Photothermal-Controlled Generation of Alkyl Radical from Organic

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Biological and Medical Applications of Materials and Interfaces

Photothermal Controlled Generation of Alkyl Radical from Organic Nanoparticles for Tumor Treatment Rui Xia, Xiaohua Zheng, Xiuli Hu, Shi Liu, and Zhigang Xie ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18953 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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ACS Applied Materials & Interfaces

Photothermal Controlled Generation of Alkyl Radical from Organic Nanoparticles for Tumor Treatment Rui Xia,†,‡Xiaohua Zheng,†,‡ Xiuli Hu,*,† Shi Liu,† and Zhigang Xie*,†,‡ †State

Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied

Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, P. R. China ‡University

of Science and Technology of China, Hefei 230026, P. R. China

ACS Paragon Plus Environment

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ABSTRACT ： The therapeutic properties of light are well known for photodynamic or photothermal therapy, which could cause irreversible photodamage to tumor tissues. Although photodynamic therapy (PDT) has been proved in the clinic, the efficacy is not satisfactory because of complicated tumor microenvironments. For example, the hypoxia in solid tumor has a negative effect on the generation of singlet oxygen. In order to address the hypoxia issues in PDT, leveraging alkyl radical is an available option due to the oxygen-independent feature. In this work, a new kind of organic nanoparticles (TPP-NN NPs) from porphyrin and radical initiator is developed. Under near infrared light irradiation, TPP-NN NPs will splitting and release alkyl radical, which could induce the obvious cytotoxicity whether in normal or hypoxia environment. The photothermal controlled generation of alkyl radical could significantly inhibit the growth of cervical cancer, and show ignorable systemic toxicity. This activatable radical therapy opens up new possibilities for application of PDT in hypoxia condition.

KEYWORDS ：Supramolecular Nanoparticles, photodynamic therapy, alkyl radical, porphyrin


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INTRODUCTION Photodynamic therapy (PDT) is a noninvasive modality for cancer therapy, and has attracted much attention for potential clinical promise.1-3 PDT has been approved for cancer treatment, and most photosensitizers (PSs) are based on porphyrin derivatives.4-5 However, the tumor hypoxia limits the therapeutic efficacy of PDT due to the oxygen-dependent nature.6-7 Many strategies have been developed to enhance the oxygen content in tumors, and then improve the therapeutic outcomes.8-9 For example, delivering oxygen into tumors could be done by using hemoglobin or perfluorocarbons.10-12 Generating oxygen in tumor cells also could be realized through the enzymes or chemical reactions.13-15 In addition, improving blood flow could also partly elevate the oxygen content in tumors via mild heating or chemotherapeutic agents.16 Alternatively, leveraging oxygen-independent radicals is interesting and promising substitutes for singlet oxygen.17-18 Alkyl radicals generated from azo compounds is not dependent on oxygen, and could be triggered under heat stimulation. Zhang and Xia et al. have reported the radical initiator loaded gold nanocages for destruction of hypoxia cancer cells.19-21 There are two things need to be considered for this oxygen-independent PDT. Firstly, shielding radical initiator is important and imperative because of their susceptibility to decomposition.22 Secondly, the precise control of the temperature is of great concern. Low temperature cannot spur initiator to generate the radicals, while high temperature may threaten the healthy tissues and cells nearby.23 So rational engineering of thermal triggered generation of alkyl radical remains ongoing challenge.24-25 Conjugating photothermal PSs with radical initiator is a direct way to tune the production of radicals.26 In order to avoid decomposition, the nanoparticle formulations are employed.27


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Considering for promising tumor treatment, the used materials must be biocompatible or biological inertia for potential clinical translations.28 Herein, the stable nanoparticles (TPP-NN NPs) were developed by using of porphyrin and 2,2'-azobis [2-(2-imidazolinI-2-yl) propane] dihydrochloride (AIBI) conjugates and pluronic F-127, as shown in Scheme 1. The aggregated porphyrin could produce heat upon irradiation,

29-32

then induce AIBI to generate alkyl radicals

for killing cancer cells.33

Scheme 1. Schematic illustrations of (A) the formation of TPP-NN NPs and the process of producing free radicals. (B) Process of treatment with laser irradiation.

RESULTS AND DISCUSSION Preparation and Characterization of TPP-NN NPs


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The light-induced free radical generator consists of two parts, porphyrin as the photothermal conversion agents and AIBI as the source of free radicals. These two parts were integrated together through the amide bond. Their chemical structures were validated by nuclear magnetic resonance hydrogen spectrum and mass spectrometry (Figures S1, S2, S3, S4). As shown in Figure S3, peak of 890.7 represents complete mass spectrum of TPP-NN molecule, peak of 863.5 represent the mass spectrum of TPP-NN after releasing of nitrogen and peak of 751.5 is corresponding to the free radical fragment after decomposition. In our design, porphyrin works as a source of heat to induce the decomposition of AIBI, leading to the generation of free radicals.19 This kind of alkyl radical can be perfectly applied to biology. Due to the good photothermal effect of porphyrin, TPP-NN monomer will be decomposed and release alkyl free radicals under light irradiation.34 This as-synthesized TPP-NN can assemble with pluronic F-127 (F127) into nanoparticles (TPP-NN NPs) in aqueous media, which could protect the AIBI from decomposing. The added F127 improve the water dispersibility and size-stability of NPs. Figure 1A and Figure S5 show the transmission electron microscopy (TEM) image in different scales. Figure 1B is the size distribution of TPP-NN NPs measured by dynamic light scattering (DLS). TPP-NN NPs possess a hydrodynamic diameter of 185 nm, which matches with the result of TEM. As control, TPP NPs were prepared in the absence of AIBI. Figure S6 shows the spherical morphology of TPP NPs and nanoscale size. In further experiment, the similar absorption spectra of TPP-NN and TPP molecules in tetrahydrofuran (THF) indicate that conjugating of AIBI does not affect the optical properties of TPP-NN (Figure 1C). TPP-NN NPs can keep stable in water and DMEM within 10 days, as evidenced by constant particle size (Figure S7), which is important for long circulation in blood.35 Figure 1C also reveals the maximum absorption of TPP-NN NPs in water has a small red shift compared to that of TPP-NN due to the aggregation.


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We further measured the fluorescence spectra (Figure 1D), and it is obvious that TPP-NN NPs in water has significant fluorescence quenching compared with TPP-NN monomer dissolved in THF. For this phenomenon, Pu's team has pointed out that the decaying fluorescence would significant contribute to the photothermal conversion efficiency,36 which is beneficial to excellent photothermal effect.37

Figure 1. Characterization of TPP-NN NPs. (A) TEM image of TPP-NN NPs. (B) DLS measurement of TPP-NN NPs in water. (C) Absorption spectra of TPP-NN, TPP dissolved in THF and TPP-NN NPs dispersed in water. (D) Fluorescence spectra of TPP-NN monomeric and TPP-NN aggregates. Photothermal Properties of TPP-NN NPs


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In previous works, porphyrin with absorption from 600 nm to 650 nm was used for photothermal therapy.38-39 Herein, we choose a 638 nm laser as the light source. First, photostability of the TPP-NN NPs was investigated by monitoring the ultraviolet spectrum with illumination. Compared with indocyanine green (ICG), TPP-NN NPs can almost keep the same absorbance after 638 nm laser irradiation (1 W/cm2) for 600 s, while absorbance of ICG had obvious attenuation (Figure S8) which indicate that TPP-NN NPs possessed good photostability. To explore its photothermal conversion ability, we measured the heating curves of TPP-NN NPs in water. TPP-NN NPs exhibited obvious increase of temperature to 55 °C upon irradiation for 5 min, while water only reached 25 °C at the same condition (Figure 2A), demonstrating that TPPNN NPs possess the ability of increasing temperature upon irradiation. And the concentrations and power intensity dependent photothermal activity was revealed in Figure 2B and 2C. Next, the photothermal conversion efficiency was monitored. First, temperature change of TPP-NN NPs with continuous irradiation by a 638 nm laser (1 W/cm2) were recorded, followed by turning off the laser (Figure 2D). Figure 2E shows the relationship between plot of cooling time and versus negative natural logarithm of the temperature driving force obtained from the cooling stage. Photothermal conversion efficiency of TPP-NN NPs was determined to be 36 %, which is close to that of porphyrin derivatives.40 In addition, we collected the changes of temperature of heating and cooling for four circulation. As shown in Figure 2F, the unchanged temperature increasing validates the possibility of reuse. All these results indicate that TPP-NN NPs have excellent photothermal activity, which could induce the generation of radicals.


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Figure 2. Photothermal properties of TPP-NN NPs. (A) Photothermal heating curves of water and TPP-NN NPs dispersed in water irradiated by 638 nm laser for 5 min. Recorded heating curves of TPP-NN NPs (B) with various concentrations under 638 nm laser irradiation (1.0 W/ cm2) for 5 min and (C) with disparate laser power densities at concentration of 0.168 µmol/mL. (D) Photothermal effect of TPP-NN NPs in water with 638 nm laser irradiation (1.0 W/m2) for 5 min, then laser was turned off and cooled for 10 min. (E) The relationship between cooling period of time and the negative natural logarithm of temperature. (F) Photothermal effect of TPPNN NPs after four cycles of heating and cooling.

Cellular Uptake of TPP-NN NPs Confocal laser scanning microscope (CLSM) was employed to investigate cellular uptake of TPP-NN NPs against HepG2 cells. Red fluorescence of TPP-NN NPs excited by 555 nm laser


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was employed to monitor the endocytosis process and blue fluorescence of 4',6-diamidino-2phenylindole (DAPI) was used to localize the nucleus. Results showed that the internalization of TPP-NN NPs by cells was time dependent. As time grows, the red fluorescence of TPP-NN NPs obviously enhanced (Figure S9). Lysosome co-localization experiment was also conducted, the green fluorescence of TPP-NN NPs matched well with the red fluorescence of endosome (Figure S10) revealing that endocytosis of TPP-NN NPs goes through lysosome. Extracellular and Intracellular free-radical detection In this part, heat-triggered generation of radical was investigated. First, we studied the freeradical generation ability of TPP-NN molecule in N, N-Dimethylformamide (DMF). TPP-NN and 1,3-diphenylisobenzofuran (DPBF)41 were dissolved in DMF, then they were heated to 45°C and their UV–vis absorption spectra were monitored with time. As shown in Figure 3A, the absorbance of DPBF at 410 nm kept continuous reduction in 4 h, indicating that DPBF was oxidized by free radicals generated from TPP-NN molecules. As a comparison, the absorption of DPBF alone dissolved in DMF show little change in the absence of TPP-NN at same condition (Figure S11). In additional, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (one of the most common free radical capture reagents) was employed to prove the generation of free radical by TPP-NN NPs with thermal stimulation.20 As shown in Figure S12, a characteristic absorbance peak at 734nm gradually increased especially in a higher temperature of 45 oC, meaning the decomposition of TPP-NN NPs and release of free radicals. Following, the intracellular production of free radicals was validated. To capture the free radicals generated by TPP-NN NPs, dichlorofluorescein diacetate (DCFH-DA) was employed to estimate the intracellular ROS level.42-43 HepG2 cells were treated with PBS, AIBI, TPP NPs, TPP-NN NPs at room temperature or 45 °C respectively. TPP-NN NPs and 45 °C treated cells show the


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strongest fluorescence than cells with other treatments, confirming that this thermosensitive material can release free radicals upon being heated in cells (Figure 3B). At last, the free radical generation ability under laser irradiation was validated in Figure 3C. HepG2 cells incubated with TPP-NN NPs upon irradiation of 638 nm laser also exhibited the brightest fluorescence whether in normal or hypoxia conditions, meaning a dependent process of the production of free radicals.44 In Xia's work, free alkyl radicals can directly destroy cells by oxidation of cell component under hypoxic conditions, and when oxygen in cells is enough, free radicals will turn into alkoxyl and peroxyl radicals with cytotoxicity too.20 All of the results above indicate that the designed TPP-NN NPs can perfectly generate free radical with a 638 nm laser irradiation under hypoxia or normoxia conditions, implying that it may have significant therapeutic effect.


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Figure 3. Extracellular and intracellular free-radical detection. (A) Detection of free radicals from TPP-NN molecules by DPBF in DMF at different time points at 45 °C. (B) Detection of free radicals induced by TPP-NN NPs with DCFH-DA against HepG2 cells. Cells were treated with AIBI (0.008 μmol/mL), TPP NPs (0.008 μmol/mL) or TPP-NN NPs (0.008 μmol/mL) and kept at 45 °C for 10 min and cells with the same condition kept at 37 °C were set as control. (C) DCFH-DA detecting the free radicals of HepG2 cells treated with AIBI (0.008 μmol/mL), TPP NPs (0.008 μmol/mL) or TPP-NN NPs (0.008 μmol/mL) with laser irradiation for 10 min under


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normoxic and hypoxic conditions (L represents 638 nm laser irradiation at a power density of 1 W/cm2 for 10 min). In Vitro Cytotoxicity of the TPP-NN NPs Herein, HepG2 cells were selected to assess cytotoxicity of TPP-NN NPs through 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assays. In Figure 4A, TPPNN NPs, TPP NPs and AIBI show negligible cytotoxicity at the tested concentrations. Then cytotoxicity under normal or hypoxia condition with 638 nm laser irradiation was collected in Figure 4B and Figure 4C, AIBI displayed ignorable cytotoxicity. TPP NPs indicated considerable cytotoxicity. As a contrast, TPP-NN NPs showed the most significant cytotoxicity, because of the synergetic effect of TPP and AIBI. After laser irradiation, porphyrin absorbed the light, then causing a temperature rise and the decomposition of AIBI. Free radicals released from decomposed AIBI lead to notable cytotoxicity. Alkyl radicals from TPP-NN NPs without oxygen can effectively cause cell apoptosis too.45-46 It is well to be reminded that even at extremely low concentration, TPP-NN NPs still shows good therapeutic effect. Figure S13 showed cytotoxicity of TPP-NN NPs against A549 cells and Hela cells under normoxic condition. They showed similar therapeutic effects as HepG2 cells. To further verify the cytotoxicity, cell live/death staining experiment were carried out in HepG2 cells. Figure 4D indicates the similar results with that of MTT above. Compared with TPP NPs, group of TPP-NN NPs with laser show the brightest red fluorescence from PI meaning the optimal cell-killing ability. The death model of cell was revealed by annexin V-FITC/PI staining assay (Figure S14).47 TPP-NN NPs induced obvious cell apoptosis while TPP NPs and AIBI alone presents negligible cytotoxicity. With further analyses, early apoptosis (Q3=52.8%) plays a major role in this process. It is likely that free radicals from TPP-NN NPs cause this result48. In a


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summary, we can find that only the laser irradiation, TPP NPs, AIBI alone have negligible cytotoxicity and cell death is mainly caused by the combined effect of TPP-NN NPs and laser irradiation. These exhilarating results show that TPP-NN NPs have excellent cell-killing ability.

Figure 4. In vitro cytotoxicity against HepG2 cells. (A) Cytotoxicity of HepG2 cells treated with TPP-NN NPs, TPP NPs and AIBI without laser irradiation. Cytotoxicity of HepG2 cells treated with TPP-NN NPs, TPP NPs and AIBI under (B) normoxic and (C) hypoxic conditions with 638 nm laser irradiation for 5 min. (D) Fluorescence images of cell death staining of HepG2 cells treated with PBS, AIBI, TPP NPs and TPP-NN NPs (0.008 μmol/mL) with or without laser irradiation under normoxic condition. Antitumor Effect and Biocompatibility of TPP-NN NPs


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All the results above showed remarkable in vitro cell-killing effect, animal experiments were carried out to validate in vivo antitumor activities of TPP-NN NPs. Kunming mice with U14 tumor xenografts were used as the model. Mice were segmented into eight groups and each group consists of 4 mice: blank, blank + Laser, TPP NPs, TPP NPs + Laser, AIBI, AIBI + Laser, TPP-NN NPs, TPP-NN NPs + Laser. Mice were intravenously injected with TPP NPs, AIBI, TPP-NN NPs separately. Firstly, the photothermal ability was evidenced by an infrared imaging camera. As shown in Figure 5A, obvious temperature rising was noticed for the group of TPP NPs + Laser and TPP-NN NPs + Laser upon irradiation for 10 min, while mice injected with PBS and AIBI only showed slight increase under the same condition. Figure S15 was the temperature plot of four groups of mice with laser irradiation for 10 minutes at different point of time. After 14 d of treatment, tumor inhibition effect was evaluated, and the best results were observed for treatment of TPP-NN NPs + Laser. In Figure 5B, tumors of mice intravenously injected with TPP-NN NPs and laser irradiation were almost completely removed after treatment for 14 d. TPP NPs + Laser treated mice indicated the moderate result because of the inner photothermal effect, and all other control groups showed negligible therapeutic effect. Figure 5C and Figure 5D is the comparison of average tumor weight and size, which is consistent with the result of tumor volume. All these results confirm that TPP-NN NPs with laser irradiation possess excellent tumor suppressor ability. At last, the hematoxylin and eosin (H&E) stained tumor sections (Figure 5E) further demonstrated the remarkable therapeutic effect of mice treated with TPP-NN NPs under laser irradiation. For the mice treated with TPP-NN NPs and laser, nuclei showed complete fragmentation and the color was shallower, meaning noticeable cell apoptosis or necrosis contrast with other groups. It is worth mentioning that all mice showed ignorable loss of body weight, indicating that NPs or laser irradiation had no obvious side effect (Figure S16).


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H&E-stained pictures of major organs further confirm their good biocompatibility (Figure 6). Groups of mice without any treatment or injected with TPP NPs, AIBI, TPP-NN NPs under 638 nm laser irradiation showed negligible inflammation lesion or organ injury. To further verify the biocompatibility, liver function, renal function and routine blood test of eight groups of mice were monitored. Results show that all the parameters fluctuate within the normal range (Figure S17). All these results suggest that TPP-NN NPs may be an effective therapeutic nanomaterial for cancer therapy.


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Figure5. In vivo antitumor effect. (A) Thermal images of U14 tumor-bearing mice without treatment or injected with TPP NPs, AIBI and TPP-NN NPs under 638 nm laser irradiation for 10 min at a power density of 1 W/cm2 recorded every two minutes. (B) Tumor volume and (C)


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tumor weight of mice without treatment or treated with L, TPP NPs, TPP NPs +L, AIBI, AIBI+ L, TPP-NN NPs, TPP-NN NPs + L (L represents 638 nm laser irradiation for 10 min at a power density of 1 W/cm2). (D) Corresponding images of tumors of 8 groups after treatment for 14 d. (E) H&E staining of tumor section of mice after treatment for 14 days.

Figure 6. H&E staining images of organ slices from four groups of mice with laser irradiation. Conclusions In conclusion, we have developed a new type of organic nanoparticles with the function of producing free radicals by photoinitiation. The photothermal effect of porphyrin could transfer to radical initiator, and induce the generation of alkyl radicals for cancer treatment. The generated radicals are not dependent on the oxygen content, which is different with traditional


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photodynamic therapy. The organic nanoparticles TPP-NN NPs could protect the initiator and promote the endocytosis, indicating the potent cytotoxicity under both normoxic and hypoxic conditions after irradiation. Experiments in vivo validated TPP-NN NPs could inhibit the growth of tumor and remove the tumor upon irradiation. We believe that this oxygen-independent radical is a great substitute of singlet oxygen in photodynamic therapy.

Experimental Section Materials: 4-formylbenzoic acid, trifluoroacetic acid, triethylamine, deuterated chloroform (CDCl3) and deuterated dimethylsulphoxide (DMSO) were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, U.S.A.). Pyrrole was purchased from Heowns Co., Ltd. 2,2'-azobis[2-(2imidazolinI-2-yl) propane] dihydrochloride (AIBI) and oxalyl chloride were obtained from Energy Chemical Co., Ltd. MTT were obtained from Beyotime Biotechnology Co., Ltd. (China). Cell viability (live dead cell staining) assay reagent was obtained from Jiangsu KeyGEN Biotechnology Co., Ltd. Synthesis of TPP-COOH: A refluxing solution of 4-formylbenzoic acid (4.05 g, 0.027 mol) and benzaldehyde (8.6 g, 0.081 mol) was heated to 135 oC for about 30 minutes under nitrogen atmosphere. Then fresh pyrrole (7.5 mL, 0.108 mol) was added in 10 min. The mixture was refluxed for another 1 h. Then it was stored at 4°C for 8 h. Purple crystals at the bottom were collected and washed with methanol. At last, they were purified by chromatography. 1H NMR (400 MHz, DMSO) δ 13.27 (s, 1H), 8.83 (s, 8H), 8.38 (d, J = 7.8 Hz, 2H), 8.29 (d, J = 7.6 Hz, 2H), 8.22 (s, 6H), 7.83 (d, J = 6.6 Hz, 9H), -2.92 (s, 2H). Synthesis of TPP-NN: To a stirred solution of TPP-COOH (200 mg, 0.3 mmol) in 20 ml dry chloroform, oxalyl chloride (0.03 mL, 0.36 mmol) and two drops of N, N-dimethyl formamide


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were added. The flask was protected with nitrogen and kept at 0° stirring for 6 h. Then the solvent and residual oxalyl chloride were removed under reduced pressure. The remaining solid was dissolved by 5ml dry chloroform and added to a stirred mixture of 2,2'-azobis[2-(2imidazolinI-2-yl) propane] dihydrochloride (97 mg, 0.3 mmol) and triethylamine (0.21 mL, 1.0 mmol) in 30ml chloroform in an ice bath under argon atmosphere. After stirring for 8 hours, the mixture was filtered. Obtained filtrate was concentrated and purified with column chromatography. 1H NMR (400 MHz, CDCl3) δ 10.43 (s, 1H), 8.94 – 8.70 (m, 8H), 8.28 (dd, J = 50.8, 7.2 Hz, 8H), 7.93 (d, J = 8.0 Hz, 2H), 7.78 (p, J = 6.4 Hz, 9H), 4.28 (t, J = 8.4 Hz, 2H), 4.13 – 3.98 (m, 6H), 1.79 (d, J = 22.5 Hz, 12H), -2.77 (s, 2H). MALDI-TOF/MS for [TPP-NN]: 891.1 calculated, 863.3 found. The decreasing 28 molecular weight may due to the instant heating leading to the break off of azo bond during testing procedure. ESI/MS also proved the formation of TPP-NN.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Additional contents including part of the experimental procedures and

1H

NMR spectrum of

TPPCOOH and TPP-NN; MALDI-TOF and ESI mass spectrum of TPP-NN; TEM image and DLS; Absorption spectra of TPP-NN NPs and indocyanine green (ICG); CLSM images; Absorption spectra of DPBF; Absorption spectra of ABTS; MTT assays against A549 and Hela cells; Cell apoptosis assay of HepG2 cells; Temperature plot with laser irradiation; Weight changes and blood biochemistry assay.

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] (X. H.). * E-mail: [email protected] (Z. X.).

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Financial support was kindly provided by the National Nature Science Foundation of China (Project No. 51773197 and 51522307).


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Photothermal-Controlled Generation of Alkyl Radical from Organic

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