been considered.9, 27-29 Nowadays, in the clinic process of PDT, patients still suffer more pain to stay at a dark room for several days to avoid the irreversible normal organ damage by the sunlight.30-31 Actually, it is better for that the intravenously injected photosensitizers are in a dormant state (“Off”) as much as possible, and


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only photosensitizers internalized by cancer cells will be activated (“On”), since most of photosensitizers need much long time for blood circulation, and only few (mostly less than 10%) accumulate on tumor sites. Thus, it is urgent to develop a “smart” photosensitizer that can be selectively activated at particular condition whether it is under strong sunlight or in the dark. In the past researches, the “smart” concept based on stimuli-responsive fragments has been widely designed for drug delivery system, where the efficiency of drug delivery has been greatly enhanced.32-34 “Smart” nanoplatform has also been utilized for delivery or activation of hydrophobic photosensitizers in PDT, which exhibits specific and superior PDT efficiency through some particular responses.35-37 Overviewing the responsive systems for the delivery of photosensitizers, a variety of works are focused upon stimuli including nucleic acid,38-42 pH,43-44 enzymes45-46 and glutathione (GSH),47-48 where photosensitizers could be released from the carriers or be activated when they arrive at tumor sites. However, these stimuli-responsive systems are endogenous, which could not be well controlled due to complex physiological environments. More recently, thermal-responsive systems are of great attraction since they can be conveniently manipulated upon external heat compress or under NIR irradiation by the introduction of photothermal agents.49-51 For example, previous studies have exhibited lower critical solution temperature (LCST) polymers could be utilized as the controlled drug-delivery system (DDS) manipulating by exogenous light irradiation.52-55 In comparison of LCST polymers, however, only few works were involved in UCST polymers for biological applications.56-58 For example, Du. et al. first reported the noncharged UCST-type DDS upon external heat compress to release hydrophobic anticancer drug, doxorubicin (DOX).57 Gong et al. further optimized the UCST-type DDS upon external light irradiation to release the drug.58 Moreover, Jin et al. reported the usage of UCST-type DDS to overcome multidrug resistance52


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and Hu et al. constructed the cancer diagnosis and treatment integrating platform using UCSTtype DDS.59 Comparatively speaking, these excellent works all focused on chemotherapy, however, to the best of our knowledge, there was no example about “smart” PDT platforms, which could regulate the photoactivity of photosensitizers by the phase transitions of thermalresponsive UCST polymers. Herein, we designed and constructed a unique NIR-manipulatable “smart” nanoplatform on the basis of a photosensitizer-containing UCST block copolymer and gold nanorods (AuNRs). As shown in Scheme 1, AuNRs were coated by porphyrin-containing distinctive nonionic UCST block copolymers to form the AuNR-based photosensitizer (AuPS) nanoplatform. The porphyrin-containing UCST block copolymer, P(AAm-co-AN-co-TPP)-b-PEG, possesses a good physiological adaptability and flexible phase transition temperature by adjusting the ratio of acrylamide (AAm) and acrylonitrile (AN). Benefiting from the outstanding surface plasmon resonance (SPR) and photothermal features, AuNRs play the roles of a fluorescent quencher of porphyrins, a heating producer and an antenna for NIR-manipulatable PDT as well. The photoactivity of the “smart” photosensitizer can be conveniently manipulated through the local heating generated by AuNRs by NIR irradiation. In general, during blood circulation, the UCST block of the copolymer, below its UCST, is hydrophobic to form a collapsed-core, resulting in the aggregation of porphyrin units and further leading to the porphyrin units “Off”. Furthermore, After the internalization of AuPS by cancer cells, porphyrin units in “Off” state can be turned “ On” through the phase transition of UCST polymers from extended to collapsed state and the breakage of the π-π stacking between the porphyrin units induced by photothermal AuNRs under NIR irradiation (Scheme 1). This process can controllably promote singlet oxygen generation and give rise to remarkable improvement of PDT efficiency, more importantly, the selective


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activation of porphyrin units could fully suppressed systemic toxicity before AuPS arrives at the tumor tissue. In this work, the synthesized porphyrin (TPP)-containing UCST polymers were confirmed using 1H NMR and FT-IR spectra. Then, the thermal responsive properties of the UCST polymers were assessed by temperature-dependent UV-Vis spectroscopy. Subsequently, the photothermal-activatable property of TPP in AuPS nanoparticles was evaluated by temperature-dependent UV-vis and fluorescence spectroscopy, respectively. The PDT efficacy of “smart” nanoplatform manipulated by NIR light was further evaluated by in vitro and in vivo studies.


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Scheme 1. Schematic illustration of preparation and “Off/On” behavior of AuPS, and the photodynamic therapy process of AuPS nanoparticles.


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EXPERIMENTAL SECTION Materials Gold (III) chloride trihydrate (HAuCl4·3H2O, ≥49.0%), N, N′-dicyclohexylcarbodiimide (DCC), 4-(dimethyl amino) pyridine (DMAP), ascorbic acid, methoxy poly(ethylene glycol) (PEG, Mn = 5000) and 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were all purchased from Aladdin Reagents of China and used directly as received. Methoxy poly(ethylene glycol) was azeotropically dried by toluene. Other reagents and solvents were all analytical grade unless mentioned. The synthesis of TPP monomer60 and chain transfer agent PEG-DDAT61 were according to the previous research works.

Synthesis of PEG-b-P(AAm-co-AN-co-TPP) block polymer The block copolymer was synthesized by a typical RAFT procedure. The molar ratio of monomers (AAm + AN), TPP monomer, chain transfer agent PEG-DDAT and AIBN as the initiator was fixed at 1000: 6: 1: 0.2 for preparing block copolymers. As an example, the procedure of the block copolymers with theoretical value of 30.0 mol% of AN was described as following. AAm (2.464 g, 34.7 mmol), AN (0.552 g, 10.4 mmol), PEG-DDAT (0.090 g, 0.063 mmol), AIBN (0.002 g, 0.0126 mmol) were dissolved in anhydrous dimethyl sulfoxide (DMSO, 3 mL) in a dried Schleck tube with a stirring bar. Three freeze-evacuate-thaw cycles were applied for the mixed solution removing oxygen, and the polymerization was operated at an oil bath of 70 °C for 24 h. Subsequently, the crude product was diluted with 8 mL DMSO and three cycles of the precipitation in excess methanol were carried out for removing the unreacted AN and AAm monomers . Then a similar precipitation process was performed in another three cycles of re-dissolution in DMSO and precipitation in excess ice ether to remove the unreacted TPP


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monomers. After dried in a vacuum oven at 40 °C for 48 h, the red solid was obtained. (Yield: 88%). The number molecular weight (Mn) and polydispersity index (PDI) of the block copolymer (denoted as PS) were measured by GPC: Mn = 87.3 kg mol-1 and PDI = 1.32. Accordingly, the control (PC) with Mn = 85.1 kg mol-1 and PDI = 1.37 was also prepared by a similar approach with the feed ratio of AN/ AAm/ TPP = 33/ 67/ 0.6.

Synthesis of Gold Nanorods (AuNRs) Silver ion-assisted seed-mediated method was used for preparing AuNRs.62 The seed solution was prepared by adding HAuCl4 (10 μL, 0.01 M) aqueous solution into CTAB solution (5 mL, 0.02 M). And then ice-cold NaBH4 solution (120 μL, 0.002 M) was added quickly with magnetic stirring (800 rpm), resulting in producing a brownish-yellow solution. The above solution was aging at 25 °C for 2 h and used as the seed solution A. In addition, another growth solution B was performed by mixing HAuCl4 solution (1.0 mL, 0.01 M) and AgNO3 (1.1 mL, 0.01 M) solution with CTAB solution (80 mL, 0.1 M) in another flask. Then, a fresh L-ascorbic acid solution (0.8 mL, 0.1 M) was subsequently added under gentle stirring until the solution B turned from yellowish to colorless. HCl solution (1.0 mL, 1.0 M) and the seed solution A (200 μL) were thereby injected into the growth solution B. Finally, the above mixed solution was setting silently in water bath at 25 °C for 18 h. The as-prepared AuNRs were purified by centrifuge (10000 rpm, 5 min) and washed with deionized water. The supernatant was discarded and the AuNRs were redispersed in 5 mL deionized water for use, assigned as AuNRs dispersion.

Preparation of AuNR-(PEG-b-(AAm-co-AN-co-TPP)) Nanoparticles


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Typically, polymer-grafted AuNRs were prepared by dropwise addition of a PEG-b-(AAm-coAN-co-TPP) block copolymer aqueous solution (2.0 mL, 5.0 mg/mL) to an 5.0 mL as-prepared AuNRs dispersion (with the AuNRs concentration about 0.1 mg/mL) under gentle magnetic stirring. After the reaction was performed for 4 h at 40 ℃ under stirring, the centrifuged mixture

was re-dispersed and washed with deionized water (8000 rpm, 7 min) for three times to remove the excess polymers. The products were denoted as AuPS and AuPC by grafting the block polymers of P(AAm980-co-AN400-co-TPP6)-b-PEG and P(AAm908-co-AN489-co-TPP6)-b-PEG, respectively.

Singlet Oxygen Production Ability of AuPS. The singlet oxygen production capability is an essential feature of the photosensitizer in PDT. Thus, the thermal-responsiveness of 1O2 production was evaluated both under and above the UCST of PS. AuPS (porphyrin concentration: 16 μg/mL) was dispersed in the mixture of 1 % DMSO and 99% PBS. After AuPS was dispersed uniformly, After the addition of DPBF solution, the mixture was irradiated by 650 nm laser in 30 °C water bath or in 60 °C water bath. The UVvis spectra of the solutions were determined at the fixed time intervals to record the absorption variation of DPBF by the capture of 1O2 produced by AuPS.

Cell Cultures and Cellular Uptake Tests Cell culture and cellular uptake tests were according to the previous work.61 Cellular uptake was characterized by flow cytometry, and the cellular localization was performed by confocal laser scanning microscope (CLSM, Nikon AIR). In a simple process, 4T1 cells at a density of 2 × 105 cells/well were cultured with 2 mL of culture medium in a Petri dish for 24 h at 37 °C. Then the


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culture medium was alternated with a fresh culture medium involving AuNR- P(AAm980-coAN400-co-TPP6)-b-PEG (AuPS) or AuNR- P(AAm908-co-AN489-co-TPP6)-b-PEG (AuPC) nanoparticles (final porphyrin concentration: 16 μg/mL) and incubated for another 24 h at 37 °C. PBS was used to wash the cells three times and the cell nucleus was stained with Hoechst for 5 min. The images of cellular uptake location were subsequently obtained using CLSM. In addition, the groups with laser irradiation were performed by the 808 nm laser irradiation upon the corresponding Petri dishes for 5 min and further taking CLSM images.

In vitro Singlet oxygen Production Ability of AuPS 4T1 cells were incubated for 24 h in Petri dishes for assessing the ROS production in living cells. Dulbecco’s modified Eagle’s (DMEM) medium containing AuPC (porphyrin concentration: 16 μg/mL), AuPS (porphyrin concentration: 16 μg/mL) were applied for incubating the cells for 24 h. Then, the cells washed with PBS were incubated with DCFH-DA (5 μM) for 30 min, after which the cells were incubated in dark or irradiation. All samples were treated with 650 nm light irradiation (1.0 W/cm2 for 100 s). The blank control, AuPS and AuPC samples were performed with 808 nm light irradiation (1.5 W/cm2 for 200 s and then 1.0 W/cm2 for 100 s) additionally.

In vitro dark cytotoxicity and phototoxicity For in vitro phototoxicity, 4T1 cells (25 000 cells mL-1, 200 μL) were incubated in 96-well plate for 24 h at 37 °C. Then the cells were incubated with a fresh culture medium containing either AuPS or AuPC nanoparticles with a series concentrations of polymers (with the porphyrin concentration at 0-16 μg/mL, respectively). After incubated at 37 °C for another 24 h, the cells


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were irradiated with 808 nm light (1.5 W cm-2 for 200 s and 1.0 W cm-2 for 100 s) and 650 nm light (500 mW cm-2 for 100 s) and then incubated for another 24 h. The culture medium was thus alternated with a fresh culture medium having MTT (5 mg/mL) for another incubation of 4 h. Finally, the culture medium with MTT was removed and alternated with 150 μL DMSO to dissolve the precipitates, followed by measuring the absorbance of solution at 560 nm by a spectrophotometric microplate reader (THERMO Multiskan MK3 spectrometer). Cell viability was calculated by the Equation (1): Cell viability (%) = (ODt - ODb) / (ODc - ODb) × 100

(1)

where ODb is the absorbance of the background; ODt and ODc are the absorbance of solutions with or without samples, respectively. For in vitro dark cytotoxicity, the process was similar to the phototoxicity described above, but without the light irradiation.

Animal Models All animal experiments were in accordance with international guidelines on the ethical use of laboratory animals and approved by the regional animal committee. The 4-6-week-old female BALB/c nude mice (17-20 g) bearing subcutaneous xenografts of 4T1 were used for in vivo studies. 4T1 cells were trypsinized and washed twice with PBS. Then, the nude mice were inoculated with 4T1 cells on their left flanks by injecting 2×106 cells in 100 μL PBS.

In Vivo Fluorescence Imaging


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AuPS dispersion (200 μL, the concentration of porphyrin was fixed at 1.0 mg/mL) was administrated into 4T1-cancer-bearing BALB /c mice by intravenously injecting. IVIS imaging systems was employed for fluorescence bioimaging at 2 h, 6 h, 12 h and 24 h.

In Vivo Tumor Growth Inhibition When the tumors grew up to a mean volume of around 200-300 mm2, all mice were randomly assigned to seven treatment groups (four mice in each group) as following: (a) PBS, (b) AuPS, without irradiation, (c) AuPC, without irradiation, (d) AuPS, with only 808 nm light irradiation, (e) AuPS, with only 650 nm light irradiation, (f) AuPC, with both 650 nm and 808 nm light irradiation, (g) AuPS, with 650 nm and 808 nm irradiation simultaneously (administered at a TPP-equivalent dose of 10 mg/kg and at a AuNR-equivalent dose of 5 mg/kg). The 808 nm irradiation was fixed at 1.0 W/cm2 and the 650 nm irradiation was fixed at 500 mW/cm2, with a spot diameter of 3-4 mm. The following equation was employed for estimating tumor volume (mm3): 1

V (mm²) = 2 × A (mm) × B (mm)²

(2)

where A and B were respectively the length and width of tumor. Tumor volume was measured every 2 days with a vernier caliper.

Histological Assays The mice were sacrificed on the 16th day after treatments. The tumors were collected, weighed and fixed with freshly prepared 4 % neutral buffered formalin, followed by hematoxylin/eosin (H&E) staining.


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RESULTS AND DISCUSSION Here, the TPP monomer was first synthesized by the acylation reaction as described in Scheme S1 and characterized by 1H NMR (Figure S1-S3), which was further used to prepare porphyrincontaining amphiphilic block copolymer, P(AAm-co-AN-co-TPP)-b-PEG via reversible addition-fragmentation chain transfer (RAFT) copolymerization by using PEG (Mn = 5000) as a macromolecular RAFT agent (Figure S4). The structure of P(AAm-co-AN-co-TPP)-b-PEG (denoted as PS) was collaboratively characterized by 1H NMR (Figure S5) and FT-IR spectra (Figure S6). According to the 1H NMR spectrum of PS, the signal at ~8.87 ppm (ascribed to β-H from TPP) testified the presence of the porphyrin units on PS chain. In view of 1H NMR results, the ratio of AAm and AN was nearly 71: 29 and about 5.8 porphyrin units were contained per PS chain. Moreover, the porphyrin content in PS was further examined by the absorption at 420 nm in UV-vis spectra with reference to a standard calibration curve of tetraphenylporphyrin in DMSO (Figure S9), where the results were basically consistent with the 1H NMR analysis. Additionally, the GPC curves of the polymers with narrow molecular weight distribution is shown in Figure S10, indicating that the RAFT copolymerization of AAm, AN and TPP was well controlled. Thus, on the basis of above results, the number-average polymerization degrees of AAm, AN and TPP units were obtained and the block copolymer was assigned as P(AAm980co-AN400-co-TPP6)-b-PEG. Similarly, the control sample, P(AAm908-co-AN489-co-TPP6)-b-PEG (denoted as “PC” ) with a higher UCST was synthesized by altering the feed ratio of three monomers (AAm/AN/TPP) (Figure S7, S8 and S10). Besides, the UV-vis spectra of prepapred PS and PC were shown in Figure S11, exhibiting the characteristic absorption peaks of porphyrins.


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Figure 1. (A) Schematic illustration of self-assembly and the thermal responsive behavior of PS. (B) Temperature-dependent changes in hydrodynamic diameter of PS. (C) Transmittance of the micellar dispersion as a function of temperature. (D) Turbidity variation of PS at 30 and 50 ℃ for several cycles in PBS.

The amphiphilic UCST polymer PS can self-assemble to micelles and exhibits outstanding thermal responsive performance in buffer solution (Figure 1A). To characterize the UCST behavior of block copolymers in PBS, DLS technology and TEM were performed to determine the size of spherical micelles obtained from self-assembly of block copolymers. As shown in Figure 1B, the hydrodynamic size of the PS micelles dramatically decreased as the temperature increased from 37 to 47 ℃, and kept constant with the temperature further increasing, since the ACS Paragon Plus Environment

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UCST block in the micelles underwent the phase transition from the highly hydrophobic to hydrophilic form. TEM images of PS micelles exhibited spherical morphology with the size about 135 nm and extremely small domains with the size about 12 nm when prepared at 35 oC and 50 oC, respectively (Figure S12 and S13). The consistence between DLS and TEM results confirmed the size variation of PS under different temperatures. To further confirm the UCST behavior of PS block copolymer in PBS, the turbidity measurement was conducted at the wavelength of 800 nm using UV-vis spectroscopy (Figure 1C). The transmittance of PS increased as the temperature increased from 38 to 43 ℃, and it basically remained constant with

the temperature beyond 43 ℃. Furthermore, the reversible transmittance changes over five cycles between 30 ℃ and 50 ℃ could be well performed (Figure 1D in PBS and Figure S14 in the

presence of fetal bovine serum), revealing the temperature-responsive reversible transition of PS. This typical UCST behavior was abundantly verified by DLS and UV-vis spectroscopy. Thus, the temperature of 43 ℃, where the transmittance began to constant, was defined as the UCST of P(AAm980-co-AN400-co-TPP6)-b-PEG. The PS with a sharp transition window for thermal

responsiveness is suitable for “smart” PDT since only gentle heating is needed from physiological temperature to 43 ℃ which exhibits negligible side effect on adjacent normal

tissues. Additionally, the UCST behavior of PC as the control in PBS was also characterized by UV-vis spectroscopy (Figure S15). However, the UCST of PC was as high as 85 ℃, which, in line with our expectation, is too high to manipulate by the photothermal effect of AuNRs under NIR irradiation in a physiological environment.


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Figure 2. (A) A TEM image of AuNRs. (B) A TEM image of AuPS nanoparticles. (C) UV-vis absorbance spectra of AuNRs, PS and AuPS nanoparticles. Fluorescent spectra of AuPS nanoparticles (D) heating from 29 to 51 ℃ and (E) cooling from 51 to 31 ℃. (F) Fluorescent intensity of AuPS at 652

nm as a function of temperature. (G) Thermal images recorded for different concentrations of AuPS nanoparticles upon 808 nm laser irradiation at different irradiation times. (H) Singlet oxygen generation of TPP, AuPS and AuPC nanoparticles in the mixture of 1% DMSO and 99 % PBS plotted according to the maximum absorption of DPBF at 365 nm via different irradiation duration with different samples AuPS at 30 ℃ (red), AuPS at 60 ℃ (black), AuPC at 30 ℃ (green), AuPC at 60 ℃ (blue), TPP at 30 ℃

(cyan) and TPP at 60 ℃ (pink). (I) Illustration of “Off/On” process AuNR-P(AAm980-co-AN400-co-TPP6)b-PEG (AuPS) nanoparticles.


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For conveniently manipulating the “smart” PDT, AuNRs, possessing an excellent photothermal conversion capability, were conjugated with PS to form hybrid AuNR/PS (AuPS) nanoparticles via the Au-S interaction between AuNRs and trithiocarbonate-bearing PS. A representative TEM image of AuNRs shown in Figure 2A demonstrated the well-dispersed character and regular rod shape of the AuNRs with the mean length and width of 64.2 ± 3.2 and 12.6 ± 2.2 nm, respectively. Similarly, AuPS nanoparticles have similar morphology with AuNRs, but no significant polymers shells were observed owing to the ultrahigh contrast ratio of AuNRs under TEM measurement (Figure 2B). Meanwhile, benefited from the coating of PS, AuPS nanoparticles were really stable and there was no significant precipitate after several days at room temperature (Figure S16). Moreover, the longitudinal surface plasmon resonance (SPR) peak of AuNRs (752 nm) in the AuPS sample showed a redshift to 796 nm for being coated with PS (Figure 2C). Additionally, for AuPS, all of peaks from porphyrin units on PS and AuNRs absorbance clearly appeared, confirming the hybrid AuPS nanoparticles were successfully prepared. The temperature-dependent (Figure S17) and laser light irradiation time-dependent (Figure S18) size changes of AuPS nanoparticles were studied by DLS. The results showed that the hydrodynamic size of AuPS increased with the increasing temperature or light-induced local heating due to the extension of the grafted PS chains. Furthermore, the photophysical property of AuPS was assessed by fluorescence spectroscopy as shown in Figure 2D-2F. The fluorescence intensity of TPP units on AuPS increased very rapidly with increasing temperature from 29 to 51 ℃ (Figure 2D) and then decreased while the temperature decreased from 51 to 31 ℃ (Figure

2E). The fluorescence intensity of AuPS nanoparticles at 652 nm in both temperature increasing

and decreasing processes was collected and plotted in Figure 2F. The fluorescence intensity


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climbed sharply in the heating range of 35 and 47 ℃, while a lagging was observed in the cooling process from 45 to 33 ℃. These results strongly demonstrate that the fluorescence

change is greatly dependent on the aggregation/extension state of porphyrin units. With the temperature increasing, the H-bonding can be broken between PS and surrounding water molecules, successfully inducing the hydrophobic-to-hydrophilic transition of PS chains, which accompanies with porphyrin units change from the agglomerated state to relatively “free” state. On the contrary, with the temperature decreasing, the H-bonding recovered, and the π-π stacking of porphyrin units was intensified to strengthen the ACQ effect. In addition, the fluorescence spectra of PS and AuPS under and above UCST were detected as well (Figure S19). A small fluorescence quenching was found both at 35 oC and 45 oC, indicating the fluorescence

quenching from AuNRs. However, for the control sample, AuPC, almost negligible fluorescence change between 37 oC and 50 oC (the temperature range of physiological temperature and AuNRs-produced local heating induced temperature) was observed for TPP units on AuPC (Figure S20, S21 and S22), indicating that porphyrin units on AuNRs were still maintained in the aggregation state, since the temperature was much lower than the UCST of PC (83 ℃).

To evaluate the in vitro NIR-induced photothermal effect of AuNRs, the temperature

elevation of the AuPS nanoparticles in PBS at different concentrations was examined upon 808 nm laser irradiation at a power density of 1.0 W/cm2. As shown in Figure 2G, a remarkable temperature elevation of AuPS (10.0 µg/mL, AuNRs equivalent) solution was observed and the temperature increased continuously within 5 min. As expected, the photothermal effect is highly dependent on the concentration of AuPS nanoparticles. The temperature of 10.0 µg/mL AuPS


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nanoparticles could be over 58 ℃ at 5-min light irradiation, moreover, for 5.0 µg/mL of AuPS solution, it still could attain 53 ℃. However, for PBS solution without AuPS, there is almost no temperature increase within 5 min. Herein, the brilliant photothermal effect was facilely achieved

for AuPS solution in a controllable manner. The temperature range (~25-60 ℃) can be modulated by the AuPS concentration and NIR laser irradiation duration to meet the needs of

“smart” PDT process. Additionally, the NIR-induced photothermal effect of AuPS as a switch of “smart” PDT was also confirmed in 96-well plates with the PBS solution of AuPS containing 3.0 µg/mL AuNRs (the same as cell viability text concentration). As shown in Figure S23 and S24, AuPS solution presented a temperature increase when it was irradiated by 808 nm NIR laser with different laser powers. Thus, to meet the “switch” temperature of PS (T> UCST), we employed the laser irradiation at a power density of 1.5 W/cm2 for 200 s and then 1.0 W/cm2 for 100 s. As shown in Figure S25, AuPS solution can maintain at 43 ℃, satisfying the “switch” temperature.

The production ability of singlet oxygen is a very important parameter for PDT. Here, the singlet oxygen induced by TPP units under 650 nm light irradiation could be evaluated by using 1, 3-diphenylisobenzofuran (DPBF) as a singlet oxygen trapper (Figure2H and Figure S26). The production of singlet oxygen was significantly dependent on the state of TPP units. For free porphyrins regardless of 30 ℃ or 60 ℃ upon light irradiation, almost negligible change of singlet

oxygen was observed. This is because that the strong hydrophobic TPP units agglomerated in aqueous solution suppressing the production of singlet oxygen. For AuPC, the DPBF degradation rate under light irradiation at 60 ℃ was only slightly higher than that at 30 ℃, revealing that there was no significant difference of singlet oxygen production. It was mainly

because most of porphyrin units were still kept in aggregation state although there was partial


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breakage of H-bonding of PC. However, the DPBF degradation in AuPS group was dramatically improved at 60 ℃, which is much higher than that at 30 ℃. These results indicated that the porphyrin units were completely extended during the hydrophobic-to-hydrophilic transition of PS chains, leading to the dramatical reduction of ACQ and remarkably increasing generation of singlet oxygen (Figure 2I).

Figure 3. (A) Confocal microscopic images of 4T1 cells incubated for 24 h with blank control, AuPS and AuPC nanoparticles without (-) or with (+) 808 nm laser irradiation (1.5 W/cm for 200 s and 1.0 W/cm2 for 100 s), scale bar: 20 µm. (B) Intracellular ROS generation in 4T1 cells mediated by different samples: blank control, AuPS and AuPC with 808 nm laser irradiation (1.5 W/cm2 for 200 s and then 1.0 W/cm2 for 100 s), and all samples were treated with 650 nm (1.0 W/cm2 for 100 s), scale bar: 20 µm. (C) Cell


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viability data of 4T1 cells with various concentrations of porphyrin units in the dark. (D) Cell viability data of 4T1 cells with various concentrations of porphyrin units after light irradiation.

We next investigated the activating capability of AuPS nanoparticles against 4T1 cells (Figure 3A). After the incubation with the AuPS or AuPC nanoparticles for 24 h, 4T1 cells were imaged by confocal laser scanning microscope (CLSM). The intracellular red fluorescence signal from porphyrin units could be clearly observed in all of these groups, but only weak red fluorescence signal was observed in cells incubated with AuPC nanoparticles, regardless of the presence or absence of 808 nm laser irradiation. For the cells incubated with the AuPS nanoparticles with 808 nm laser irradiation, it had the strongest fluorescence in these four groups, meaning that the nanoparticles were internalized by tumor cells and TPP units on AuPS were efficiently activated by the increasing temperature under the NIR laser irradiation. The producing 1O2 capacity of AuPS nanoparticles in cancer cells was further assessed by intracellular 1O2 sensor, 2′, 7′-dichlorofluorescein diacetate (DCFH-DA). As shown in Figure 3B, the untreated cells showed a weak green fluorescence even with the presence of 650 nm and 808 nm laser irradiation. For cells treated by AuPC nanoparticles, only slightly enhanced green fluorescence was observed with or without 808 nm laser irradiation. However, it is worthy to note that the cells treated with AuPS nanoparticles with 650 and 808 nm laser irradiation showed the extremely bright green fluorescence, which illustrated that the 1O2 production of AuPS nanoparticles can be conveniently manipulated by the NIR laser due to the state variation of porphyrin units. Thus, the manipulatable AuPS nanoparticles can effectively boost the PDT efficiency under dual laser irradiation in tumor tissues and minimize the potential systemic toxicity in blood circulation.


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To evaluate the therapeutic efficacy of AuPS nanoparticles, the in vitro experiments on dark cytotoxicity and phototoxicity were performed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) assays in 4T1 cells. As shown in Figure 3C, in the dark, there was still more than 90 % cell survival ratio even when 4T1 cells incubated with AuPS or AuPC nanoparticles solution even with 16 µg/mL TPP units, which suggested AuPS and AuPC nanoparticles are good biocompatible and almost no toxic to cells. For phototoxicity (Figure 3D), 4T1 cells were incubated with AuPS nanoparticles and then suffered from 808 nm laser irradiation, it can be seen that only slight phototoxicity was generated by AuPS nanoparticles, which is attributed to the photothermal effect of AuNRs. Here, AuNRs were introduced to the system mainly for providing the thermal source, so the photothermal therapy of AuNRs was controlled as low as possible. When the cells were incubated with AuPS nanoparticles and only exposed to 650 nm laser irradiation, the phototoxicity of AuPS nanoparticles clearly appeared with the concentration-dependent feature, but the high cell viability still remained even at high concentration of nanoparticles because of the insufficient singlet oxygen produced by the π-π stacking porphyrin units. However, the phototoxicity of AuPS nanoparticles was greatly enhanced for the cells irradiated with both 808 and 650 nm, the cell viability could be less than 20 % with the concentration of 16 µg/mL TPP. This is because that TPP units on AuPS nanoparticles were activated by the phase transition of PS chains from hydrophobic to hydrophilic state when AuPS nanoparticles were irradiated upon the 808 nm laser to induce the increasing temperature higher than the UCST of PS. Additionally, cells incubated with AuPC nanoparticles and irradiated with 808 and 650 nm laser showed a quite low cell viability, which was similar to that of the groups of AuPS nanoparticles only exposed to 650 nm laser, since PC as the control has a higher UCST and porphyrin units were still in aggregated state.


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Figure 4. (A) Time-dependent in vivo NIR fluorescence images of 4T1 tumor-bearing mice after intravenous injection of AuPS nanoparticles. (B) IR thermal images of 4T1-tumor-bearing mice injected with AuPS nanoparticles or PBS during 5-min NIR laser irradiation. (C) Representative mice after 16 days treated with AuPS nanoparticles and different laser irradiation conditions, and their H&E staining images of tumor tissue sections after 16 days of treatment. The scale bar represents 100 μm. (D) Average tumor weight of different groups; *p < 0.05, **p < 0.01, and ***p < 0.001, determined by a Student’s ttest. (E) Digital photos of four groups mice after 16-day treatment. (F) Tumor volumes of mice after different treatments. All data are presented as mean ± SD (n = 4).


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To explore the tumor preferential accumulation of AuPS nanoparticles and further guide PDT in vivo, NIR fluorescence imaging system was used to track the fluorescence of TPP units. After intravenously injected AuPS nanoparticles for 2 h, the tumor sites of BALB/c mice bearing subcutaneous 4T1 xenografts appeared weak fluorescence. The tumor became much clearer after 12 h post-intravenous injection of AuPS, and then a high contrast signal of the tumor with tumor margin could be well distinguished after 24 h post-intravenous injection (Figure 4A). The excellent AuPS accumulation in the tumor sites benefited from the PEGylation and their suitable small size facilitating the passive-target approach via the enhanced permeability and retention (EPR) effect. Furthermore, the photothermal capacity of AuPS accumulated in tumor region was evaluated to guide the strategy of 808 nm laser irradiation (Figure 4B). At 24 h post-injection of AuPS nanoparticles, the temperature of tumor region can be increased rapidly to approximately 65 ℃ upon the irradiation of 808 nm laser at 1.0 W/cm2 for 5 min. These results indicated the photothermal capacity of the accumulated AuPS was sufficient to meet the need of PDT activation. The in vivo PDT efficiency was further assessed by using 4T1-tumor-bearing mice via the tail vein injection of AuPS nanoparticles. The fluorescence of porphyrin units in AuPS nanoparticles was turned “On” under the 808 nm laser irradiation, and then the hybrid photosensitizer AuPS extensively generated singlet oxygen with 650 nm laser irradiation. The phototoxicity of AuPS nanoparticles to the mice bearing 4T1 tumor was evaluated by monitoring the tumor volumes and tumor weights. In regard to tumor cell death, the treatment efficacy was also assessed by hematoxylin and eosin (H&E) staining of tissue sections (Figure 4C). According to the NIR fluorescence imaging of AuPS nanoparticles accumulating at tumor sites in vivo, experimental groups were irradiated with NIR laser at 24 h post-intravenous injection. As shown in Figure


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4C-4F, Figure S27 and S28, an approximately 15-fold increase of tumor volumes and no tumor tissue necrosis were observed in PBS group as a blank control. As expected, the groups of AuPS and AuPC in the absence of irradiation also exhibited no tumor growth inhibition or tumor tissue necrosis. And the mice group injected with AuPS under 808 nm laser irradiation exhibited no apparent therapeutic effect in tumor tissues due to the short irradiation time where AuNRs were only used as a tool to wake up porphyrin units. For PDT treatment, the tumor growth only could be suppressed to some extent for the group of the mice injected with AuPS under 650 nm laser irradiation, so the average tumor volume on day 16 was around 3 folds larger than that on day 0. In marked contrast, the tumor growth in mice injected with AuPS under both 808 and 650 nm laser irradiation was notably suppressed. Moreover, the tumor tissue displayed apparent necrosis, revealing that the aggregation of porphyrin units in AuPS nanoparticles could be suppressed by 808 nm laser irradiation and further be effectively excited by 650 nm laser irradiation to produce powerful tumor phototoxicity and achieve PDT in “On” state. However, for the mice injected AuPC under both 808 and 650 nm laser irradiation, the therapeutic effect was almost similar to that of the mice injected with AuPS only under 650 nm laser irradiation. As far as we know, it is the first time that PDT efficiency has been successfully modulated in vivo by a porphyrincontaining UCST polymer. To evaluate the potential systemic toxicity and side effects, the body weight loss of all groups was measured and H&E staining of their major organs including liver, spleen, lung, and kidney was analyzed. There were negligible differences in body weight loss among various groups (Figure 5A). No distinct signs of physiological morphology abnormalities were detected in the H&E stained organ slices, which confirms that AuPS nanoparticles are safe for in vivo PDT application (Figure 5B). Thus, AuPS nanoparticles could be well used to enhance PDT


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efficiency in vivo by manipulating the phase transition of PS chains from hydrophobic to hydrophilic state.

Figure 5. (A) Mean body weights of mice in different groups after treatment (n = 4). (B) H&E staining images of major organ tissues of various samples. Scale bar 100 μm.

CONCLUSION In summary, we have presented a strategy using NIR-light manipulating the PDT efficiency by an UCST block copolymer to balance activation and inactivation of photosensitizers for minimizing systemic toxicity and enhancing PDT efficiency. The block copolymer with a suitable UCST of 43 ℃ can be utilized to construct AuPS nanoparticles. During long blood

circulation, porphyrin photosensitizers in AuPS nanoparticles were in “Off” state resulted from

π-π stacking and hydrogen bonding interactions, where the phototoxicity of photosensitizers was greatly minimized to meet the avoiding light requirement in the process of photosensitizer delivery. With AuPS nanoparticles immigrated into the tumor sites and internalized by tumor cells, porphyrin photosensitizers can be turned “On” to significantly improve the PDT efficiency


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by the photothermal effect of AuNRs irradiated under by NIR light. Thus, this NIR-light manipulatable “Off/On” PDT nanoplatform based on an UCST block copolymer would open new horizons for clinical application of PDT. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] Notes The authors declare no competing financial interest. Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Synthesis of PS, 1H NMR spectra of TPP-OH, TPPC6-OH, TPPC6A, DDAT-PEG, PS and PC, FT-IR of PS and PC, GPC curves, photos of AuNRs and AuPS in PBS, fluorescent intensity variety of AuPC upon heating or cooling, laser power-dependent temperature elevation of deionized water and AuPS, H&E staining images of tumor tissues (PDF) ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (No. 51803058, 21875063) and the Fundamental Research Funds for the Central Universities (No. 222201814018) REFERENCES


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NIR-activated “OFF/ON” Photodynamic Therapy by a Hybrid Nanoplatform with Upper Critical Solution Temperature Block Copolymers and Gold Nanorods Baoxuan Huang, Jia Tian*, Dawei Jiang, Yun Gao and Weian Zhang*


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ONâ Photodynamic Therapy by a Hybrid

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