Fenton Reaction-Assisted Photodynamic Therapy for Cancer with

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Biological and Medical Applications of Materials and Interfaces

Fenton reaction assisted photodynamic therapy for cancer with multifunctional magnetic nanoparticles Huabo Hou, Xuehui Huang, Guoqing Wei, Funeng Xu, Yi Wang, and Shaobing Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09671 • Publication Date (Web): 30 Jul 2019 Downloaded from pubs.acs.org on July 30, 2019

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ACS Applied Materials & Interfaces

Fenton reaction assisted photodynamic therapy for cancer with multifunctional magnetic nanoparticles Huabo Hou,† Xuehui Huang,† Guoqing Wei,† Funeng Xu,† Yi Wang,‡,* and Shaobing Zhou†,* † Key Laboratory of Advanced Technologies

of Material, Minister of Education, School

of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, Sichuan, P.R. China ‡

School of Life Science and Engineering, Southwest Jiaotong University, Chengdu

610031, P.R. China KEYWORDS: Fenton reaction, mitochondria-targeting, photodynamic therapy, magnetic resonance imaging, nanoparticle

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ABSTRACT Tumor hypoxia and the short half-life of reactive oxygen species (ROS) with small diffusion distance have greatly limited the therapeutic effect of photodynamic therapy (PDT). Here, a multifunctional nanoplatform is developed to enhance the PDT effect through increasing the oxygen concentration in tumor cells by Fenton reaction and reducing the distance between the ROS and the target site by mitochondria-targeting. Fe3O4@Dex-TPP nanoparticles are firstly prepared by co-precipitation in the presence of triphenylphosphine (TPP) grafted-dextran (Dex-TPP) and Fe2+/Fe3+, which have a magnetic resonance imaging effect. Next, the photosensitizers of protoporphyrin (PpIX) and glutathione-responsive mPEG-ss-COOH are grafted on Fe3O4@Dex-TPP to form Fe3O4@Dex/TPP/PpIX/ss-mPEG nanoparticles. After the nanoparticles internalized, part of Fe3O4 are decomposed into Fe2+/Fe3+ in the acidic lysosome, and then the Fe2+/Fe3+ diffused into the cytoplasm, subsequently the Fe2+ reacted with the overproduced H2O2 to produce O2 and •OH. The undecomposed nanoparticles enter the cytoplasm by photo induced internalization and targeted to the mitochondria, leading to ROS direct generation around the mitochondria. Simultaneously, the produced O2 by the Fenton reaction can serve as a raw material for PDT to continuously exert PDT effect. As a result, the Fenton reaction assisted PDT can significantly improve the therapeutic efficacy of tumors.

INTRODUCTION

In recent years, PDT has gradually become one of the most attractive methods for cancer treatment,1-4 because of its non-invasiveness, minimal damage to normal tissues


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and inactivation of multi-drug resistance.3-7 PDT is a new type of tumor treatment by irradiating the tumor with a laser of corresponding wavelength in the presence of both photosensitizer and O2 to produce singlet oxygen (1O2),6,

8

further damaging the

biological macromolecules and deoxyribonucleic acid (DNA),9-11 and finally leading to cell apoptosis. Therefore, the therapeutic effect of PDT is greatly affected by the oxygen concentration.12 However, the O2 is scarce in the tumor microenvironment due to the rapid proliferation of tumor cells and the non-normalized growth of tumor blood vessels.12-14 As a result, the therapeutic effect is still unsatisfying when applying PDT alone.4, 5, 15 Naturally, researchers tried to combine PDT and other therapies to enhance the therapeutic effect, such as chemotherapy5, 16-23 and radiotherapy.24-28 However, the introduced chemotherapy and radiotherapy have brought some new problems, such as systemic toxicity, multi-drug resistance, complications and so on.16, 24, 25, 29 To avoid these problems and enhance the PDT effect, to increase the oxygen concentration at the tumor site could be an ideal strategy for cancer treatment. Currently, several strategies have been employed to increase the oxygen concentration at the tumor site, e.g., delivering the oxygen-rich reagents to the tumor site by nanocarrier,14, 30-35 reducing tumor oxygen consumption,36, 37 generating oxygen in situ at the tumor site.38-43 Compared with other strategies, in situ generation of oxygen have received more and more attention because it does not require complex synthesis and avoid the premature leakage of oxygen. Contrary to normal cells, cancer cells produce more ROS due to metabolic and peroxisome activity, increasing cellular receptor signaling, oncogene activity and mitochondrial malfunction. Among all ROS



in cancer cells, H2O2 is generally regarded as the most abundant non-radical reactive oxygen species.44 It is well known that the amount of hydrogen peroxide (H2O2) is higher (100 μM -1 mM)8, 45 in tumor cells than that in normal cells. Therefore, in situ decomposing the H2O2 in tumor cells to produce oxygen becomes a promising approach to increase the oxygen concentration in cancer cells. The decomposition methods of H2O2 usually include catalase43 and Fenton reaction.40, 46, 47 Researchers prefer the latter method rather than the former as the catalase activity in tumor cells is very low.48 The equations of the Fenton reaction are as follows: 49 Fe2+ + H2O2 → Fe3+ + ·OH + OHFe3+ + H2O2 → Fe2+ + ·OOH + H+ Fe3+ + ·OOH → Fe2+ + O2 + H+ The equations show that Fenton reaction can produce ·OH and O2, which can increase the oxygen concentration in tumor cells, further enhancing the PDT effect. In addition, the ·OH itself has a potent oxidation potential, which can oxidize and destroy the organelle of tumor cells, leading to tumor cells apoptosis. Fe3O4 nanoparticles have high biocompatibility in vivo, and when they enter cancer cells by endocytosis, they can be effectively decomposed to Fe2+ and Fe3+ in acidic environment of cancer cells, so they usually be used for cancer treatment by the Fenton reaction.41,50 On the other hand, the intracellular distribution of 1O2 also affects the therapeutic effect of PDT.4 To the best of our knowledge, 1O2 has a short half-life of less than 40 ns and a low diffusion radius of less than 20 nm,6, 51, 52 which limit its oxidative damage effect.53, 54 So, it is necessary to directly target the organelles or biomacromolecules that are important to tumor cells and sensitive to ROS, such as mitochondria,11, 55-57 to produce ROS in situ. For this reason, targeting mitochondria is a good choice. And the


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mitochondria are also vital to tumor cells, once it is damaged, it will lead to the occurrence of mitochondrial apoptosis pathway, causing tumor cell death. Accordingly, in this work a multifunctional nanoplatform was developed to enhance the PDT effect through increasing the oxygen concentration in tumor cells by Fenton reaction and reducing the distance between the ROS and the target site by mitochondria-targeting. As shown in Scheme 1a, Fe3O4@Dex-TPP was firstly synthesized by a co-precipitation method in the presence of iron (III) chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), ammonium hydroxide (NH3·H2O) and Dex-TPP. The obtained Fe3O4@Dex-TPP possess magnetic cores (Fe3O4), making it be suitable for magnetic resonance imaging (MRI). TPP functional groups on these nanoparticles endow them a mitochondria-targeting ability. Later, the photosensitizer PpIX and the GSH-responsive mPEG-ss-COOH were grafted on Fe3O4@Dex-TPP to obtain the desired nanoparticle (Fe3O4@Dex-TPP/PpIX/ssmPEG) with photo- and GSH-responsive capabilities. After Fe3O4@Dex-TPP/PpIX/ssmPEG were administrated in vivo via tail vein injection, they were firstly enriched in the tumor site by enhanced permeability and retention (EPR) effect, and then internalized by tumor cells (Scheme 1b). Part of the Fe3O4 were decomposed to produce Fe2+/Fe3+ in the acidic environment of lysosome, and the generated Fe2+/Fe3+ quickly diffused into the cytoplasm. Then the Fe2+ reacted with the overexpressed H2O2 in cytoplasm to produce ·OH and O2, which could destroy tumor organelles. After that, the tumor was irradiated with a 637 nm laser, the residual Fe3O4@Dex-TPP/PpIX/ssmPEG escaped from lysosomes and entered cytoplasm through photo induced



internalization (PCI).4 Once Fe3O4@Dex-TPP/PpIX/ss-mPEG entered the cytoplasm, the outer mPEG component fell off from the nanoparticles in response to the excess GSH, and the TPP on the nanoparticles was exposed, which help the nanoparticles to rapidly target mitochondria because of the positivity of triphenylphosphine cation (TPP+). After that, under the illumination with 637 nm laser, ROS was generated in situ on the mitochondrial surface, which leads to an increase of mitochondrial membrane permeability and a decrease of membrane potential.58-60 Moreover, the cytochrome C was released from mitochondria, causing the occurrence of mitochondrial apoptosis pathways and cell death.59, 61 As a result, the Fenton reaction assisted photodynamic therapy brought a significant improvement in the therapeutic efficacy of tumors. RESULTS AND DISCUSSION Synthesis and Characterization of the Nanoparticles. The multifunctional magnetic nanoplatform was synthesized according to the routes in Scheme 1a. The formation mechanism of Fe3O4@Dex-TPP/PpIX/ss-mPEG was briefly illustrated in Figure 1a. Firstly, Fe2+/Fe3+ were attracted by the oxygen atoms of the Dex-TPP molecular chains and gradually formed the crystal nucleus of Fe3O4 and finally became the Fe3O4@DexTPP under an alkaline condition. Next, PpIX and mPEG-ss-COOH were linked to the nanoparticles to form the resultant nanoparticles (Fe3O4@Dex-TPP/PpIX/ss-mPEG). The chemical structure of TPP-COOH, mPEG-COOH, mPEG-ss-COOH and Dex-TPP were confirmed by 1H nuclear magnetic resonance (1H-NMR) spectra in Figure S1-S4 and Fourier Transform Infrared (FT-IR) spectrums in Figure S5-S7. The grafting yield of TPP was calculated to be 12.6% by 1H-NMR of TPP-Dex.


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The size and morphology of the nanoparticles were measured by transmission electron microscope (TEM), atomic force microscope (AFM) and dynamic light scattering (DLS). As shown in Figure 1b and 1c, the particle size of Fe3O4@Dex-TPP was detected to be 11.13 ±1.23 nm while the size of Fe3O4@Dex-TPP/PpIX/ss-mPEG was about 64.34 ± 2.63 nm with the core size of approximately 37.33 ± 1.46 nm. The size of Fe3O4@Dex and Fe3O4@Dex-TPP/PpIX was also measured by TEM to be 12.32 ±2.33 nm and 21.12 ±1.26 nm, respectively, as shown in Figure S8a and b. The AFM images further confirm the morphology of the nanoparticles. As shown in Figure S9ae, the particle sizes of Fe3O4@Dex, Fe3O4@Dex-TPP, Fe3O4@Dex-TPP/PpIX, Fe3O4@Dex-TPP/PpIX/mPEG and Fe3O4@Dex-TPP/PpIX/ss-mPEG were determined to be 25.63 ±4.27 nm, 26.9 ±2.04 nm, 31.13 ±5.31 nm, 58.91 ±7.91 nm and 59.78 ± 5.73 nm, respectively. The results showed no significant difference between AFM and TEM. The particle size distribution of all nanoparticles was shown in Figures 1d, and the average sizes of Fe3O4@Dex, Fe3O4@Dex-TPP, Fe3O4@Dex-TPP/PpIX, and Fe3O4@Dex-TPP/PpIX/ss-mPEG were 25.81 ± 2.12 nm, 34.607 ± 3.53 nm, 49.107 ± 4.74 nm and 76.91 ±5.33 nm, while the corresponding polydispersity index (PDI) were 0.125, 0.166, 0.201 and 0.223, respectively. Compared with the results of TEM and AFM, the hydrodynamic diameters of all nanoparticles were slightly increased, because the mPEG chains were extended in phosphate buffer saline (PBS).62, 63 All the particle size was less than 200 nm, enabling the particles to be effectively enriched at the tumor site by EPR effect.64 The zeta potential of all the nanoparticles was presented in Figure 1e. The zeta potential of Fe3O4@Dex was measured to be 7.94 ±0.72 mV, and increased



to 23.1 ± 1.91 mV after the TPP was grafted because of the positive polarity of TPP+. The zeta potential of Fe3O4@Dex-TPP/PpIX decreased slightly to 13.2 ± 1.23 mV, which is probably due to the exposure of the carboxyl group of PpIX. The zeta potential of Fe3O4@Dex-TPP/PpIX/mPEG and Fe3O4@Dex-TPP/PpIX/ss-mPEG were -6.23 ± 0.92 mV and -5.73 ±0.65 mV, respectively. The negative charges on the surface of the nanoparticles was helpful for the long circulation in the blood. The ultraviolet (UV) absorption spectrum of all materials was further examined by UV-visible spectrophotometer (UV-vis). It could be seen in Figure 1f, compared with Dex, a wide UV absorption of Fe3O4@Dex and Fe3O4@Dex-TPP were detected in the range of 300-800 nm because of the broad absorption of Fe3O4. After PpIX was grafted, the Soret band peak at 400 nm of PpIX appeared in the UV absorption peak of Fe3O4@Dex-TPP/PpIX and Fe3O4@Dex-TPP/PpIX/ss-mPEG. At the same time, unlike the narrow Soret band peak of PpIX, the Fe3O4@Dex-TPP/PpIX and Fe3O4@Dex-TPP/PpIX/ss-mPEG possessed a broad Soret band.60, 64 In addition, the absorption peaks of Fe3O4@Dex-TPP/PpIX and Fe3O4@Dex-TPP/PpIX/ss-mPEG were similar to PpIX at 637 nm. Thus, once the Fe3O4@Dex-TPP/PpIX/ss-mPEG stimulated by 637 nm laser, the nanoparticles could absorb lasers and produce PDT effect. To further characterize the structure of the nanoparticles, the crystal structure of all nanoparticles was measured by X-ray Diffraction (XRD). As presented in Figure 1g, six distinct diffraction peaks in the XRD pattern of Fe3O4@Dex, Fe3O4@Dex-TPP, Fe3O4@Dex-TPP/PpIX, and Fe3O4@Dex-TPP/PpIX/ss-mPEG were detected at 2θ =


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30.1°, 35.6°, 43.3°, 53.5°, 57.2°and 62.9°, respectively, representing the six crystal faces of (220), (331), (400), (422), (511) and (440). The results were consistent with the crystal plane of Fe3O4 in the Joint Committee on Powder Diffraction Standards (JCPDS) [85-1436] standard card, indicating that the core of nanoparticle is Fe3O4 crystal. All the nanoparticles were analyzed by thermal gravimetric analyzer (TGA) to determine the content of each component of all nanoparticles. As shown in Figure 1h, the contents of Fe3O4, Dex-TPP, PpIX and mPEG-ss-COOH in Fe3O4@DexTPP/PpIX/ss-mPEG were calculated to be 21.202%, 26.461%, 10.782% and 41.554%, respectively. To further explore the loading content of PpIX, different concentrations of materials were measured by UV-vis, then the standard UV absorption curve of PpIX was established through the absorption intensity at 400 nm as shown in Figure S10a. The UV absorption of 200 μg/mL Fe3O4@Dex-TPP/PpIX/ss-mPEG which was decomposed with acid and dialyzed firstly was measured by UV-vis as shown in Figure S10b, the loading content of PpIX was calculated to be ~9.43%, which is almost in agreement with the result calculated by TGA analysis. Subsequently, the saturation magnetic strength of all nanoparticles was analyzed by Vibrating Sample Magnetometer (VSM). As shown in Figure 1i, the saturation magnetic strength of Fe3O4@Dex,

Fe3O4@Dex-TPP,

Fe3O4@Dex-TPP/PpIX

and

Fe3O4@Dex-

TPP/PpIX/ss-mPEG were measured to be 32.71 emu/g, 28.68 emu/g, 24.45 emu/g and 20.60 emu/g, respectively, with no residual magnetic strength, indicating that all the nanoparticles were superparamagnetic, which endowed them a better MR imaging



effect. Finally, to detect whether the NPs can fracture mPEG and expose TPP for targeting mitochondria, the response performance of disulfide bond was investigated by DLS as shown in Figure 1j. The particle size of Fe3O4@Dex-TPP/PpIX/ss-mPEG decreased from 67.32 nm to 57.68 nm in the presence of GSH. And the zeta potential increased from -5.7 mV to +13.3 mV with time, which is better for targeting mitochondria as the mitochondrial membrane potential is negative.66 In Vitro Investigation of Fenton Reaction Enhanced PDT. First of all, the 1O2 was detected by UV-vis through the absorption change of 1,3-diphenylisobenzofuran (DPBF) as shown in Figure 2a. Compared with Fe3O4@Dex-TPP/PpIX/ss-mPEG, the absorption of DPBF for Fe3O4@Dex-TPP/PpIX/ss-mPEG + H2O2 group decreased from 95.3% to 89.1% due to the produced ·OH by Fenton reaction, while the Fe3O4@Dex-TPP/PpIX/ss-mPEG + H2O2 + L group decreased to 63.8%, which was lower than that of Fe3O4@Dex-TPP/PpIX/ss-mPEG and Fe3O4@Dex-TPP/PpIX/ssmPEG + H2O2 groups because there was more ROS produced by PDT effect in the existence of both the residual O2 in the solution and the 637 nm laser irradiated photosensitizer. The UV absorption of DPBF of Fe3O4@Dex-TPP/PpIX/ss-mPEG + H2O2 + L group decreased to 48.5%, indicating that the ROS produced by PDT was enhanced by Fenton reaction. Then, the ·OH generated by Fenton reaction was analyzed by UV-vis through the UV absorption change of methylene blue (MB). As shown in Figure 2b, there was almost no change of the UV absorption of MB in Fe3O4@Dex, Fe3O4@Dex + H2O2 and Fe3O4@Dex + H2O2 + L groups because Fe3O4 could not be decomposed at pH 7.4. The UV absorption of MB in Fe3O4@Dex + H2O2 group at pH


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6.5 decreased to 94.8%, which was lower than that of the above groups. The reason can be mainly ascribed to that the Fe3O4 was decomposed into Fe2+/Fe3+, which reacted with H2O2 to produce ·OH, further reacted with MB. Compared with FDPSP NPs + H2O2 + L group (decreased to 85.9%), the UV absorption of MB in FDPSP NPs + H2O2 + L group at pH 6.5 decreased to 75.39%, which is mainly due to the fact that the Fe3O4 was decomposed under acidic conditions to produce Fe2+, which could react with H2O2 to produce not only ·OH but also O2. The ·OH was highly oxidative and the produced O2 can serve as the raw material for PDT to overcome the tumor oxygen consumption. To further validate this point of view, the electron spin resonance (ESR) was used to verify the exact type of ROS generated in the PDT process under different conditions in Figure S11. Thus, the Fenton reaction could enhance the PDT effect. The ROS generation efficiency of all the materials in the cell was then confirmed by ROS probe, 2′,7′-Dichlorofluorescin diacetate (DCFH-DA). From the flow cytometric (FCM) evaluation in Figure 2c, it can be seen that the mean fluorescence intensity, which stands for ROS concentration, is sharply increased in mouse breast tumor cells (4T1) treated by Fe3O4@Dex, Fe3O4@Dex-TPP/PpIX/mPEG and Fe3O4@Dex-TPP/PpIX/ss-mPEG in contrast to the control group without the treatment of the nanoparticles, which can be ascribed to the generation of ·OH by Fenton reaction. Moreover, the mean fluorescence intensity of ROS in Fe3O4@Dex-TPP/PpIX/mPEG + L and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group was approximately 1.5 times higher than PpIX + L group, and was 4-5 times higher than that of the Fe3O4@Dex, Fe3O4@Dex-TPP/PpIX/mPEG and Fe3O4@Dex-TPP/PpIX/ss-mPEG groups without



laser irradiation due to the generation of both ·OH and 1O2. The result further indicates that the Fenton reaction with the multifunctional magnetic nanoparticles can enhance the PDT effect through the generation of more ROS. The fluorescence microscopy (FM) images in Figure 2d also show that the related ROS intensity of Fe3O4@DexTPP/PpIX/mPEG + L and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group was significantly stronger than that of other groups, which was consistent with the results obtained by FCM (Figure 2c). Cellular Uptake and Lysosomal Escape Assays. The endocytosis and PCI effect of these nanoparticles in tumor cells were further studied. Frist of all, the endocytosis effect was determined by flow cytometry (FCM). As shown in Figure 3a, it can be seen that the fluorescence intensity of PpIX reached the maximum after four-hour coincubation, providing a time reference for the subsequent cell characterization. For the mitochondria targeting of nanoparticles, it is a critical step to escape from the lysosome into the cytoplasm. In Figure 3b, FM was employed to investigate the lysosome escape. In Fe3O4@Dex-TPP/PpIX/ss-mPEG group, the large overlapping area of red (PpIX) and green (LysoTracker) fluorescence suggests that the nanoparticles still existed in lysosomes without the laser irradiation. In Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group, few overlapping areas can be observed, which is owing to that the nanoparticles successfully escaped into cytoplasm with the destruction of lysosomes after exposed to 637 nm laser, exhibiting a good PCI effect. The red fluorescence of Fe3O4@DexTPP/PpIX/ss-mPEG + L group presented as granular probably because Fe3O4@DexTPP/PpIX/ss-mPEG targeted to the mitochondrial after internalized into the cytoplasm.


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For further semi-quantitative analysis of lysosomal escape effect, the merged images were analyzed by Image Pro Plus, as shown in Figure 3c. The data showed that the Pearson coefficient of red and green fluorescence of Fe3O4@Dex-TPP/PpIX/ss-mPEG and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group were 0.7986 and 0.2972, respectively, the good PCI effect was proved more accurately. Mitochondrial Targeting and Damage Analysis. To verify whether the nanoparticles can target mitochondria after they enter cells, FM was used to analyze them. As shown in Figure 4a, the red fluorescence was dispersed in 4T1 cells of Fe3O4@Dex-TPP/PpIX/mPEG + L group which has no mitochondria-targeted ability, while the red and green fluorescence of Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group was coincident well, suggesting that the mitochondrial targeting effect of Fe3O4@DexTPP/PpIX/ss-mPEG is good. The co-localization effect of Fe3O4@Dex-TPP/PpIX/ssmPEG and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group was analyzed by Image Pro Plus (Figure 4b). The Pearson coefficient of red and green fluorescence were calculated to be 0.2317 and 0.7692, respectively, indicating that Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group has a good mitochondrial targeting effect. After that, the mitochondrial damage was detected by 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1). When the mitochondrial membrane potential was high, JC-1 would aggregate into the matrix of the mitochondria to form a polymer (J-aggregate), and red fluorescence could be observed by FM. Once the mitochondria were destroyed, JC-1 could not aggregate in mitochondria matrix, green fluorescence could be observed for JC-1 monomer. In Figure 4c, compared with the control group, the red fluorescence



gradually weakened and the green fluorescence gradually increased in Fe3O4@DexTPP/PpIX/ss-mPEG and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group, indicating that the mitochondria were destroyed by PDT, especially in the Fe3O4@Dex-TPP/PpIX/ssmPEG + L group. The result also confirmed the mitochondrial targeting effect of Fe3O4@Dex-TPP/PpIX/ss-mPEG. To further investigate the mitochondrial apoptosis, the relevant protein content in the tumor cells was analyzed by western blot (WB). As shown in Figure 4d, compared with the control group, the procaspase-3 of Fe3O4@Dex-TPP/PpIX/mPEG + L and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L groups was down-regulating, while the cleaved caspase-3 was up-regulating in Fe3O4@Dex-TPP/PpIX/mPEG + L and Fe3O4@DexTPP/PpIX/ss-mPEG + L groups, especially in Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group. These results demonstrated that the ROS produced by the Fenton reaction assisted by PDT led to the occurrence of mitochondrial apoptosis pathway, moreover, a better therapeutic efficacy could be achieved due to the mitochondria targeting of the nanoparticles. The cytochrome C expression was up-regulating especially in Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group, indicating that the mitochondrial membrane permeability was increased and the cytochrome C was released into the cytoplasm due to the mitochondrial apoptosis. The released cytochrome C can induce tumor cells death. In Vitro Cytotoxicity assays. To assess the biocompatibility of the nanoparticles, the cytotoxicity was analyzed using normal human endothelial cells (EC) and murine breast tumor (4T1) cells. As shown in Figure 5a and 5b, the cytotoxicity of Fe3O4@Dex-


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TPP/PpIX/ss-mPEG against EC and 4T1 cells with/without 637 nm laser was analyzed, respectively. The results show that the toxicity of Fe3O4@Dex-TPP/PpIX/ss-mPEG without 637 nm laser irradiation was negligible (cell viability > 90%) for EC, but it was toxic for 4T1 cells as the concentration increased. When the concentration of Fe3O4@Dex-TPP/PpIX/ss-mPEG was higher than 200 μg/mL (concentration of PpIX was 20 μg/mL), the cell viability of 4T1 cells was ~77%, which might be due to the higher level of H2O2 in tumor cells than that in normal cells.8, 45 A higher concentration of H2O2 means that a greater amount of Fenton reaction occurred, causing more damage to the 4T1 cells. The cell viability of tumor cells with 637 nm laser irradiation group was lower than 50% when the material concentration was higher than 25 μg/mL (concentration of PpIX was 2.5 μg/mL). The result suggested that the cytocompatibility of Fe3O4@Dex-TPP/PpIX/ss-mPEG was good for normal cells, while it was toxic for tumor cells especially had highly phototoxic. Furthermore, the live/dead staining of Fe3O4@Dex-TPP/PpIX/ss-mPEG for EC and 4T1 cells with/without laser illumination was observed by FM. As shown in Figure 5c, almost no dead cells were observed at all different concentrations in EC group, while a small amount of dead 4T1 cells were observed without laser irradiation when the PpIX concentration was over 10 μg/mL and a large number of dead cells were observed with laser irradiation when the PpIX concentration was over 2.5 μg/mL. These results also demonstrated that the multifunctional nanoparticles have an efficient Fenton reaction in tumor cells and can be employed to enhance the photodynamic therapy for cancer. The half maximal inhibitory concentration (IC50) values of PpIX, PpIX + L, Fe3O4@Dex-TPP/PpIX,



Fe3O4@Dex-TPP/PpIX/mPEG, Fe3O4@Dex-TPP/PpIX/mPEG + L, Fe3O4@DexTPP/PpIX/ss-mPEG and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L were then determined by Alamar Blue (AB) assay. The results were summarized in Figure 5d and Table S1. We could find that the IC50 values of Fe3O4@Dex-TPP/PpIX, Fe3O4@DexTPP/PpIX/mPEG and Fe3O4@Dex-TPP/PpIX/ss-mPEG were 30.829 μg/mL, 35.722 μg/mL and 32.250 μg/mL, respectively, indicating that the Fenton reaction alone was toxic to tumor cells with a high concentration (> 30 μg/mL). And such a high concentration cannot be achieved in the body. While the IC50 values of PpIX + L, Fe3O4@Dex-TPP/PpIX/mPEG + L and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L were 11.352 μg/mL, 8.375 μg/mL and 2.6167 μg/mL, respectively. The IC50 of Fe3O4@DexTPP/PpIX/ss-mPEG + L group (combination of Fenton reaction and PDT) was ~12.32fold lower than that in Fe3O4@Dex-TPP/PpIX/ss-mPEG group (single Fenton reaction), indicating that light irradiation can produce ROS, which could improve anti-tumor effect of Fe3O4@Dex-TPP/PpIX/ss-mPEG. In addition, compared with PpIX + L group, the IC50 of Fe3O4@Dex-TPP/PpIX/mPEG + L group was lower, demonstrating that Fenton reaction could enhance the therapeutic effect of PDT. While the IC50 value of Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group was even lower, suggesting that once the nanoparticles targeted to mitochondria, the PDT effect would be further improved. MR Imaging Effect Analyses. Magnetic resonance scanner was employed to investigate whether the multifunctional magnetic nanoparticles (Fe3O4@DexTPP/PpIX/ss-mPEG) still remain the MRI effect of the superparamagnetic Fe3O4.67 As shown in Figure 6a, the longitudinal relaxation rate (r1) and the transverse relaxation


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rate (r2) were calculated to be 3.364 mM-1s-1 and 72.998 mM-1s-1, respectively. The MR behavior of nanoparticles depended on the relaxivity ratio. If r2/r1 ≥ 10, the material was more suitable for T2 contrast, and T1 contrast was widely used when r2/r1 < 2.68 The relaxivity ratio of Fe3O4@Dex-TPP/PpIX/ss-mPEG was about 21.7, indicating that they can be used for T2 imaging. Then, the intracellular MR imaging was studied as shown in Figure 6b. MR signal intensity was gradually reduced to half of the original within the first four hours, exhibiting a good MRI effect of Fe3O4@Dex-TPP/PpIX/ssmPEG in tumor cells. After that, the in vivo MR imaging of tumor model was observed. As shown in Figure 6c, it was clear that the MR signal of 4T1 tumor model gradually decreased with time after post intravenous injection, and reduced to the minimum at 6 h, indicating that Fe3O4@Dex-TPP/PpIX/ss-mPEG had a good MR imaging effect. In Vivo Anti-tumor Efficacy Study. The in vivo anti-tumor effect of the materials were studied on 4T1 tumor-bearing Balb/c mice. The images of tumor-bearing mice at day 0 and 14th day were showed in Figure S12 and Figure 7a. It could be clearly seen that the tumor size of all mice were almost same at day 0, while the growth of the tumor in Fe3O4@Dex-TPP/PpIX/mPEG + L group and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group were inhibited obviously at 14th day compared with other groups. After the tumors were stripped from the mice at 14th day, the tumor volume can be visually observed as shown in Figure 7b. And the results were consistent with Figure 7a. It could be clearly seen that the tumor volume of Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group was the smallest, indicating that the therapeutic effect was better than other groups. The tumor volume and body weight of all mice were recorded every two days after the first



injection as shown in Figure 7c and 7d. Compared with saline group (increased ~8 times), the tumor volume of Fe3O4@Dex-TPP, Fe3O4@Dex-TPP/PpIX/mPEG, Fe3O4@Dex-TPP/PpIX/ss-mPEG group were increased ~5.6 times, confirming the weak anti-tumor effect of the Fenton reaction alone. While the tumor volume of Fe3O4@Dex-TPP/PpIX/mPEG + L and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L groups were only increased 3.55 and 2.43 times, indicating that the therapeutic effect was greatly increased when the Fenton reaction was combined with PDT. And once the nanoparticles targeted to the mitochondria, the therapeutic effect would be further improved. There were no significant differences between different groups in body weight (p > 0.05), suggesting that all materials had minimal systemic toxicity. After that, the tumor growth inhibition rate was calculated in Figure 7e. The inhibition rate of Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group was almost 4 times than that of Fe3O4@Dex-TPP and Fe3O4@Dex-TPP/PpIX/mPEG (p < 0.001) group, and about 1.2 times than that of Fe3O4@Dex-TPP/PpIX/mPEG + (p < 0.01) group, also indicating that the Fe3O4@Dex-TPP/PpIX/ss-mPEG with laser irradiation exhibits the best antitumor effect among all groups. After that, the anti-tumor effect was further analyzed through histological and immunohistochemical analyses. The tumor tissue sections were analyzed by hematoxylin-eosin (H&E) staining, transferase mediated UTP end labeling (TUNEL) and Ki67 staining, respectively, as presented in Figure 7f. It could be clearly seen that tumor cells of the saline group were dense, indicating that the growth of tumors was not interfered in the absence of treatment. The results showed no significant difference


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between

Fe3O4@Dex-TPP,

Fe3O4@Dex-TPP/PpIX/mPEG

and

Fe3O4@Dex-

TPP/PpIX/ss-mPEG groups. However, to Fe3O4@Dex-TPP/PpIX/mPEG + L and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group, a large amount of tumor cells were found to be apoptotic, nuclear contraction and decomposition, especially in the Fe3O4@DexTPP/PpIX/ss-mPEG + L group. The main organs of heart, liver, spleen, lung and kidney were also analyzed by H&E staining to verify the toxicity of materials to normal tissues in vivo. As shown in Figure S13, no significant difference in H&E staining of the main organs of heart, liver, spleen, lung and kidney were observed, indicating that there were no physiological and morphological changes for all groups. Therefore, it can be concluded from the above data that all the groups have no systemic toxicity to major organs. In the TUNEL and Ki67 analysis, a small number of proliferative cells and many apoptotic cells were observed in Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group. All the results suggested that Fe3O4@Dex-TPP/PpIX/ss-mPEG with 637 nm laser illumination had the strongest anti-tumor ability compared to other groups.

CONCLUSION

In summary, we have developed a new multifunctional nanoplatform for enhanced photodynamic therapy for cancer. This nanoplatform can catalyze the in situ decomposition of the over-expressed H2O2 in tumor cells via Fenton reaction to produce ·OH and O2, increasing the oxygen concentration as raw material for continuously exerting PDT. It has also a great capacity of overcoming the drawback of the short half-life of reactive oxygen species (ROS) through the mitochondrial targeting,



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highly effectively prompting mitochondrial apoptosis, finally significantly improving the PDT effect. In addition, the excellent Magnetic Resonance Imaging ability was still remained, making it be able to real-time monitor the tumor. Therefore, this multifunctional

nanoplatform

overcomes

the

limitations

of

the traditional

photodynamic therapy, provides a new strategy for enhanced cancer therapy.

EXPERIMENTAL SECTION Materials. Iron (III) chloride hexahydrate (FeCl3·6H2O), ferrous chloride tetrahydrate (FeCl2·4H2O), ammonium hydroxide (NH3·H2O) and dextran (Dex, Mn=70000 Da) were purchased from Aladdin-Reagent Co. Ltd. (China). TPP, 3,3'disulfanediyldipropionic acid, succinic anhydride and 6-Bromohexanoic acid were bought from Biokem Chemical Reagent Co. (Chengdu, China). Methoxypolyethylene glycols (mPEG, Mn=1900 Da) was purchased from Shanghai Ponsure Biotech, Inc. (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl), Protoporphyrin IX (PpIX), N, N'-diisopropylcarbodiimide (DIC) and 4dimethylaminopyridine (DMAP) were obtained from Sigma-Aldrich, America. 2',7'Dichlorodihydrofluorescein

diacetate

(DCFH-DA),

Mito-Tracker

Green

and

LysoTracker® Green DND-26 were purchased from Beyotime Biotechnololy (Shanghai, China). All other reagents were obtained from Kelong Chemical Reagent Ltd. (Chengdu, China) without further process. Synthesis of (5-Carboxypentyl) Triphenylphosphonium Bromide (TPP-COOH). 4.00 g 6-bromohexanoic acid (21.50 mmol) and 5.60 g TPP (21.49 mmol) were


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weighed and put into a 250 mL round bottom. Then, 80 mL of acetonitrile was added with stir at 90 ℃ for 24 h. The reaction process was checked by thin-layer chromatography (TLC). Once the reaction was completed, the product was precipitated by ethyl acetate, and then dried in vacuo later. The product was white power. (Yield: 98%) Synthesis of mPEG-COOH. 7.60 g mPEG1900 (4.00 mmol) and 2.00 g succinic anhydride (20.00 mmol) were dissolved in dichloromethane (80 mL), and then stirred and heated to 30 ℃. Checking the progress of the reaction by TLC. Once the mPEG was consumed completely, the unreacted succinic anhydride was washed by water. The organic phase of the extraction was condensed by rotary evaporator, and then the product (white powder) was precipitated by ether. (Yield: 93%) Synthesis of mPEG-ss-COOH. 2.50 g mPEG1900 (1.30 mmol) and 0.56 g 3,3'disulfanediyldipropionic acid (2.60 mmol) were added to a 50 mL round bottom flask with 30 mL dichloromethane. Then 0.34 g DIC and 0.35 g DMAP were added, stirred to dissolve and reacted at room temperature for 48 h. The progress of the reaction was investigated by TLC. Once the mPEG-ss-COOH was exhausted, the DMAP and 3,3'disulfanediyldipropionic acid was removed by weak acidic water (pH = 5.0). The product was extracted by dichloromethane, and then it was precipitated by diethyl ether. The product was light yellow powder and it was dried in vacuo. (Yield: 78%) Synthesis of Dex-TPP. 5.00 g Dex was added into a 150 mL round bottom flask, 50 mL of dimethyl sulfoxide (DMSO) was added and stirred to dissolve. Then 5.36 g TPPCOOH, 3.16 g DIC and 3.25 g DMAP were added. The reaction was checked by TLC.



Once the reaction finished, the reaction solution was dialyzed (molecular weight cut off (MWCO) = 3500) for 2 days. After that, the dialysate was lyophilized to obtain the product (white powder). (Yield: 80%) Preparation of Fe3O4@Dex-TPP. 2.00 g Dex-TPP was dissolved in 100 mL water under nitrogen atmosphere with mechanical stir (500 rpm/min) at room temperature. After the reactant was dissolved, raise the temperature to 60 ℃ and keep it for 10 mins to eliminate the dissolved oxygen. Then, 0.40 g FeCl3·6H2O and 0.16 g FeCl2·4H2O were added in 10 mL of NH3·H2O (w/w 7.5%) with the agitator was opened one min every 10 mins for 1 h. Then, the product was centrifuged at 4000 rpm for 5 mins, repeated three times. The larger particles were discarded. Finally, the product was collected by dialysis (MWCO = 50000) and lyophilize. Preparation of Fe3O4@Dex-TPP/PpIX. 100.00 mg Fe3O4@Dex-TPP was dispersed in 100 mL DMSO. Then, 40.00 mg PpIX, 7.69 mg EDC·HCl, 7.92 mg DMAP and 100 μL triethylamine were added with stir at room temperature for 48 h. Then it was dialyzed (MWCO = 3500) for 2 days and lyophilized. Preparation of Fe3O4@Dex-TPP/PpIX/mPEG. 50.00 mg Fe3O4@Dex-TPP/PpIX was dispersed in 50 mL DMSO. After that, 20.00 mg mPEG-COOH, 2.30 mg EDC·HCl and 2.40 mg DMAP were added. After reacted at room temperature for 48 h, the liquid was dialyzed (MWCO = 3500) for 2 days and lyophilized. Preparation of Fe3O4@Dex-TPP/PpIX/ss-mPEG. The preparation procedure was similar to the synthesis process of Fe3O4@Dex-TPP/PpIX/mPEG. Characterizations of Materials. 1H-NMR of each sample was detected by Nuclear


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magnetic resonance spectrometer (Bruker AM 300 apparatus) with tetramethylsilane (TMS) as internal reference. FT-IR spectra were obtained by infrared spectrometer (FTIR, Nicolet 5700). The hydrodynamic diameter and zeta potential of nanoparticles were determined by DLS (Zeta-sizer Nano-ZS90, Malvern Instruments, U.K.) at 25 ℃. TG curve of the nanoparticle was obtained by Thermal Gravimetric Analyzer (TGA, Netzsch STA 449C, Bavaria, Germany), at the condition of 0 ℃ to 800 ℃ with a heating rate of 10 ℃/min under nitrogen atmosphere. TEM (H-700H, HITACHI) observation of all nanoparticles were operated at 200 kV. The morphology of all the nanoparticles was further determined by AFM (CSPM5000, Being). Drug-loading content (DLC) of the materials was measured by UV-vis spectrophotometer (UV-2550, Shimadzu, Japan). ESR spectrum were acquired by JEOL JES-FA200 (ESR/EPR). Saturation magnetic intensity of all the nanoparticles were measured at 300 K by VSM (JDM-13, Quantum Design). The crystal structure of the material was measured by XRD (XL-30, Phlips X’Pert PRO). Cell Culture. Mouse breast cancer cell (4T1) and endothelial cells (EC) were obtained from Sichuan University (China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) at 37 ℃ in a 5% CO2 atmosphere and 100% humidity. Animals. The animals were SPF female mice Balb/c (5-6 weeks old and body weight = 20 ± 2 g), which were purchased from the Animal Experimental Center of Sichuan University. To establish a tumor model, in vitro cultured 4T1 cells were injected into the fat pad



of the mouse breast in the number of 1 × 106 cells in 100 μL. After 10 days of inoculation, the breast model of the mouse was visible and the volume was approximately 50 mm3 (the long diameter (a) and the short diameter (b) of the tumor were measured by a Vernier caliper, the volume was calculated according to the volume formula of the tumor: V = a*b2/2), and this day was set as day 0. Characterization of ROS. The 1O2 and ·OH produced by the materials were tested by the methods reported in the previous literature.45,69,70 DPBF was used to measure extracellular 1O2 produced by PDT. 1 μL of 1 M H2O2 and 40 μL of 10 mM DPBF ethanol solution were dispersed in 4 mL of 200 μg/mL Fe3O4@Dex-TPP/PpIX/ss-mPEG solution. The pH value of each group was adjusted to 6.5, then irradiated with/without 637 nm laser (100 mW/cm2) for different time. The change in UV absorbance of DPBF was measured by UV-vis. The ·OH generated by Fenton reaction was detected by methylene blue (MB). 0.30 mg Fe3O4@Dex and 0.60 mg Fe3O4@Dex-TPP/PpIX/ss-mPEG were separately added to 3 mL of 8 μg/mL MB solution. Then, each group was irradiated with/without 637 nm laser (100 mW/cm2) for different time, the change in UV absorbance at 663 nm of MB was measured by UV-vis to semi-quantitative analysis the amount of ·OH. With trapping agent 2,2,6,6-Tetramethylpiperidine (TEMP), different types of ROS generation were detected by ESR. 10 mM H2O2 was dispersed in 4 mL of 200 μg/mL Fe3O4@Dex-TPP/PpIX/ss-mPEG solution. The pH values of some groups were adjusted to 6.5, then irradiated with/without 637 nm laser (100 mW/cm2). Characterization of Intracellular ROS Production. Intracellular ROS production


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was then characterized by FCM and FM. 4T1 cells were inoculated in two 6-well plates at a density of 5 x 104 cells per well. After 24 h, they were treated with PBS, 40 μg/mL of PpIX, 400 μg/mL of Fe3O4@Dex-TPP, Fe3O4@Dex-TPP/PpIX/mPEG and Fe3O4@Dex-TPP/PpIX/ss-mPEG for 6 h. Then, ROS probe of DCFH-DA was added for 30 mins. After that, the Fe3O4@Dex-TPP/PpIX/mPEG + L and Fe3O4@DexTPP/PpIX/ss-mPEG + L group were irradiated with 637 nm laser (100 mW/cm2) for 10 mins. One of the 6-well plates was measured by FCM, and another one was observed by FM. Cellular Uptake Study. To investigate cellular uptake of Fe3O4@Dex-TPP/PpIX/ssmPEG, the 4T1 cells were inoculated in 6-well plates at a density of 1 x 105 cells/well for 24 h. The cells were treated with 200 μg/mL Fe3O4@Dex-TPP/PpIX/ss-mPEG for 0, 1, 2 and 4 h, respectively. Then, the fluorescence intensity of PpIX was measured by FCM. Investigation of Lysosome Escape. After Fe3O4@Dex-TPP/PpIX/ss-mPEG were internalized by endocytosis, the lysosome escape behavior with/without illumination was investigated. First of all, the 4T1 cells were planted in 6-well plates for 24 h at a density of 5 x 104 cells per well. Then, 200 μg/mL of materials was co-cultured for 2 h with the Fe3O4@Dex-TPP/PpIX/ss-mPEG + L group illuminated by 635 nm laser (100 mW/cm2) for 10 min. Thereafter, all the cells were stained with LysoTracker Green (50 nM) and analyzed by FM. The co-localization effect was analyzed by Image Pro Plus. Investigation of Mitochondrial Targeting. Once the nanoparticles escaped from lysosome, the mitochondrial targeting behavior was investigated. After co-cultured



with different materials for 4 h, all the cells were stained by MitoTracker Green (50 nM) and analyzed by FM. Investigation of Mitochondrial Membrane Potential. Once the nanoparticles targeted to mitochondrial membrane, the mitochondrial damage effect was analyzed through the change of mitochondrial membrane potential which were demonstrated by JC-1. The cell culture process was as same as the investigation of mitochondrial targeting. Finally, all the group were stained by JC-1 (2.5 µg/mL) for 30 min and analyzed by FM. WB Analysis. 4T1 cells were inoculated at a density of 1 x 106 cells/well in 6-well plates and incubated for 24 h. Then, cells were treated with saline, 100 μg/mL Fe3O4@Dex-TPP/PpIX/mPEG + L and Fe3O4@Dex-TPP/PpIX/ss-mPEG + L for 3 h, respectively. Then, the Fe3O4@Dex-TPP/PpIX/mPEG + L and Fe3O4@DexTPP/PpIX/ss-mPEG + L groups were exposed to 637 nm laser (100 mW/cm2) for 10 mins. After continuing incubated for 4 h, the cells were washed by PBS for 3 times, and then the cells were trypsinized to be samples. In Vitro Cytotoxicity. The in vitro cytotoxicity of Fe3O4@Dex-TPP/PpIX/ss-mPEG was detected by the AB assay and live/dead staining. 4T1 cells and EC were inoculated in 96-well plates at a density of 1 x 103 cells/well for 24 h. Then, different concentrations of Fe3O4@Dex-TPP/PpIX/ss-mPEG were added into the 96-well plate. After co-cultured for 3 h, one of the 96-well plate was exposed to 637 nm (100 mW/cm2) laser for 10 mins and then cultured for 24 h. After that, the medium was removed from the 96-well plate, the residual nanoparticles were washed away by PBS for 3 times.


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Then, 300 μL of AB solution was added into the 96-well plate for 4 h, and then detected by Enzyme-labeled Instrument. For live/dead staining, 4T1 cells and EC were inoculated in 6-well plates at a density of 1 x 105 cells/well and incubated for 24 h. The plate was also treated in the same way as mentioned in the part of in vitro cytotoxicity. Then, the live cells were stained by 600 μL of 2 mM calcein acetoxymethylester (calcein-AM) for 15 mins (green fluorescence). After that, calcein-AM was removed from the plate, the dead cells were stained by 600 μL of 2 mM propidium Iodide (PI) for 15 mins (red fluorescence). Finally, the live and dead cells were observed by FM. MR Imaging. First of all, T2 relaxation rate (1/ T2) and T1 relaxation rate (1/ T1) of the Fe3O4@Dex-TPP/PpIX/ss-mPEG with different concentrations of 0, 0.1, 0.2, 0.4, and 0.8 mM (Fe concentration) were measured by MR (1.5T, Seimens Sonata, Germany). The parameters were: TR=5000 ms, TE=8.7 ms. 4T1 cells were inoculated in 6-well plates at a density of 1 x 105 cells/well and incubated for 12 h. After that, 200 μg/mL of Fe3O4@Dex-TPP/PpIX/ss-mPEG were added into the 6-well plate at different time points (0, 2, 3, 3.5 and 4 h). Subsequently, the residual nanoparticles were removed from the 6-well plate, and washed with PBS for 3 times. 800 μL trypsin was added in each well and digested for 2 mins in the incubator. After that, 1 mL medium was added to terminate digestion, and then centrifuged at 1200 rpm for 4 mins. The supernatant was discarded, 500 μL agarose (w/w 1%) was added to disperse cells, and then the samples were measured by MRI. The parameters were: TR=2500 ms, TE=40.3 ms. Finally, in vivo MRI was examined. 200 μg/mL of Fe3O4@Dex-TPP/PpIX/ss-mPEG



Page 28 of 44

were injected into four mice intravenously. The MR imaging of mice were observed at 0, 0.5, 1, 2, 4 and 6 h. The T2-weighted SE PRPELLER sequence was subjected to T2weighted scan of the tumor site, and the signal intensity of the tumor site was semiquantitatively analyzed. The parameters were: TR=3000 ms, TE=93 ms, slice thickness= 1 mm, slice number = 11. In Vivo Antitumor Activity. Once the tumor volume grows to 50 mm3, all the mice were randomly assigned into six groups (n = 5). Then, different groups of mice were treated with saline, Fe3O4@Dex-TPP, Fe3O4@Dex-TPP/PpIX/mPEG, Fe3O4@DexTPP/PpIX/mPEG

+

L,

Fe3O4@Dex-TPP/PpIX/ss-mPEG

and

Fe3O4@Dex-

TPP/PpIX/ss-mPEG + L (at the dose of 5 mg/kg of PpIX), respectively. Fe3O4@DexTPP/PpIX/mPEG

+

L,

Fe3O4@Dex-TPP/PpIX/ss-mPEG

and

Fe3O4@Dex-

TPP/PpIX/ss-mPEG + L groups were exposed to 637 nm laser for 10 mins after 6 h of injection. All the mice were treated in the same way at 0th, 3th, 6th and 9th days. The body weight and the volume of the tumor were recorded every 2 days after the first injection. The results were processed by Origin 2018. Histological Analyses. After 14 days of treatments, all the mice were dissected. The heart, liver, spleen, lung, kidney and tumors were fixed by paraffin wax. Then, the tissues were cut into 5-μm thick sections and the tumor sections were stained by hematoxylin and eosin (H&E), Ki-67 and TUNEL assay while the tissue sections were only stained by H&E. After that, all the sections were observed by optical microscope. Statistical Analysis. Statistic Package for Social Science (SPSS) was used to analysis the statistical data. Data was expressed as means ±SD. One-way ANOVA was


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performed to determine the statistical significance of the data. Survival analyses were done according to the Kaplan-Meier method. The differences were considered significant for p values: # > 0.05, * < 0.05, **

Fenton Reaction-Assisted Photodynamic Therapy for Cancer with

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