NIR-Activated Polymeric Nanoplatform with Upper Critical Solution

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A NIR-activated Polymeric Nanoplatform with Upper Critical Solution Temperature for Image-guided Synergistic Photothermal and Chemotherapy Jia Tian, Baoxuan Huang, Haiquan Li, Hongliang Cao, and Weian Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00321 • Publication Date (Web): 24 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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A NIR-activated Polymeric Nanoplatform with Upper Critical Solution Temperature for Imageguided Synergistic Photothermal and Chemotherapy

Jia Tian#, Baoxuan Huang#, Haiquan Li, Hongliang Cao, Weian Zhang*

Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237, China

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Abstract Premature and uncomplete drug release are the typical bottlenecks of drug release in traditional chemotherapy. Synergistic therapies are high desirable in medicine and biology since they can compensate for the drawbacks of single therapy and significantly enhance the therapeutic efficacy. Herein, a novel NIR-activated polymeric nanoplatform with upper critical solution temperature (UCST) was constructed for image-guided synergistic photothermal and chemotherapy. UCST-responsive amphiphilic block copolymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization and then co-assembled with IR780 and cabazitaxel (Cab) to form spherical nanoparticles. IR780/Cab dual-loaded UCST polymeric nanoparticles can produce local heating upon NIRlaser irradiation and further lead to the dissociation of cargo-loaded nanoparticles and controlled release of Cab. IR780 acted as both the roles of heating generator and the activator for “on-demand” drug release. The investigation of in vivo fluorescence and photothermal imaging demonstrated clearly tumor targeting. Notably, both in vitro and in vivo studies illustrated that the synergistic photothermal and chemotherapy presented better anticancer efficacy than the simple plus of photothermal therapy and chemotherapy. Thus, the welldefined polymeric nanoplatform opens a versatile and effective path to develop image-guided synergistic therapies platforms for tumor treatment.

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Introduction The development of anticancer treatments with high therapeutic efficacy has been devoted considerable efforts since cancer is still one of the most hazardous disease to human health. However, conventional chemotherapy always encounters various challenges, for instance, most of anticancer drugs are hydrophobic, which leads to the poor biostability in blood circulation and further results in short circulation time, low cellular uptake, premature and uncomplete drug release. Additionally, the frequently high amount anticancer drug injection induces the undesirable multidrug resistance. In the past decades, drug delivery system (DDS) has been explored to be a promising and elegant strategy to address the problems of short circulation time and mononuclear phagocyte system elimination.1–6 Recently, stimuliresponsive DDSs7–11 have been demonstrated to prolong the circulation time and obtain the controlled drug release upon endogenous or exogenous triggers, such as pH,12,13 cytosolic GSH,14 glucose,15 reactive oxygen species,16 gas,17 light18 and magnetic field.19 Practically, among these DDSs, thermo-responsive DDSs have attracted much attention because a certain range of temperature deviation around physiological temperature (about 37 oC) in a specific tissue or organ, particularly in tumor-lesion tissue, almost has no influence on the whole body.20–23 For instance, poly(N-isopropylacrylamide) (PNIPAM) as the most studied thermoresponsive polymer, has been well known with the lower critical solution temperature (LCST) at about 32 oC.24–26 However, the shrink of PNIPAM chains at the temperature above LCST leads to the uncomplete drug release. Additionally, it has been reported that PNIPAM chains with low biocompatibility and high toxicity are not suitable for using in cancer treatment. Thus, the development of a new class of more biocompatible thermo-responsive

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materials is in an urgent need for the applications in biomedical area. Up critical solution temperature (UCST) responsive polymers will undergo the phase transformation from collapsed to stretched state when the temperature increases up to UCST, leading to the satisfied drug release efficiency.27–30 However, it is well known that most of UCSTresponsive polymers are based on polyzwitterions which are sensitive to polymer concentration and ionic strength. In recent years, the copolymer of acrylonitrile (AN) and acrylamide (AAm) (P(AAm-co-AN)) has been reported to be a novel UCST-responsive biocompatible uncharged polymer.31–35 Synergistic therapies by the combination of chemotherapy and phototherapy could be promising to address the drawbacks of chemotherapy and express the merits of each therapy.36–38 Phototherapy is considered as a powerful therapeutic strategy for treating cancers due to its invasiveness, time and site-specific control, high treatment efficacy and low side effects.39–41 Accordingly, photothermal therapy (PTT) depends on the PTT agent which could efficiently convert light energy to local heating and further induce thermal ablation of cancer cells. To date, much effort has been focused on the development of inorganic PTT agents42–44 including gold nanorods, black phosphorus, CuS nanoparticles and MoS2 nanosheets. However, the potential long-term toxicity and low biodegradability limit the future clinic translation of inorganic PTT agents. Consequently, various NIR-absorbing organic PTT agents have been attracted increasing interest as potential alternatives in the past decade.45–47 Heptamethine cyanine dyes, as an ideal candidate for PTT cancer treatment, possess strong absorbance in NIR region, good biocompatibility and multifunctional characteristics.48–53 IR780, a typica heptamethine cyanine dye, exhibits higher fluorescence efficiency and higher

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stability in blood circulation.54,55 Despite the promise, IR780 frequently suffers from the low tumor-targeting efficacy, poor water-solubility and remarkable photobleaching leading to remarkable PTT efficiency reduction. Even though varieties of vehicles have been exploited to improve the water-solubility and photothermal conversion efficiency, there are almost few vehicles that can simultaneously achieve on-demand translocation of co-encapsulated chemotherapeutic drug molecules from vehicles to cytoplasm and the excellent PTT efficiency. Therefore, the rational design of smart drug release platform is highly desirable for the maximization of synergistic thermo-chemotherapy by simultaneous release of chemotherapeutic drug. Based on the above considerations, in this work, we developed a novel drug release system for cancer synergistic thermo-chemotherapy by “programmed” PTT and drug release. UCSTtriggered amphiphilic PEG-b-P(AAm-co-AN) block copolymer was synthesized by RAFT polymerization and further used as the “smart” vehicle. Cargoes co-encapsulated nanoparticles (NPs) were prepared by the co-assembly of IR780, cabazitaxel (Cab) and PEGb-P(AAm-co-AN) block copolymer. IR 780, which could produce local heating under NIR irradiation, was utilized as both the generator of local heating and the “antenna” of drug release. Correspondingly, along with the temperature increase, PEG-b-P(AAm-co-AN) block copolymer was used as the “executor” of drug release, in which the stretched P(AAm-co-AN) chains would induce the dissociation of cargo-encapsulated nanoparticles and subsequently release Cab molecules. Therefore, IR780/Cab-encapsulated PEG-b-P(AAm-co-AN) NPs can accomplish spatiotemporally controlled PTT-induced drug release and achieve synergistic photothermal and chemotherapy. Confocal laser scanning microscopy (CLSM) and flow cytometry were employed to evaluate the cellular uptake and distribution. The toxicity of

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IR780/Cab-encapsulated PEG-b-P(AAm-co-AN) NPs against 4T1 cells was evaluated by standard methyl thiazolyl tetrazolium (MTT) assay in vitro. Additionally, IR780/Cabencapsulated PEG-b-P(AAm-co-AN) NPs were injected intravenously into 4T1 tumorbearing nude mice for evaluate bioimaging and therapeutic efficacy in vivo.

Scheme 1. Illustration for the preparation of IR780/Cab dual-loaded PEG-b-P(AAm-co-AN) NPs and synergistic photothermal and chemotherapy. Experimental Section Materials

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S-1-Dodecyl-S′-(R, R′-dimethyl-R′′-aceticacid)trithiocarbonate (DDAT) was synthesized according to a previous method.57 Poly(ethylene glycol) monomethyl ether (PEG-OH, Mn = 5000) was purchased from Sigma-Aldrich. 4-Dimethylaminopyridine-p-toluenesulfonate (DPTs),

triphenylphosphine

(PPh3),

diisopropyl

azodiformate

(DIAD),

2,3,3-

trimethylindolenine, 1,3-diphenylisobenzofuran (DPBF), 1-alkynyl-propyl butanedioate, azodiisobutyronitrile (AIBN), acrylamide (AAm), acrylonitrile (AN) and 1,4-dioxane were all purchased from Aladdin Reagents of China. Cabazitaxel (Cab), Hochest33342 and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Beyotime Biotechnology Institute. Toluene, methanol, dichloromethane (DCM), N,Ndimethylformamide (DMF), dimethyl sulfoxide (DMSO) and tetrahydrofuran (THF) were dried over calcium hydride and distilled before use. The cyanine dye IR780 was synthesized according to the previous publication.56 Synthesis of PEG-DDAT A typical procedure for the synthesis of PEG-DDAT is briefly described as follows. PEG-OH (1.00 g, 0.20 mmol), DDAT (87.5 mg, 0.24 mmol) and PPh3 (62.9 mg, 0.24 mmol) was dissolved in 25 mL of dry THF and stirred in ice-water bath. And then, DIAD (48.5 mg, 0.24 mmol) was dissolved in 5 mL dry THF and dropwise added into the above solution. After being stirred at room temperature for 20 h, the reaction solution was transferred into 40 oC oil bath for another 5 h. The solution was concentrated on a rotary evaporator and then diluted with DCM and washed with NaHCO3 solution and distilled water. The collected organic phase was dried over MgSO4 and concentrated on a rotary evaporator. The residue was precipitated in cold diethyl ether and dried in vacuum, the yield of PEG-DDAT is about 75 %.

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1H

NMR (400 MHz, Chloroform-d) δ 4.28 (s, 2H), 3.59-3.71 (s, 458H), 3.40 (s, 3H), 3.31 (d,

2H), 2.03 (t, 2H), 1.69 (m, 6H), 1.26 (s, 20H), 0.88 (t, 3H). Synthesis of PEG-b-P(AAm-co-AN) block copolymer via RAFT polymerization. PEG-bP(AAm-co-AN) block copolymer was prepared by RAFT polymerization with PEG-DDAT as the macromolecular chain transfer agent. PEG-DDAT (0.12 g, 0.022 mmol), AIBN (1.2 mg, 0.007 mmol), AAM (1.08 g, 14.3 mmol) and AN (0.28 mL, 4.4 mmol) were added into a Schlenk flask and dissolved in 1.5 mL 1,4-dioxane. The reaction mixture was degassed by three freeze-pump-thaw cycles and the polymerization was conducted at 65 °C for 10 h. The polymerization solution was diluted in DMSO and precipitated in methanol and diethyl ether for twice to remove the unpolymerized monomers. The monomer conversion is about 93 %. 1H

NMR (400 MHz, Chloroform-d) δ 6.70-7.83 (m, 1160H), 3.51 (s, 450H), 3.21 (s, 3H),

1.18-2.41 (m, 1940H). Co-assembly of Cab, IR780 and PEG-b-P(AAm-co-AN) block copolymer PEG-b-P(AAm-co-AN) micelles, and Cab/IR780-loaded NPs were prepared via a membrane dialysis method. Cab (1.33 mg), IR780 (1.33 mg) and copolymers (10 mg) were dissolved in 2 mL of mixture solvent (THF/DMF (v/v) = 2:3). The mixture was dropwise added into 3 mL of deionized water under magnetic stirring for 30 min. Then the solution was dialyzed against deionized water for 24 h to remove solvent and free Cab and IR780. Free micelles were prepared by the similar method without Cab and IR780. Cab/IR780-loaded NPs were dissolved in DMF after freeze drying, and determined the amount of Cab by high performance liquid chromatography (HPLC) and IR780 by the absorption intensity by UV-vis spectrometer according to the calibration curve. The drug 8


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loading content (DLC) and drug encapsulation efficiency (EE) were calculated according to the following formula, respectively:

DLC (%) =

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑚𝑖𝑐𝑒𝑙𝑙𝑒𝑠 × 100% 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑒𝑑 𝑚𝑖𝑐𝑒𝑙𝑙𝑒𝑠

EE (%) =

𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑚𝑖𝑐𝑒𝑙𝑙𝑒𝑠 × 100% 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑓𝑒𝑒𝑑

In vitro Release of Cab. Cab release behavior was explored by adding Cab/IR780-loaded NPs solution into dialysis tube (MWCO 3.5 kDa) and immersing in 50 mL of PBS solution at 37 °C with magnetic stirring. 2.0 mL of the dialyzed solution was taken out at certain time intervals and lyophilized, an equal volume of fresh PBS solution was supplied into dialysis tube. The release rate of Cab was calculated according to the HPLC analysis of the collected and lyophilized samples. Cell Culture. Murine breast cancer cells (4T1) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics (50 units per mL penicillin and 50 units per mL streptomycin) at 37°C in humidified atmosphere containing 5% CO2. In vitro cytotoxicity The cells were incubated for 24 h on 96-well plates at a seeding density of 4×103 cells/well followed by overnight attachment. The cell medium was replaced with IR780, Cab, IR780loaded NPs, Cab-loaded NPs, IR780/Cab dual-loaded NPs with different concentrations of IR780 ranging from 0 to 20.0 g/mL and Cab ranging from 0 to 24.0 g/mL. After incubation for another 24 h, the cell viability was evaluated by the standard MTT (5 mg/mL) 9



assay and the results were utilized to evaluated the dark cytotoxicity. The absorbance at the wavelength of 560 nm was determined by a SpectraMax spectrometer. The following equation was used to calculate the cell viability: cell viability (%) = (ODsample – ODblank) / (ODcontrol – ODblank) × 100, where ODsample represents the absorbance with sample solutions, ODcontrol represents the absorbance without treatment, and ODblank is the absorbance of empty plates. Similarly, for the phototoxicity, after the incubation of different doses of IR780, Cab, IR780loaded NPs, Cab-loaded NPs, Cab/IR780-loaded NPs, 4T1 cells in plates were applied for 5 min light irradiation (808 nm, 500 mW/cm2). The cell viability was incubated for additional 24 h and valuated by the standard MTT (5.0 mg/mL) assay. Cellular uptake Cellular uptake behavior and distribution of free IR780 and Cab/IR780-loaded NPs were determined with 4T1 cells by cytometry and confocal laser scanning microscopy (CLSM). For flow cytometry analysis, 4T1 cells suspension was seeded in 6-well plate at a density of 1 × 106 cells per well and cultured for 24 h. The cells were treated with free IR780 and Cab/IR780-loaded NPs with the same concentration of IR780, and incubated for the predicted time respectively. the cells were detached with trypsin from the wells and washed with PBS solution for three times. The cells were analyzed using flow cytometry to illustrate the fluorescence intensity via a BD FACS Calibur flow cytometer. For CLSM measurements, 4T1 cells were seeded on glass-bottomed dishes at a density of 6 × 103 cells/dish and cultured at 37 oC for 24 h. Free IR780 and Cab/IR780-loaded NPs containing culture medium were added and cultured for 24 h at the IR780 concentration of 14 10


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g/mL. The culture medium was removed and the cells were washed with PBS for three times. The cells were fixed with 4% paraformaldehyde in PBS for 25 min, further labeled by Hoechst 33342 for 25 min. The stained cells were thoroughly washed by PBS (pH = 7.4) for three times and imaged by a laser scanning confocal fluorescence microscope (Nikon AIR). In vivo fluorescence imaging Female BALB/c mice (4-6 weeks) were purchased from Jiesijie Animal Technology Co. Ltd. (Shanghai, China). All animal experiments were in accordance with international guidelines on the ethical use of laboratory animals approved by the regional animal committee. Tumorbearing mice were established by subcutaneously injecting 1×106 4T1 cells in a 100 L of PBS. Mice with tumor size of about 100 mm3 were used to in vivo fluorescence imaging and Infrared thermal imaging. Cab/IR780-loaded NPs in PBS solution (200 L) was intravenously injected to the tumor-bearing mice at the IR780 concentration of 500 g/mL. The kinetic fluorescence images were captured at 0.5 h, 2 h, 6 h, 12 h, and 24 h using an IVIS in vivo imaging instrument (Lumina XR Series III, PerkinElmer). In vivo Photothermal evaluation The tumor-bearing mice were intravenously injected with 200 L Cab/IR780-loaded NPs at IR780 concentration of 500 g/mL. Twelve hours post injection, the mice were exposed to the 808 nm laser at 500 mW/cm2 for 5 min. The temperature increasing was recorded by an infrared thermal imaging camera (Fluke Ti27). In vivo efficacy study The samples (200 L) of free IR780 (0.5 mg/mL), Cab (0.6 mg/mL), IR780-loaded NPs

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(IR780 0.5 mg/mL), Cab-loaded NPs (Cab 0.6 mg/mL), IR780/Cab dual-loaded NPs (IR780 0.5 mg/mL, Cab 0.6 mg/mL) were injected intravenously from the tail vein (n=4). The IR780 and Cab dose were fixed at about 5.0 mg/kg and 6.0 mg/kg for all samples, respectively. The tumor volume was measured every other day after treatments and calculated by (length of tumor) × (width of tumor)2/2. For photothermal or synergistic therapy, free IR 780, IR780loaded NPs and IR780/Cab-loaded NPs injected mice were exposed to the 808 nm laser at 500 mW/cm2 for 5 min. Thereafter, the mice tumor volume and body weight of all groups were monitored every other day. At the end of experiment, all the mice were sacrificed. The tumors were weighted and the collected tumor tissues were fixed and analyzed by hematoxylin-eosin (H&E) staining assay. Statistical Analyses Student’s t test was used for statistical analysis between different treated groups. Differences were considered statistically significant, and marked as *, **, ***, when p < 0.05, p < 0.01, p < 0.001, respectively.

Results and Discussion Synthesis of UCST-triggered polymer Recently, the copolymers of AAm and AN have been reported as a novel kind polymer with an UCST feature. Compared with the common UCST-triggered polyzwitterions, there are no ions in P(AAm-co-AN) chains which minimize the influence of external ions, particularly in the blood circulation and tumor sites. Amphiphilic UCST-triggered PEG-b-P(AAm-co-AN)

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block copolymer was synthesized by RAFT polymerization with PEG-DDAT as the macromolecular chain transfer agent (Figure 1a), and the 1H NMR spectrum of PEG-DDAT is shown in Figure S1. It is convenient to modulate the UCST of PEG-b-P(AAm-co-AN) by adjusting the molar ratio of AAM and AN. The 1H NMR spectrum of PEG-b-P(AAm-co-AN) in d6-DMSO is shown in Figure 1b. The peaks in the range of 6.67-7.82 ppm are assigned to the protons of amino group in AAm units and the peaks from 1.19 ppm to 2.40 ppm are ascribed to the protons of -CH2-CH- in the polymer backbone. Based on the peak area ratio of the above characteristic peaks in 1H NMR results, the polymerization degree of AAm and AN were calculated as 580 and 165, respectively. Therefore, the molar ratio of AAm and AN in PEG-b-P(AAm-co-AN) block copolymer is about 79/21. According to SEC analysis (Figure S1B), the number-average molecular weight and polydispersity index of the obtained PEG-bP(AAm-co-AN) block copolymer was 57.3 kDa and 1.16, respectively. Fourier transform infrared absorption (FTIR) spectrum of PEG-b-P(AAm-co-AN) is shown in Figure 1c. The bands around 1655 cm-1 and 3300 cm-1 were attributed to AAm units, and the characteristic bands of AN at about 2241 cm-1 could be observed.

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Figure 1. (a) Synthesis of UCST-triggered PEG-b-P(AAm-co-AN) block copolymer, (b) 1H NMR spectrum and (c) FT-IR spectrum of PEG-b-P(AAm-co-AN) block copolymer.

UCST property measurements of PEG-b-P(AAm-co-AN) block copolymer In order to explore the thermo-responsive behavior of PEG-b-P(AAm-co-AN) block copolymer, dynamic light scattering (DLS) technology was utilized to monitor the hydrodynamic diameter changes upon the temperature variation. At 25 oC, the amphiphilic PEG-b-P(AAm-co-AN) chains self-assemble to micelles with the size about 280 nm (Figure 2a) in aqueous solution. However, the hydrodynamic diameter of PEG-b-P(AAm-co-AN) block copolymer decreases to about 5 nm when the temperature increases to 60 oC (Figure 2b). The photographs of the self-assembly of PEG-b-P(AAm-co-AN) block copolymer in

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aqueous solution under different temperatures are displayed in the inner of Figure 2a and Figure 2b. Obviously, the cloudy solution turns to transparent while the temperature increases to 60 oC. The dependence of the hydrodynamic diameter of PEG-b-P(AAm-co-AN) agglomeration on the temperature was studied by DLS technology with gradually increasing temperature. The hydrodynamic diameter sharply drops from 280 nm to 5 nm in the range of 38 oC to 48 oC (Figure 2c). Thus, the PEG-b-P(AAm-co-AN) block copolymer exhibits an UCST at about 43 oC, which is very suitable to drug delivery in a low demand of temperature increase from the physiological temperature (about 37 oC). On the other hand, the transmittance of the self-assembly of PEG-b-P(AAm-co-AN) block copolymer in aqueous solution with the variations of temperature was monitored by UV-Vis spectrometer (Figure 2d). A remarkable increase of transmittance was observed with the temperature increasing from 37 oC to 45 oC. Similarly, the transmittance reduced to almost zero while it cooled from 45 oC to 36 oC. It is worthy of note that there was a small lag during cooling process, which suggests that drug release would continue for a while even after removing the heat producer.

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Figure 2. (a) DLS curve of PEG-b-P(AAm-co-AN) micelles at 25 oC, (b) DLS curve of PEGb-P(AAm-co-AN) chains at 60 oC, the photographs of PEG-b-P(AAm-co-AN) nanoparticles at different temperatures are displayed inset; The logo is used with permission from East China University of Science and Technology. (c) the dependence of hydrodynamic diameter and the variation of temperature, (d) the dependence of transmittance and the variation of temperature. Self-assembly of PEG-b-P(AAm-co-AN) block copolymer and encapsulation of IR780 and Cab The self-assembly of PEG-b-P(AAm-co-AN) block copolymer and encapsulation of IR780 and Cab were prepared by a nanoprecipitation method. DMF solution of PEG-b-P(AAm-co-

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AN) or the mixture of PEG-b-P(AAm-co-AN), IR780 and Cab were added into 10-fold excess PBS solution and further dialyzed in a dialysis tubing (MWCO 14 kDa) for 2 days. Figure 3a shows the TEM image of the morphology of PEG-b-P(AAm-co-AN) block copolymer in PBS solution at room temperature. The thermo-responsive block copolymer self-assembled into uniform micelles with collapsed P(AAm-co-AN) chains as the core and PEG block as the shell. Based on the TEM image, the size of PEG-b-P(AAm-co-AN) micelles in dry state is calculated to be about 155 ± 31 nm. The TEM image of PEG-bP(AAm-co-AN) micelles after a heating-cooling circle is shown in Figure S1C, which almost keeps the same with that in Figure 3a. Additionally, the average hydrodynamic diameter (Dh) of single Cab-encapsulated NPs and IR780-encapsulated NPs were found to be 310 nm (Figure S2) and 270 nm (Figure S3), respectively. Moreover, after the encapsulation of both IR780 and Cab, the micellar structures turn to be a smaller size about 86 ± 11 nm (Figure 3b) due to the additional hydrophobic IR780 and Cab. The Dh of IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs in PBS solution is about 150 nm (Figure 3d). Compared with the TEM results, the Dh of these micelles is a little bigger in PBS solution owing to the stretched hydrophilic PEG chains and the hydration shell. Furthermore, Figure 3c shows the TEM image of IR780/Cab-loaded PEG-b-P(AAm-coAN) NPs upon the treatment of 808 nm irradiation (500 mW/cm2) for 5 min. Irregular aggregations were observed, indicating the dissociation of cargo-loaded polymeric nanoparticles and the aggregation of hydrophobic cargoes. Additionally, the UV-vis and fluorescence spectra of IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs are shown in Figure 3e. A strong band near 800 nm was observed in both UV-vis and fluorescence spectra owing to the unique photochemical and photophysical property of IR780. The results suggest that 17



the local heating generated by the loaded IR780 is high enough up to the UCST of PEG-bP(AAm-co-AN) block copolymer, leading to the “programmed” drug release.

Figure 3. TEM images of (a) PEG-b-P(AAm-co-AN) micelles, (b) IR780/Cab-loaded PEGb-P(AAm-co-AN) NPs, (c) IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs with the treatment of 808 nm laser irradiation 500 mW/cm2 for 5 min. (d) A DLS curve of IR780/Cabloaded PEG-b-P(AAm-co-AN) NPs prepared at room temperature, (e) UV-vis and fluorescence spectra of IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs prepared at room temperature. Photothermal property In order to evaluate the photothermal properties of IR780/Cab-loaded PEG-b-P(AAm-coAN) NPs, the real-time temperature changes of IR780/Cab-loaded NPs and PBS solution are 18


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identified. All samples are studied under laser irradiation (808 nm, 500 mW/cm2) for 5 min. Obviously, a gradual irradiation time-dependent temperature increasing was observed for IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs. The increased temperature of IR780/Cabloaded NPs is about 20 oC after 5 min irradiation while the temperature of PBS solution increases only 3.2 oC (Figure 4). The results indicate that the loaded IR780 in PEG-bP(AAm-co-AN) NPs can serve as an outstanding photothermal agent to be the heat producer in order to “activate” the UCST-triggered polymer.

Figure 4. (a) Photothermal property of IR780/Cab-encapsulated PEG-b-P(AAm-co-AN) NPs and PBS solution as the control sample. (b) The changes in temperature of IR780/Cabencapsulated PEG-b-P(AAm-co-AN) NPs and PBS solution during laser irradiation (808 nm). 19



(c) Temperature- and NIR laser irradiation-triggered release of Cab from cargo-loaded PEGb-P(AAm-co-AN) NPs. Values are mean ± SD (n= 4). Drug release The release of Cab was studied in different temperatures in PBS solution with 0.5 wt % tween 80 in order to reveal the thermo-responsive behavior of PEG-b-P(AAm-co-AN). As shown in Figure 4c, only 19 % of Cab was released at 37 oC in around 24 h. However, the release of Cab was greatly accelerated to 92 % when the temperature increased to 50 oC owing to the stretched P(AAm-co-AN) chains and the dissociation of nanoparticles. It indicates that the temperature of nanoparticles solution plays a crucial role in drug release. Additionally, the cumulative drug release profile (triangle plots in Figure 4c) exhibits rapid release of Cab after 808 nm laser irradiation for 5 min at 4 h. Therefore, the IR780/Cabloaded PEG-b-P(AAm-co-AN) NPs show thermo-responsive drug release upon NIR irradiation-induced temperature increasing while possessing good biostability at normal body temperature. Based on the above, the thermo-responsive polymeric nanoparticles could serve as a “smart” drug release platform by spatiotemporally “programmed” drug release upon NIR light irradiation. Cellular Uptake In order to explore whether IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs could be effectively absorbed by cancer cells endocytosis, CLSM was utilized to qualitatively investigated the cellular uptake behavior and distribution. 4T1 cells were cultured with IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs for 4 h and 24 h before observation, respectively. As shown in Figure 5, the nuclei were stained by Hochest33342 for 15 min to

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locate the pending cells. The red fluorescence generated by IR780 was observed in the cytoplasm both with the treatment of IR780 and IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs, and became brighter while prolonging the culture time to 24 h. Furthermore, a much stronger red fluorescence was detected in 4T1 cells incubated with IR780/Cab-loaded NPs in comparison to those cells treated with free IR780 after both 4 h and 24 h, which is mainly because of the more uptake of IR780/Cab-loaded NPs than free IR780. Furthermore, the cellular uptake behavior of drug loaded NPs was qualified by flow cytometric examination. In order to qualitatively examine the effect of PEG-b-P(AAm-coAN) NPs on cellular uptake, 4T1 cells were incubated by free IR780 and IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs for 4 h and 24 h, respectively before analysis. As shown in Figure S4, after 4 h incubation of IR780/Cab dual-loaded PEG-b-P(AAm-co-AN) NPs, an obvious enhancement of cell fluorescence intensity was revealed, which indicated the rapid uptake of micelles by 4T1 cells. Additionally, further prolonging the incubation time to 24 h led to another 3-fold higher fluorescence intensity. Moreover, compared with the free IR780, much stronger fluorescence intensity was presented from IR780/Cab dual-loaded PEG-bP(AAm-co-AN) NPs due to the difference between endocytosis process for PEG-corona NPs and passive diffusion for small molecules. According to the fluorescence of flow cytometry, it illustrated that the PEG-b-P(AAm-co-AN) encapsulation could significantly enhance its cell uptake by cancer cells and would further improve the therapeutic efficiency.

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Figure 5. Cellular uptake of free IR780 and IR780/Cab-loaded NPs after incubated with 4T1 cells for 4 h and 24 h (stained with Hoechst 33342). Scale bar: 20 µm. In vitro Cytotoxicity Studies The cytotoxicity of IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs was evaluated against 4T1 cell by MTT essay in vitro. As depicted in Figure 6a, when the free IR780 and Cab incubated with 4T1 cells, the proliferation of these 4T1 cells was remarkably inhibited. For example, the group of free IR780 displayed obvious cytotoxicity with only 18% cell viability at the IR780 concentration of 20 g mL-1. Similarly, the cytotoxicity of free Cab was up to 16% when its concentration was about 24 g mL-1. However, the native PEG-b-P(AAm-co-AN) micelles, IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs did not show significant cytotoxicity, in which the cell viability remains as high as 91 % after 24 h treatment with the concentration of Cab as high as 24.0 g mL-1 and IR780 20.0g mL-1. The excellent 22


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cytocompatibility of IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs would remarkably prevent the premature drug release. The phototoxicity of IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs was also further verified by MTT assay. In Figure 6b, all samples exhibited obviously concentrationdependent cytotoxicity after laser irradiation or heating compared with the cytotoxicity of IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs in dark. IR780-loaded NPs performed a high phototoxicity owing to the photothermal effect under NIR irradiation with 808 nm laser irradiation for 5 min. Additionally, it is worthy of note that the sample of Cab-loaded NPs was conducted on the heating block at 50 oC for 5 min to accelerate the release of Cab. The cell viability of Cab-loaded NPs decreased to 67 % with the concentration of Cab about 6.0 g mL-1 due to the high cell uptake and the external heating induced release of Cab. Moreover, IR780/Cab dual-loaded NPs led to higher cytotoxicity than either single IR780 or Cab-loaded NPs because of the greatly enhanced photothermal efficacy of IR780 and complete NIR-triggered release of Cab. Notably, IR780 acted as the dual roles of the heating generator and the activator for “on-demand” drug release. In addition, it has been reported that cyanine dyes can produce both local heating and singlet oxygen upon the NIR irradiation.52,57,58 A small amount of singlet oxygen will lead to the damage of endosome membrane and further induce high cellular uptake of drugs instead of drug efflux.59–62 Furthermore, the cytoablation induced by local heating could simultaneously enhance the translocation of Cab to deeper regions in cancer site. In order to investigate the synergistic effect of IR780/Cab-loaded PEG-b-P(AAm-co-AN) NPs, the therapeutic efficacy of the combination of each group was calculated carefully according to the following equation 31,63: Tadditive (%) = 100 - (fcab × fIR780) ×100 23



Where, fcab and fIR780 are the fraction of surviving cells after the treatment of Cab-loaded NPs and IR780-loaded NPs upon heating or NIR irradiation, respectively. As shown in Figure 6c, the therapy efficacy of IR780/Cab dual-loaded NPs was higher than the calculated Tadditive, indicating the excellent synergistic effect of photothermal and chemotherapy.

Figure 6. Cell viability against 4T1 cells in dark and under light irradiation, (a) Relative viabilities of the 4T1 cells after incubation with different concentrations of free IR780, free Cab, PEG-b-P(AAm-co-AN) micelles and Cab/IR780-loaded PEG-b-P(AAm-co-AN) NPs for 24 h. (b) Relative viabilities of 4T1 cells after incubation with different concentrations of IR780-loaded PEG-b-P(AAm-co-AN) NPs, Cab-loaded PEG-b-P(AAm-co-AN) NPs and Cab/IR780-loaded PEG-b-P(AAm-co-AN) NPs for 24 h followed by exposure to the 808 nm 24


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NIR laser for 5 min. (c) Therapeutic efficacy evaluation of Cab-loaded PEG-b-P(AAm-coAN) NPs, IR780-loaded PEG-b-P(AAm-co-AN) NPs with NIR irradiation, and Cab/IR780 dual-loaded PEG-b-P(AAm-co-AN) NPs with NIR irradiation. Values are mean ± SD (n= 4), *p < 0.05; **p < 0.01. (d) Illustration of producing NIR-activated synergistic photothermal and chemotherapy. In vivo fluorescence imaging To examine whether Cab and IR780 dual-loaded PEG-b-P(AAm-co-AN) NPs could accumulate in tumor tissues successfully for diagnosis and therapy, in vivo fluorescence imaging experiments was conducted to examine the in vivo biodistribution against a 4T1 tumor-bearing mice model after tail vein injection. IR780, as a typical NIR imaging probe, has been used in bioimaging due to the deep penetration and low biological tissue damage of NIR irradiation. Figure 7a shows the in vivo fluorescence images of tumor-bearing mice to illustrate the biodistribution of the IR780/Cab dual-loaded NPs. The IR780/Cab dual-loaded NPs were firstly enriched in the liver at 0.5 h post injection. With the blood circulation, after 12 h of the administration of IR780/Cab dual-loaded NPs, the maximum fluorescence signal was observed in tumor region, whereas the fluorescence signal reduced at 24 h post injection. These results reveal that the outstanding stability during blood circulation, perfect tumortargeting and accumulation properties of IR780/Cab dual-loaded NPs. Moreover, motivated by the excellent thermal imaging ability, photothermal imaging performance of IR780/Cab dual-loaded NPs was investigated in vivo. 4T1 tumor-bearing mice were exposed to 808 nm laser irradiation. The surface temperature of tumor site under NIR irradiation was real-time recorded and the images of in vivo photothermal imaging were shown in Figure 7b. The highest temperature in the irradiation center was collected and 25



displayed in Figure S5. The results illustrated that the average surface temperature of tumor site after injecting intravenously IR780/Cab dual-loaded NPs can increase to about 50 oC by the NIR irradiation (500 mW/cm2) for 3 min and then kept around 50 oC upon longer irradiation. Since the UCST of PEG-b-P(AAm-co-AN) block copolymer is about 43 oC, the temperature was high enough to activate the phase transition of UCST-triggered chains and the controlled release of Cab. For comparison, the temperature of PBS buffer solution injected mouse, as a control, only increased 3 oC under the same NIR irradiation dose and duration.

Figure 7. (a) In vivo fluorescence image of IR780/Cab dual-loaded PEG-b-P(AAm-co-AN) NPs in 4T1 tumor-bearing mice, at intervals of 0.5, 2, 6, 12 and 24 h after administration, and (b) in vivo photothermal image at intervals of 0, 60, 120, 180 and 240 s after administration. 26


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In vivo Tumor Growth Inhibition The antitumor effect of IR780/Cab dual-loaded PEG-b-P(AAm-co-AN) NPs was then explored by tracking the relative tumor size, tumor weight and volume after different treatments. Nude mice bearing 4T1 (breast cancer cells) tumor were selected as the animal models and divided into six groups randomly, as following: Group 1, Control (treated by PBS saline only); Group 2, free Cab treated; Group 3, IR780/Cab dual-loaded PEG-bP(AAm-co-AN) NPs treated; Group 4, Cab-loaded PEG-b-P(AAm-co-AN) NPs treated with 50 oC water bath for 5 min; Group 5, IR780-loaded PEG-b-P(AAm-co-AN) NPs upon NIR irradiation (808 nm, 500 mW/cm2) for 5 min; Group 6, IR780/Cab dual-loaded PEG-bP(AAm-co-AN) NPs upon NIR irradiation (808 nm, 500 mW/cm2) for 5 min. The tumor sizes and body weights of each group were measured and recorded every other day (Figure 8a and Figure 8d). The groups upon treatments with PBS or IR780/Cab-loaded NPs without irradiation presented negligible anticancer effect. In contrast, moderate tumor growth was displayed in the groups of Cab-loaded NPs with 50 oC water bath for 5 min and IR780-loaded NPs with NIR laser irradiation after 16 days post-injection, whereas chemotherapy or photothermal therapy alone exhibited moderate tumor growth inhibition. Notably, the tumor growth was significantly inhibited after the synergistic photothermal and chemotherapy of the IR780/Cab dual-loaded NPs with 808 nm laser irradiation. Furthermore, the average tumor weights of Group 6 were calculated to significant statistical difference (Figure 8b) compared with that of IR780-loaded NPs with NIR irradiation and Cab-loaded NPs with 50 oC water bath. The tumor photos of all mice groups after 16 days post-injection was displayed in Figure 8c. With the aim of histological analysis of tumor tissues, the hematoxylin and eosin (H&E) staining of the slices from major organs of treated mice was investigated. As shown in

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Figure 8e, IR780-loaded PEG-b-P(AAm-co-AN) NPs upon NIR irradiation group exhibited the best suppression of tumor cell proliferation, while tumor cells in the other five groups mainly retained the normal morphology. Taken together, these results demonstrated that the synergistic photothermal and chemotherapy provides the strongest anticancer effect from IR780/Cab dual-loaded NPs, which is prominent better than either PTT from IR780-loaded NPs or chemotherapy from Cab-loaded NPs alone.

Figure 8. In vivo antitumor efficacy using IR780/Cab-encapsulated PEG-b-P(AAm-co-AN) 28


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NPs. (a) Relative tumor volume of the mice; (b) Average tumor weights at the 16th day of post-treatments; (c) Relative body weight of the mice treated with different agents of PBS, free Cab, Cab-loaded NPs, IR780-loaded NPs and Cab/IR780-loaded PEG-b-P(AAm-co-AN) NPs with or without NIR irradiation 808 nm (500 mW/cm2, 5 min); (d) Representative photos of mice bearing 4T1 tumors 18 day after treatments; (e) H&E staining of tumor tissues after treating with different formulations. Scale bar: 100 µm. n=4, *p > 0.05, **p < 0.05, and ***p < 0.001, determined by a Student’s t-test. The safety of materials is crucial in the application of biomedicine. Thus, the mice body weight in all groups and histological analysis of major organ tissues were studied to evaluate the safety of IR780/Cab dual-loaded PEG-b-P(AAm-co-AN) NPs. No obvious abnormal behaviors from the treated mice were observed during experiments. Furthermore, the body weight loss is supposed to be an indicator for the evaluation of the nanomedicine’s safety. From Figure 8d, it shows that no obvious weight loss is found in the treated groups and there is no obvious difference in body weight between all groups after various treatments. The histological analysis of organ tissues was carried by H&E stain of the slices from major organs of treated mice. As shown in Figure S6, there is no significant inflammatory lesion or organ damage in the stained organs, which indicates the low body cytotoxicity of cargoloaded NPs. All these results suggest that the IR780/Cab dual-loaded PEG-b-P(AAm-co-AN) NPs is an outstanding candidate in cancer therapy by the synergistic photothermal and chemotherapy, possessing low side effect to the normal organs.

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Conclusions In summary, we have developed a NIR-activated UCST-triggered drug release platform for synergistic photothermal and chemotherapy. The UCST of PEG-b-P(AAm-co-AN) was set at 43 oC by manipulating the molar ratio of AAm and AN. Spherical micelles have been obtained by the co-assembly of IR780, Cab and PEG-b-P(AAm-co-AN) block copolymer. Consequently, the high loaded IR780 efficiently converted the light energy to local heating by photothermal effect, which further induced micellar dissociation and the on-demand release of Cab. Meanwhile, in vitro and in vivo studies have been confirmed that IR780/Cab dual-loaded NPs possessed outstanding anticancer efficacy by the synergistic photothermal and chemotherapy. This work provides a novel and multifunctional anticancer system, and we believe that it would have broad prospects to stimuli-responsive controlled release and synergistic therapy in cancer therapy.

Supporting Information 1H

NMR spectra of PEG-DDAT, SEC curves, DLS curves of IR780-loaded NPs and Cab-

loaded NPs, the highest surface temperature plots in the irradiation center during photothermal imaging against irradiation duration, flow cytometric examination, H&E staining images of major organ tissues (PDF) Author Contributions #These

authors contributed equally to this work.

Corresponding Author *Email: [email protected].

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Notes The authors declare no competing financial interest. Acknowledgments 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).

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NIR-Activated Polymeric Nanoplatform with Upper Critical Solution

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