Comparing Effects of Redox Sensitivity of Organic Nanoparticles to

Subscriber access provided by Fudan University

Article

Comparing Effects of Redox Sensitivity of Organic Nanoparticles to Photodynamic Activity Wei Zhang, Wenhai Lin, Xiaohua Zheng, Shasha He, and Zhigang Xie Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.7b00207 • Publication Date (Web): 01 Feb 2017 Downloaded from http://pubs.acs.org on February 4, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Chemistry of Materials is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Chemistry of Materials

Comparing Effects of Redox Sensitivity of Organic Nanoparticles to Photodynamic Activity Wei Zhang,†, ‡ Wenhai Lin,†, ‡ Xiaohua Zheng,†, ‡ Shasha He,†, ‡ and Zhigang Xie†, * †

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China ‡

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

ABSTRACT: Smart responsive nanomaterials, which are sensitive to biological signals, are promising therapeutic formulations. It has been well studied that redox potential is much different at both tissue and cellular level. In this work, three organic nanoparticles with variable redox sensitivity were fabricated, and their redox sensitivity was evaluated and compared in detail. Firstly, diselenide, disulfide and carbon-carbon bond containing porphyrin dimers TPP-SeSe/ TPP-SS/ TPP-CC were synthesized. Then, the corresponding self-assembled nanoparticles (TPP-SeSe/ TPP-SS/ TPP-CC NPs) were prepared via nanoprecipitation method. As-synthesized nanoparticles were utilized to systematically compare the stimuli responsiveness to reductive agents (e.g. glutathione) in different conditions, especially in the living cells, through the singlet oxygen generation ability and the cytotoxicity. A series of experimental results demonstrated diselenide bond shows better superiority in reduction sensitivity than disulfide or carbon-carbon bond, which contributed to rapider delivery of the photosensitizer and facilitated exerting better PDT activity. These results highlight the potential of diselenide bond to be developed as novel and effective platform to fabricate more functional stimuli-responsive nanomaterials.

In recent years, nanomaterials responding to specific biological triggers attract much more attention for biomedical applications.1-6 A large number of data indicate that the intracellular and extracellular environment are of great differences, especially in tumor tissues, which includes lower pH, special enzymes, tissue hypoxia and so on.7,8 In addition to these differences, the redox potential is another very important signal in the cancer cells compared with the normal cells.9 Thus, a series of smart redox-sensitive nanomedicines based on disulfide bond were synthesized in past decades.10-17 Selenium (Se), which lists in the same family of sulfur (S), also gradually becomes a research hotspot in recent years and is considered as potential candidates to fabricate smart nanomaterials.18-20 On account of the special electronegativity and atom radius, selenium possesses many different chemical properties in contrast with carbon or sulfur. As reported, the bond energy of Se-Se bond is much weaker in comparison with C-C and S-S bond and such property results in the more reductive sensitivity of diselenidecontaining compounds than disulfide bond linked materials theoretically.21 However, there still exists some controversy over the redox responsiveness between the diselenide and disulfide bond. For example, according to Gu’ group, diselenide-bond cross-linked oligoethylenimine (OEI-SeSe) was not that sensitive to intracellular reductive environment.22,23 However, contrary to Gu’s finding, Wang reported that diselenide bond was more easier to be broken than disulfide bonds even at low con-

centration of reductive agents.24 Thus, it is necessary to clarify the stimuli responsiveness of diselenide and disulfide bond. As a kind of minimally noninvasive, controllable and safe method for cancer treatment, PDT has become one of the most promising protocols for cancer therapy both in experiment and clinical practice.25-30 During the typical PDT process, a photosensitizer (PS), light and tissue oxygen are necessarily involved.31 In the case of appropriate light irradiation, the activated-photosensitizer will transfer excited-state energy to the oxygen and reactive oxygen species (ROS) are generated, which further induces the cellular dysfunction and the necrosis or apoptosis.32,33 To obtain better anti-cancer effect, a series of photosensitizers with high singlet oxygen (1O2) quantum yields with favorable photostability have been extensively developed, including TiO2,34 C60,35 methylene blue,36 BODIPY,37-39 ect.. Porphyrin and its derivatives, have aroused more and more attention to be used as an effective and crucial PS in PDT process.40-42 For example, Cai and his coworkers developed polyglycol-coated nanomicelles with Ce6 as a PDT agent, which was used as theranostic nanomedicine with both excellent antitumor activity and fluorescence and PET imaging.25 Our group also reported the design and synthesis of porphyrin-containing PDT nanomaterials, which could effectively kill cancer cells.43,44 Furthermore, in order to achieve ideal PDT activity, the accurate and fast release of PSs was also of great importance because of the fluorescence resonance energy transfer

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(FRET)-mediated intramolecular photoquenching between the proximal two PSs molecules.45 Due to the big difference in the concentration in the cytosol (2-10 mM) and extracellular environment (2-20 μM), glutathione (GSH) could be an ideal endogenous stimulus to rapidly trigger the nanocarriers to release the PSs.46 In the past few decades, a serious of GSH activated photosensizing systems had been reported, such as PEG-ss-Ce6-MMP2 conjugate, mPEG-(ss-pheophorbide a)2 conjugates and hyaluronic acid-ss-Ce6 conjugate.47-49 These redoxresponsive nanocarriers showed ability for selective release and efficient activation of the PSs in tumor cells, which could enhance the intracellular dose of PSs to achieve higher cytotoxicity, thereby enhancing the treatment efficacy.

Page 2 of 9

knowledge, there are few literatures which systematically compare the effects of redox sensitivity to photodynamic activity via organic nanoparticles so far. RESULTS AND DISCUSSION Synthesis and characterization of the nanoparticles (TPP-SeSe/ TPP-SS/ TPP-CC NPs). The synthesis routes of TPP-SeSe/ TPP-SS/ TPP-CC were shown in Figure S1 (Supporting Information). Simply, 6,6’diselanediyldihexanoic acid (SeSeCOOH) and 5-(4hydroxylphenyl)-10,15,20-triphenylporphyrin (TPP-OH) were prepared according to the literature methods.43,50 Subsequently, TPP-SeSe/ TPP-SS/ TPP-CC were synthesized through one-step condensation reaction between TPP-OH and SeSeCOOH, 3,3’-dithiodipropionic acid (SSCOOH) and suberic acid (C8COOH), respectively. After purification by silica gel column, all the products were obtained and well characterized by proton nuclear magnetic resonance (1H NMR) and matrix-assisted laser desorption/ionization time-of-light mass spectrometry (MALDI-TOF MS). As shown in Figure S2, all the protons could be clearly resolved in 1H NMR spectra, corresponding to the targeted compounds. Moreover, the peak at m/z 1614.5, 1434.46 and 1398.55 in the MOLDI-TOF MS spectra were the same to the theoretical molecular weight of TPP-SeSe/ TPP-SS/ TPP-CC, further confirming the successful synthesis of the compounds. Probably due to the symmetric structure and the

Scheme 1. (a) Synthesis routes of TPP-SeSe/ TPP-SS/ TPP-CC and the corresponding self-assembled nanoparticles TPP-SeSe/ TPP-SS/ TPP-CC NPs via nanoprecipitation method. (b) Schematic illustration of the cellular internalization, intracellular disassembly and in vitro photodynamic therapy processes of the nanoparticles. In this study, diselenide, disulfide and carbon-carbon bond linked porphyrin dimers (TPP-SeSe/ TPP-SS/ TPPCC) were synthesized via condensation reactions. These small organic molecules could self-assemble into nanoparticles in aqueous media without adding any surfactants or adjuvants (Scheme 1). They were designed in order to systematically test and compare the reduction responsiveness differences in intracellular environment and the corresponding influence on PDT activity. To our best

Figure 1. Morphology of the nanoparticles. TEM images and size distribution results of (a)-(b) TPP-SeSe NPs, (c)(d) TPP-SS NPs and (e)-(f) TPP-CC NPs measured by DLS in aqueous solution. Scale bar, 500 nm. strong π-π interactions between the porphyrin,51,52 TPPSeSe/ TPP-SS/ TPP-CC could self-assemble into nanoparticles TPP-SeSe/ TPP-SS/ TPP-CC NPs in aqueous solution via nanoprecipitation method. Briefly, a tetrahydrofuran solution of TPP-SeSe/ TPP-SS/ TPP-CC was added dropwise into the deionized water with vigorous stirring. After fully evaporation of THF, the nanoparticles were obtained. The size distribution and morphology of the nanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The TEM images demonstrated TPP-SeSe/ TPPSS/ TPP-CC could self-assemble into perfect spherical


Page 3 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nanoparticles in aqueous solution (Figure 1). The average diameter, polydispersity index (PDI) and zeta potential results measured by DLS of TPP-SeSe/ TPP-SS/ TPP-CC NPs were listed in Table S1. As shown in Figure S3, all the nanoparticles could keep their diameter and PDI for around a week, indicating their favorable stability in water. Moreover, negligible changes of the size and PDI after being incubated in phosphate buffer saline (PBS, pH 7.4) containing 10% fetal bovine serum (FBS) for 24 h demonstrated the nice stability in physiological environment. The optical properties of the TPP-SeSe/ TPP-SS/ TPPCC and TPP-SeSe/ TPP-SS/ TPP-CC NPs were recorded by UV-vis absorption and photoluminescence spectra. As shown in Figure 2 and Figure S4, TPP-SeSe/ TPP-SS/ TPPCC in DMF/H2O= 49:1 (v/v) exhibited the same absorption spectra with maximum absorption at 416.5 nm. However, the maximum absorption of TPP-SeSe NPs, TPP-SS NPs and TPP-CC NPs were red-shifted about 12.5 nm, 10 nm and 9 nm compared to the results of TPP-SeSe, TPPSS and TPP-CC, respectively. The bathochromic shift absorption of the nanoparticles was possibly ascribed to the formation of nano-aggregation in aqueous solution.53,54 In Figure 2b, the maximum emission wavelength of the small molecules was mainly centered at 653 nm, however, almost no fluorescence could be seen for the nanoparticles because the Dexter-type excitonic migration between porphyrins results in fluorescence quenching.55 Taking TPP-SeSe and TPP-SeSe NPs for example, to visually observe the phenomenon, photos of the TPP-SeSe in DMF/H2O= 49:1 (v/v) solution and TPP-SeSe NPs in aqueous solution were taken under 365 nm light irradiation. As shown in Figure 2c, TPP-SeSe showed a strong red fluorescence nevertheless no fluorescence was observed for TPP-SeSe NPs.

Figure 2. Representative normalized UV-vis absorption (a) and fluorescence spectra (b) of TPP-SeSe in DMF/H2O = 49:1 (v/v) and TPP-SeSe NPs in aqueous solution. (c) Photos of TPP-SeSe and TPP-SeSe NPs in bright field and under 365 nm light irradiation.

Figure 3. Singlet oxygen generation and photostability of (a) TPP-OH, (b) TPP-SeSe, (c) TPP-SS and (d) TPP-CC (TPP: 10 μM) in DMF monitored by disappearance of UV absorption of ICG (5 μg/mL) at 790 nm with an LED lamp at power density of 12 mW/ cm2 over 8 min. (e) Photostability of ICG in DMF solution under the light irradiation in the absence of any photosensitizer. (f) The quantification of the 1O2 generation ability of according to the decrease of absorbance at 790 nm for ICG blank, TPP-OH, TPP-SeSe, TPP-SS and TPP-CC and the corresponding mixture under light irradiation (12 mW/ cm2). The singlet oxygen generation ability of TPP-OH, TPPSeSe, TPP-SS and TPP-CC and the corresponding nanoparticles upon irradiation was chemically determined by using indocyanine green (ICG) as scavenger and monitored by time-dependent electronic absorption spectroscopy.56,57 As shown in Figure 3e, ICG in DMF solution was irradiated with an LED lamp (630 nm, 12 mW/ cm2) for 8 min, and negligible decrease was founded at the absorption of 790 nm (the maximum absorption of ICG). Nevertheless, irradiation of the ICG solution in the presence of TPP-SeSe/ TPP-SS/ TPP-CC caused a steady generation of 1 O2, as evidenced by the decrease in the absorption band at 790 nm (Figure 3a-d). Moreover, TPP-SeSe/ TPP-SS/ TPP-CC possessed the similar 1O2 generation ability as free porphyrin (TPP-OH) while simultaneously maintained their photostability under the irradiation, indicating the functionalization of free porphyrin would not affect the generation of ROS. Finally, as shown in Figure 3f, the 1O2 generation abilities of these porphyrin-containing molecules are nearly unanimous under the same concentration of porphyrin in DMF and light irradiation. Subsequently, we also tested the 1O2 generation ability of TPPSeSe/ TPP-SS/ TPP-CC NPs in aqueous solution. As demonstrated in Figure S5, the nanoparticles formulation still kept the ability of producing 1O2 upon irradiation, but the efficiency was far below than that of the small organic molecules. Therefore, the dissociation of nanoparticles is imperative to exert their PDT function effectively, especially in living cells. Thus, the comparison of redox responsiveness of the diselenide, disulfide and carboncarbon bond to GSH is meaningful for developing ideal PDT system.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 9

ther demonstrate the different redox-sensitivity of Se-Se, S-S and C-C bond, we collected oxidation potentials of the small organic molecules (TPP-SeSe/ TPP-SS/ TPP-CC), the corresponding self-assembled nanoparticles (TPPSeSe/ TPP-SS/ TPP-CC NPs) after treatment of 10 mM GSH via cyclic voltammetry (CV) method.59 As shown in Figure S7, the oxidation potentials of TPP-SeSe, TPP-SS and TPP-CC were 0.9933 V, 0.90 V and 0 V, respectively. Moreover, the corresponding oxidation potentials of TPPSeSe, TPP-SS and TPP-CC NPs were 0.9344V, 0 V and 0 V, respectively. All the results showed that diselenidecontaining compounds could make faster response in cells compared with disulfide or carbon-carbon containing compounds, indicating the superior redox-sensitivity of diselenide bond. Figure 4. (a) and (e) Summary of diameter and PDI changes of TPP-SeSe/ TPP-SS/ TPP-CC NPs over different time period with or without treatment of 10 mM GSH monitored by DLS. Size distribution changes of (b) TPPSeSe NPs, (c) TPP-SS NPs and (d) TPP-CC NPs after incubation with GSH for 1.5 h. (f) Photos of the nanoparticles in the absence and presence of GSH after 24 h. For comparison of the reductive sensitivity between TPP-SeSe NPs, TPP-SS NPs and TPP-CC NPs, they were treated with 10 mM GSH to mimic the intracellular GSH level at 37 oC for different time. The time-dependent size and PDI changes were monitored by DLS. As shown in Figure 4a and Figure 4e, ignorable diameter and PDI changes were observed for the nanoparticles in the absence of reductive agents. When the nanoparticles were incubated with 10 mM GSH, obvious size and PDI increase were detected for TPP-SeSe NPs and TPP-SS NPs. According to DLS results in Figure 4, little size and PDI changes were observed for TPP-CC NPs and the average diameter of TPP-SS NPs increased from 192 nm to 396.7 nm with concomitant increasing of PDI from 0.124 to 0.223 with 10 mM GSH for 1.5 h. It is noteworthy that TPPSeSe NPs swelled rapidly from 193.5 nm to 970 nm with increasement of PDI from 0.115 to 0.298 after being incubated with GSH for the same period. The continuous increase of incubation time with GSH led to the further disruption of the diselenide and disulfide bonds in the presence of GSH. As shown in Figure 4a, after being incubation with 10 mM GSH for 24 h, TPP-SeSe NPs swelled more rapidly to a final size of about 3745 nm, which is much bigger than that of TPP-SS NPs. Little influence was observed on the particle size of TPP-CC NPs (Figure S6). The above phenomena further indicated the hyper reduction sensitivity of diselenide bond compared with disulfide or carbon-carbon bond. The reductive dissociation of the nanoparticles could also be observed visually. As shown in the picture in Figure 4f, the solution changed from clear to muddy and a large number of aggregations were founded at the bottom of the glass bottle upon incubation for 24 h, which might be attributed to the disassembly of the nanoparticles and the rearrangement of the hydrophobic parts into large aggregating form.58 To fur-

Figure 5. (a) Confocal laser scanning microscopy of HepG2 cells being incubated with TPP-SeSe NPs, TPP-SS NPs and TPP-CC NPs (TPP: 2 μM) for 10 h at 37 oC. For each panel, the images from left to right show cell nuclei stained by DAPI (blue), porphyrin fluorescence in cells (red), and overlays of both images. Scale bar: 20 μm. (b) Flow cytometry analyses of the cellular uptake of TPPSeSe NPs, TPP-SS NPs and TPP-CC NPs (from top to bottom) with the same concentration of porphyrin (25 μM) over different time (4 h and 24 h) at 37 oC. Cellular internalization was carried out before comparing their photodynamic activity in reductive cellular conditions. Liver hepatocellular carcinoma (HepG2) cells were used to investigate the cellular uptake of TPP-SeSe/ TPP-SS/ TPP-CC NPs by confocal laser scanning microscopy (CLSM). HepG2 cells were incubated with TPP-SeSe NPs, TPP-SS NPs and TPP-CC NPs (TPP: 2 μM), respectively. The cell nuclei were stained with 4’,6-diamidion-2phenylindole (DAPI). As shown in Figure 5a, obvious red fluorescence could be seen in cell plasma, indicating that the nanoparticles could endocytosed by cancer cells. To further compare and quantify the cellular uptake of the nanoparticles, flow cytometry was carried out. It could be observed that all internalization of the nanoparticles improved with the incubation time increasing from 4 h to 24 h, demonstrating time-dependent endocytosis (Figure 5a and Figure S8). Moreover, we discovered that TPP-SeSe


Page 5 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NPs exhibited stronger red fluorescence compared with that of TPP-SS NPs and TPP-CC NPs at the same concentration of porphyrin and incubation time according to the relative mean/media fluorescence intensity (Figure S8). We hypothesized this phenomenon might be attributed to the faster stimuli responsiveness of TPP-SeSe NPs to the reductive agents in cells than that of TPP-SS NPs and TPP-CC NPs. Then, we investigated the intracellular 1O2 production ability of the nanoparticles under light irradiation and 2’, 7’-dichlorofluorescin diacetate (DCFH-DA) was used as a ROS sensor, which could be oxidized to emit green fluorescence.60 As shown in Figure 6, stronger green fluorescence could be observed for the cells pretreated with TPP-SeSe NPs after light irradiation compared with that of cells treated with TPP-SS NPs and TPPCC NPs, which was almost two times larger for the relative fluorescence intensity than that of TPP-SS NPs and TPP-CC NPs (Figure S10a). The cells with treatment of the nanoparticles in dark were set as the controls (Figure S9 and S10b). These results validated that diselenide bond possesses faster response to the intracellular reductive environment and more porphyrin could be released, resulting in more ROS production. In order to compare the photodynamic efficiency of TPP-SeSe NPs, TPP-SS NPs and TPP-CC NPs, in vitro dark and photocytotoxicity at various concentrations (TPP: 550 µM) on HepG2 cells were performed by methyl tetrazolium (MTT) viability assays. We first studied the

Figure 6. Intracellular DCF fluorescence detection by CLSM of HepG2 cells after incubation with TPP-SeSe NPs, TPP-SS NPs and TPP-CC NPs (TPP: 50 μM) for 10 h at 37 o C, and then under light irradiation (12 mW/ cm2) for 60 min. The cells without any treatment were set as the controls. Scale bar, 20 μm.

Figure 7. In vitro cytotoxicity of TPP-SeSe NPs, TPP-SS NPs and TPP-CC NPs against HepG2 cells: (a) dark and (b) phototoxicity at different concentrations of porphyrin (5-50 μM) with an LED lamp at a power density of 12 mW/ cm2 for 60 min. (c) Typan blue stained HepG2 cells with PBS, TPP-SeSe NPs, TPP-SS NPs and TPP-CC NPs (TPP: 50 μM) after incubation for 10 h and then being exposed to 630 nm light irradiation, with blue color indicating dead cells. Scale bar, 100 μm. dark toxicity of the nanoparticles against the cancer cells, and no obvious cytotoxicity was observed even the at a porphyrin concentration of 50 µM, indicating their pronounced cytobiocompatibility without light irradiation (Figure 7a). We then incubated HepG2 cells with the nanoparticles for 10 h and subsequently irradiated the cells using an LED lamp at a power density of 12 mW/ cm2 for 1 h. As demonstrated in Figure 7b, TPP-SeSe NPs showed a dramatic photocytotoxicity toward HepG2 cells compared with TPP-SS NPs and TPP-CC NPs. With the increase of concentration to 50 µM, the cell viability decreased to less than 30% for TPP-SeSe NPs. However, the cell viability of TPP-SS NPs and TPP-CC NPs only reduce about 30% and 20%. The IC50 values of the three kinds of nanoparticles are 25.7 µM, >50 µM and >50 µM, respectively. We hypothesized that such difference in cytotoxicity of TPPSeSe NPs, TPP-SS NPs and TPP-CC NPs was mainly due to the different redox sensitivity of the nanoparticles in cells. As reported in the article before, the bond length of diselenide bond is little shorter than that of disulfide bond (232 pm and 206 pm, respectively).61 Therefore, diselenide bond is more responsive to reductive agents than disulfide bond. To further visualize the cell death caused by PDT, the cells were stained with Trypan blue. In Figure S11, cells in the control group and incubated



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

with TPP-SS NPs and TPP-CC NPs without irradiation showed no color, confirming that the nanoparticles treated alone were not harmful to the cells. Upon light irradiation, most cells incubated with TPP-SeSe NPs after irradiation were killed, as evidenced by the large areas of homogeneous blue color (Figure 7c). However, no obvious influence was observed for the cells treated with TPP-SS NPs and TPP-CC NPs. Furthermore, the PDT efficacy was qualitatively demonstrated via the live/dead staining and the cells were stained with calcein-AM and propidium iodide (PI) to differentiate live (green) and dead/late apoptotic (red) cells, respectively. Figure S12 exhibited that most cells treated the nanoparticles in dark were alive with strong green fluorescence after incubation for 48 h, indicating fascinating biocompatibility of the nanoparticles. However, majority HepG2 cells were dead with a large number of red color with treatment with TPP-SeSe NPs under LED irradiation compared with that treated with TPP-SS NPs and TPP-CC NPs. To further quantitatively determine the cell death induced by PDT, the cells were stained with Annexin V-FITC and PI and analyzed by flow cytometry. As show in Figure 8 and Figure S13, the late apoptosis ratios were 20.0% and 24.3% for TPP-SS NPs and TPP-CC NPs after irradiation, respectively. While after being incubated with TPP-SeSe NPs, the late apoptosis ratio increased to 64.7%. These results were consistent with that of MTT assays and substantiated the superiority of TPP-SeSe NPs as potential PDT agents. All

Page 6 of 9

the results above further showed that the diselenide linker is more favorable and sensitive for the dissociation of nanoparticles, release of porphyrin in cellular reductive environment and inducement of more cancer cells’ death compared with disulfide or carbon-carbon bond.

CONCLUSION In summary, we presented diselenide bond linked porphyrin TPP-SeSe for used as reduction-sensitive drug delivery nanocarriers. The corresponding analogs disulfide and carbon-carbon bond linked molecules, TPP-SS and TPP-CC, were prepared and compared. All the small organic molecules could self-assemble into uniform nanoparticles (TPP-SeSe NPs, TPP-SS NPs and TPP-CC NPs), which showed favorable stability in aqueous solution and physiological environment. Compared with TPP-SS NPs and TPP-CC NPs, TPP-SeSe NPs exhibited hyper-redox sensitivity under the reductive environment. This responsive property contributes to more rapid delivery of porphyrin and facilitates exerting the PDT activity. Thus, TPP-SeSe NPs exhibited distinct advantages over TPP-SS NPs and TPP-CC NPs no matter in the cell toxicity assays or intracellular singlet oxygen production ability assays. These results indicate that diselenide bond could be used as effective redox-sensitive group to fabricate promising drug delivery systems and diselenide-based selfassembling nanoparticles provide an important platform to develop novel functional nanomaterials.

ASSOCIATED CONTENT Supporting Information. Experimental details, synthesis 1 routes, H NMR and MOLDI-TOF MS spectra, the average diameter, polydispersity index and zeta potential, UV-vis absorption and fluorescence spectrum, singlet oxygen generation and photostability, TEM images, cyclic voltammograms, intracellular fluorescence intensity of DCF, flow analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author

*corresponding authors: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Figure 8. Flow cytometry analysis of apoptosis cells induced by incubation with TPP-SeSe NPs, TPP-SS NPs and TPP-CC NPs after light irradiation toward HepG2 cells: Annexin V-FITC/PI staining detects apoptosis in cells (top-right panel indicating late apoptotic cells) after treatment for 48 h at a porphyrin concentration of 50 μM .

This work was supported by the National Natural Science Foundation of China (Project. No. 51522307). We are very thankful for Jie Chen for the test of flow cytometry and Mingbo Ruan for the test of the oxidation potential of the small molecules and the nanoparticles.


Page 7 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES 1. Thornton, P. D.; Mart, R. J.; Ulijn, R. V. Enzyme-Responsive Polymer Hydrogel Particles for Controlled Release. Adv. Mater. 2007, 19, 1252-1256. 2. Tai, W.; Mo, R.; Di, J.; Subramanian, V.; Gu, X.; Buse, J. B.; Gu, Z. Bio-Inspired Synthetic Nanovesicles for Glucose-Responsive Release of Insulin. Biomacromolecules 2014, 15, 3495-3502. 3. Chang, B.; Chen, D.; Wang, Y.; Chen, Y.; Jiao, Y.; Sha, X.; Yang, W. Bioresponsive Controlled Drug Release based on Mesoporous Silica Nanoparticles Coated with Reductively Sheddable Polymer Shell. Chem. Mater. 2013, 25, 574-585. 4. Iyisan, B.; Kluge, J. R.; Formanek, P.; Voit, B.; Appelhans, D. Multifunctional and Dual-Responsive Polymersomes as Robust Nanocontainers: Design, Formation by Sequential PostConjugations, and pH-Controlled Drug Release. Chem. Mater. 2016, 28, 1513-1525. 5. Meng, H.; Xue, M.; Xia, T.; Zhao, Y.-L.; Tamanoi, F.; Stoddart, J. F.; Zink, J. I.; Nel, A. E. Autonomous in vitro Anticancer Drug Release from Mesoporous Silica Nanoparticles by pH-Sensitive Nanovalves. J. Am. Chem. Soc. 2010, 132, 12690-12697. 6. Sun, W.; Jiang, T.; Lu, Y.; Reiff, M.; Mo, R.; Gu, Z. Cocoon-Like Self-Degradable DNA Nanoclew for Anticancer Drug Delivery. J. Am. Chem. Soc. 2014, 136, 14722-14725. 7. An, X.; Zhu, A.; Luo, H.; Ke, H.; Chen, H.; Zhao, Y. Rational Design of Multi-Stimuli-Responsive Nanoparticles for Precise Cancer Therapy. ACS Nano 2016, 10, 5947–5958. 8. Jhaveri, A.; Deshpande, P.; Torchilin, V. Stimuli-Sensitive Nanopreparations for Combination Cancer Therapy. J. Controlled Release 2014, 190, 352-370. 9. Yeh, C. C.; Hou, M. F.; Wu, S. H.; Tsai, S. M.; Lin, S. K.; Hou, L. A.; Ma, H.; Tsai, L. Y. A Study of Glutathione Status in the Blood and Tissues of Patients with Breast Cancer. Cell Biochem. Funct. 2006, 24, 555-559. 10. Hu, H.; Li, Y.; Zhou, Q.; Ao, Y.; Yu, C.; Wan, Y.; Xu, H.-B.; Li, Z.; Yang, X. Redox-Sensitive Hydroxyethyl Starch-Doxorubicin Conjugate for Tumor Targeted Drug Delivery. ACS Appl. Mater. Interfaces 2016, 8, 30833–30844. 11. Li, S.; Zhang, J.; Deng, C.; Meng, F.; Yu, L.; Zhong, Z. RedoxSensitive and Intrinsically Fluorescent Photoclick Hyaluronic Acid Nanogels for Traceable and Targeted Delivery of Cytochrome C to Breast Tumor in Mice. ACS Appl. Mater. Interfaces 2016, 8, 21155-21162. 12. Liu, X.; Wu, M.; Hu, Q.; Bai, H.; Zhang, S.-Q.; Shen, Y.; Tang, G.; Ping, Y. A Redox-Activated Light-Up Nanomicelle for Precise Imaging-Guided Cancer Therapy and Real-Time Pharmacokinetic Monitoring. ACS Nano 2016,10, 11385-11396. 13. Yen, H.-C.; Cabral, H.; Mi, P.; Toh, K.; Matsumoto, Y.; Liu, X.; Koori, H.; Kim, A.; Miyazaki, K.; Miura, Y. Light-Induced Cytosolic Activation of Reduction-Sensitive Camptothecin-Loaded Polymeric Micelles for Spatiotemporally Controlled In Vivo Chemotherapy. ACS Nano 2014, 8, 11591-11602. 14. Bauhuber, S.; Hozsa, C.; Breunig, M.; Göpferich, A. Delivery of Nucleic Acids via Disulfide-Based Carrier Systems. Adv. Mater. 2009, 21, 3286-3306. 15. Zhang, Z.; Yin, L.; Tu, C.; Song, Z.; Zhang, Y.; Xu, Y.; Tong, R.; Zhou, Q.; Ren, J.; Cheng, J. Redox-Responsive, Core Cross-linked Polyester Micelles. ACS Macro Lett. 2012, 2, 40-44. 16. Lin, C.-W.; Lu, K.-Y.; Wang, S.-Y.; Sung, H.-W.; Mi, F.-L. CD44-Specific Nanoparticles for Redox-Triggered Reactive Oxygen Species Production and Doxorubicin Release. Acta Biomater. 2016, 35, 280-292. 17. Wang, H.; Tang, L.; Tu, C.; Song, Z.; Yin, Q.; Yin, L.; Zhang, Z.; Cheng, J. Redox-Responsive, Core-Cross-Linked Micelles Capable of On-Demand, Concurrent Drug Release and Structure Disassembly. Biomacromolecules 2013, 14, 3706-3712.

18. Xu, H.; Cao, W.; Zhang, X. Selenium-Containing Polymers: Promising Biomaterials for Controlled Release and Enzyme Mimics. Acc. Chem. Res. 2013, 46, 1647-1658. 19. Mugesh, G.; Singh, H. B. Synthetic Organoselenium Compounds as Antioxidants: Glutathione Peroxidase Activity. Chem. Soc. Rev. 2000, 29, 347-357. 20. Cao, W.; Wang, L.; Xu, H. Selenium/tellurium Containing Polymer Materials in Nanobiotechnology. Nano Today 2015, 10, 717-736. 21. Kildahl, N. K. Bond Energy Data Summarized. J. Chem. Educ. 1995, 72, 423-424. 22. He, Y.; Nie, Y.; Cheng, G.; Xie, L.; Shen, Y.; Gu, Z. Viral Mimicking Ternary Polyplexes: A Reduction-Controlled Hierarchical Unpacking Vector for Gene Delivery. Adv. Mater. 2014, 26, 15341540. 23. Yue, D.; Cheng, G.; He, Y.; Nie, Y.; Jiang, Q.; Cai, X.; Gu, Z. Influence of Reduction-Sensitive Diselenide Bonds and Disulfide Bonds on Oligoethylenimine Conjugates for Gene Delivery. J. Mater. Chem. B 2014, 2, 7210-7221. 24. Li, W.; Zhang, P.; Zheng, K.; Hu, Q.; Wang, Y. RedoxTriggered Intracellular dePEGylation Based on DiselenideLinked Polycations for DNA Delivery. J. Mater. Chem. B 2013, 1, 6418-6426. 25. Cheng, L.; Kamkaew, A.; Sun, H.; Jiang, D.; Valdovinos, H. F.; Gong, H.; England, C. G.; Goel, S.; Barnhart, T. E.; Cai, W. DualModality Positron Emission Tomography/Optical Image-Guided Photodynamic Cancer Therapy with Chlorin e6-Containing Nanomicelles. ACS Nano 2016, 10, 7721-7730. 26. Celli, J. P.; Spring, B. Q.; Rizvi, I.; Evans, C. L.; Samkoe, K. S.; Verma, S.; Pogue, B. W.; Hasan, T. Imaging and Photodynamic Therapy: Mechanisms, Monitoring, and Optimization. Chem. Rev. 2010, 110, 2795-2838. 27. Liu, K.; Xing, R.; Zou, Q.; Ma, G.; Möhwald, H.; Yan, X. Simple Peptide‐Tuned Self‐Assembly of Photosensitizers towards Anticancer Photodynamic Therapy. Angew. Chem. Int. Ed. 2016, 55, 3036-3039. 28. Schmitt, J.; Heitz, V.; Sour, A.; Bolze, F.; Ftouni, H.; Nicoud, J. F.; Flamigni, L.; Ventura, B. Diketopyrrolopyrrole‐Porphyrin Conjugates with High Two-Photon Absorption and Singlet Oxygen Generation for Two-Photon Photodynamic Therapy. Angew. Chem. Int. Ed. 2015, 54, 169-173. 29. Lu, K.; He, C.; Lin, W. A Chlorin-Based Nanoscale MetalOrganic Framework for Photodynamic Therapy of Colon Cancers. J. Am. Chem. Soc. 2015, 137, 7600-7603. 30. Dolmans, D. E.; Fukumura, D.; Jain, R. K. Photodynamic Therapy for Cancer. Nat. Rev. Cancer 2003, 3, 380-387. 31. Wang, C.; Cheng, L.; Liu, Z. Upconversion Nanoparticles for Photodynamic Therapy and Other Cancer Therapeutics. Theranostics 2013, 3, 317-330. 32. Li, Y.; Tan, C.-P.; Zhang, W.; He, L.; Ji, L.-N.; Mao, Z.-W. Phosphorescent Iridium (III)-bis-N-heterocyclic Carbene Complexes as Mitochondria-Targeted Theranostic and Photodynamic Anticancer Agents. Biomaterials 2015, 39, 95-104. 33. Ge, J.; Lan, M.; Zhou, B.; Liu, W.; Guo, L.; Wang, H.; Jia, Q.; Niu, G.; Huang, X.; Zhou, H. A Graphene Quantum Dot Photodynamic Therapy Agent with High Singlet Oxygen Generation. Nat. Commun. 2014, 5, 4596. 34. Idris, N. M.; Lucky, S. S.; Li, Z.; Huang, K.; Zhang, Y. Photoactivation of Core-Shell Titania Coated Upconversion Nanoparticles and Their Effect on Cell Death. J. Mater. Chem. B 2014, 2, 7017-7026. 35. Liu, X.; Zheng, M.; Kong, X.; Zhang, Y.; Zeng, Q.; Sun, Z.; Buma, W. J.; Zhang, H. Separately Doped Upconversion-C60 Nanoplatform for NIR Imaging-Guided Photodynamic Therapy of Cancer Cells. Chem. Commun. 2013, 49, 3224-3226.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

36. Chen, H.; Tian, J.; He, W.; Guo, Z. H2O2-Activatable and O2Evolving Nanoparticles for Highly Efficient and Selective Photodynamic Therapy Against Hypoxic Tumor Cells. J. Am. Chem. Soc. 2015, 137, 1539-1547. 37. Wang, W.; Wang, L.; Li, Z.; Xie, Z. BODIPY-Containing Nanoscale Metal-Organic Frameworks for Photodynamic Therapy. Chem. Commun. 2016, 52, 5402-5405. 38. Kamkaew, A.; Lim, S. H.; Lee, H. B.; Kiew, L. V.; Chung, L. Y.; Burgess, K. BODIPY Dyes in Photodynamic Therapy. Chem. Soc. Rev. 2013, 42, 77-88. 39. Zhang, Q.; Cai, Y.; Wang, X.-J.; Xu, J.-L.; Ye, Z.; Wang, S.; Seeberger, P. H.; Yin, J. Targeted Photodynamic Killing of Breast Cancer Cells Employing Heptamannosylated β-CyclodextrinMediated Nanoparticle Formation of an AdamantaneFunctionalized BODIPY Photosensitizer. ACS Appl. Mater. Interfaces 2016, 8, 33405-33411. 40. Ethirajan, M.; Chen, Y.; Joshi, P.; Pandey, R. K. The Role of Porphyrin Chemistry in Tumor Imaging and Photodynamic Therapy. Chem. Soc. Rev. 2011, 40, 340-362. 41. Bonnett, R. Photosensitizers of the Porphyrin and Phthalocyanine Series for Photodynamic Therapy. Chem. Soc. Rev. 1995, 24, 19-33. 42. Xiang, Z.; Zhu, L.; Qi, L.; Yan, L.; Xue, Y.; Wang, D.; Chen, J.F.; Dai, L. Two-Dimensional Fully-Conjugated Polymeric Photosensitizers for Advanced Photodynamic Therapy. Chem. Mater. 2016, 28, 8651-8658. 43. Zheng, X.; Li, Z.; Chen, L.; Xie, Z.; Jing, X. Self-Assembly of Porphyrin-Paclitaxel Conjugates into Nanomedicines: Enhanced Cytotoxicity due to Endosomal Escape. Chem. - Asian J. 2016, 11, 1780-1784. 44. Li, Y.; Zheng, X.; Zhang, X.; Liu, S.; Pei, Q.; Zheng, M.; Xie, Z. Porphyrin-Based Carbon Dots for Photodynamic Therapy of Hepatoma. Adv. Health. Mater. 2016. DOI: 10.1002/adhm.201600924. 45. Campo, M. A.; Gabriel, D.; Kucera, P.; Gurny, R.; Lange, N. Polymeric Photosensitizer Prodrugs for Photodynamic Therapy. Photochem. Photobiol. 2007, 83, 958-965. 46. Gamcsik, M. P.; Kasibhatla, M. S.; Teeter, S. D.; Colvin, O. M. Glutathione Levels in Human Tumors. Biomarkers 2012, 17, 671691. 47. Hou, W.; Xia, F.; Alves, C. S.; Qian, X.; Yang, Y.; Cui, D. MMP2-Targeting and Redox-Responsive PEGylated Chlorin e6 Nanoparticles for Cancer Near-Infrared Imaging and Photodynamic Therapy. ACS Appl. Mater. Interfaces 2016, 8, 1447-1457. 48. Kim, W. L.; Cho, H.; Li, L.; Kang, H. C.; Huh, K. M. Biarmed Poly(ethylene glycol)-(pheophorbide a)2 Conjugate as a Bioactivatable Delivery Carrier for Photodynamic Therapy. Biomacromolecules 2014, 15, 2224-2234. 49. Kim, H.; Mun, S.; Choi, Y. Photosensitizer-Conjugated Polymeric Nanoparticles for Redox-Responsive Fluorescence Imaging and Photodynamic Therapy. J. Mater. Chem. B 2013, 1, 429-431. 50. Zhang, W.; Lin, W.; Pei, Q.; Hu, X.; Xie, Z.; Jing, X. RedoxHypersensitive Organic Nanoparticles for Selective Treatment of Cancer Cells. Chem. Mater. 2016, 28, 4440-4446. 51. Xu, L.; Liu, L.; Liu, F.; Cai, H.; Zhang, W. PorphyrinContaining Amphiphilic Block Copolymers for Photodynamic Therapy. Polym. Chem. 2015, 6, 2945-2954. 52. Kasai, H.; Murakami, T.; Ikuta, Y.; Koseki, Y.; Baba, K.; Oikawa, H.; Nakanishi, H.; Okada, M.; Shoji, M.; Ueda, M. Creation of Pure Nanodrugs and Their Anticancer Properties. Angew. Chem. Int. Ed. 2012, 51, 10315-10318. 53. Liu, K.; Xing, R.; Li, Y.; Zou, Q.; Möhwald, H.; Yan, X. Mimicking Primitive Photobacteria: Sustainable Hydrogen Evolution Based on Peptide-Porphyrin Co-Assemblies with a SelfMineralized Reaction Center. Angew. Chem. Int. Ed. 2016, 55, 12503-12507.

Page 8 of 9

54. Nam, Y. S.; Shin, T.; Park, H.; Magyar, A. P.; Choi, K.; Fantner, G.; Nelson, K. A.; Belcher, A. M. Virus-Templated Assembly of Porphyrins into Light-Harvesting Nanoantennae. J. Am. Chem. Soc. 2010, 132, 1462-1463. 55. Scholes, G. D. Long-Range Resonance Energy Transfer in Molecular Systems. Annu. Rev. Phys. Chem. 2003, 54, 57-87. 56. He, S.; Qi, Y.; Kuang, G.; Zhou, D.; Li, J.; Xie, Z.; Chen, X.; Jing, X.; Huang, Y. Single-Stimulus Dual-Drug Sensitive Nanoplatform for Enhanced Photoactivated Therapy. Biomacromolecules 2016, 17, 2120-2127. 57. Tang, C.-Y.; Wu, F.-Y.; Yang, M.-K.; Guo, Y.-M.; Lu, G.-H.; Yang, Y.-H. A Classic Near-Infrared Probe Indocyanine Green for Detecting Singlet Oxygen. Int. J. Mol. Sci. 2016, 17, 219-226. 58. Zhao, J.; Liu, J.; Xu, S.; Zhou, J.; Han, S.; Deng, L.; Zhang, J.; Liu, J.; Meng, A.; Dong, A. Graft Copolymer Nanoparticles with pH and Reduction Dual-Induced Disassemblable Property for Enhanced Intracellular Curcumin Release. ACS Appl. Mater. Interfaces 2013, 5, 13216-13226. 59. Li, T.; Li, F.; Xiang, W.; Yi, Y.; Chen, Y.; Cheng, L.; Liu, Z.; Xu, H. Selenium-Containing Amphiphiles Reduced and Stabilized Gold Nanoparticles: Kill Cancer Cells via Reactive Oxygen Species. ACS Appl. Mater. Interfaces 2016, 8, 22106-22112. 60. Han, K.; Lei, Q.; Wang, S. B.; Hu, J. J.; Qiu, W. X.; Zhu, J. Y.; Yin, W. N.; Luo, X.; Zhang, X. Z. Dual-Stage-Light-Guided Tumor Inhibition by Mitochondria-Targeted Photodynamic Therapy. Adv. Funct. Mater. 2015, 25, 2961-2971. 61. Blom, R.; Haaland, A. A Modification of the SchomakerStevenson Rule for Prediction of Single Bond Distances. J. Mol. Struct. 1985, 128, 21-27.


Page 9 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC


9

Comparing Effects of Redox Sensitivity of Organic Nanoparticles to

Recommend Documents