Multifunctional co-loaded magnetic nanocapsules for enhancing targeted MR imaging and in vivo photodynamic therapy

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a Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China Institute of Nano Biomedicine and Engineering, Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, China c Shanghai Engineering Research Center for Intelligent Diagnosis and Treatment Instrument, Shanghai, China d School of Biomedical Engineering, Institute for Personalized Medicine, Shanghai Jiao Tong University, Shanghai, PR China Revised 13 June 2019

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Yuhui Zhang, MSc a, b , Qian Zhang, PhD b, c,⁎, Amin Zhang, PhD b , Shaojun Pan, PhD b , Jin Cheng, PhD b, c , Xiao Zhi, PhD d , Xianting Ding, PhD d , Lixin Hong, MSc a, b , Mei Zi, MSc a , Daxiang Cui, PhD b, c , Jinghua He, PhD a,⁎⁎

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Multifunctional co-loaded magnetic nanocapsules for enhancing targeted MR imaging and in vivo photodynamic therapy

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Abstract

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Drug delivery nanocarriers based on magnetic nanoparticles have attracted increasing attention due to their potential applications in magnetic resonance imaging, photodynamic therapy and targeted drug delivery. Herein, we have fabricated the multifunctional co-loaded magnetic nanocapsules (MNCPs) using a microemulsion process for enhancing targeted magnetic resonance imaging and in vivo photodynamic therapy. MNCPs were synthesized by co-loading Co@Mn magnetic nanoparticles and chlorin e6 into the matrix of an amphiphilic polymer, and further surface covalently coupled with target molecules. This work demonstrates that MNCPs have uniform sizes (dc: ~150 nm), favorable biocompatibility, long-term stability, excellent T2 relaxation values, and high drug loading efficiency. These advantages offer MNCPs successfully applied in targeted magnetic resonance imaging, real-time fluorescent labeling, and photodynamic therapy. The research results will contribute to rationally design novel nano-platform and provide a promising approach for further clinical integration of diagnosis and treatment in the near future (Graphical Abstract: Figure 1). © 2019 Elsevier Inc. All rights reserved.

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Key words: Magnetic nanoparticles; Co-loaded magnetic nanocapsules; Fluorescent labeling; Magnetic resonance imaging; Photodynamic therapy

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Recently, the population of cancer patients has been gradually increasing, while cancer-induced death rates remain at a high level. 1,2 The standard cancer treatment strategies, such as surgery,

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Notes: The authors declare no competing financial interest. Acknowledgments: We thank the support from Shanghai Sailing Program (No. 19YF1422300), Shanghai Jiao Tong University (No. 18X100040044), Shanghai Jiao Tong University “Transformation Medicine Cross Research Fund” (No. ZH2018QNB25, ZH2018QNB26), and Shanghai Engineering Research Center for Intelligent Diagnosis and Treatment Instrument (No. 15DZ2252000). ⁎Correspondence to: Q. Zhang, Institute of Nano Biomedicine and Engineering, Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, China. ⁎⁎Corresponding author. E-mail addresses: [email protected], (Q. Zhang), [email protected]. (J. He).

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chemotherapy and radiotherapy, can significantly increase fiveyear survival rate, yet many disadvantages associated with these procedures, including high pain levels, limited tumor enrichment, and high rate of relapse, 3,4 have limited their clinical applications. Therefore, many novel therapeutic strategies, such as gene therapy, photothermal therapy (PPT), immunotherapy and photodynamic therapy (PDT), 5–7 have attracted widespread attentions by researchers. As an emerging clinical treatment that could be widely applied in the biomedical field, PDT exhibits several merits, including high enrichment efficiency of tumor region, simplicity of operation, and minimal damage to normal tissues in peripheral tissues. Under near-infrared laser irradiation, the photosensitizer molecules could be stimulated to produce singlet oxygen, thereby irreversible damage to the tumor tissue, and finally achieve therapeutic purposes. 8–10 Several studies have revealed that early gastric cancer is difficult to be detected and can easily lead to the development of

https://doi.org/10.1016/j.nano.2019.102047 1549-9634/© 2019 Elsevier Inc. All rights reserved. Please cite this article as: Zhang Y., et al., Multifunctional co-loaded magnetic nanocapsules for enhancing targeted MR imaging and in vivo photodynamic therapy.... Nanomedicine: NBM 2019;xx:1-11, https://doi.org/10.1016/j.nano.2019.102047

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Methods

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Synthesis of Co@Mn MNPs and MNCPs

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The fabrication of MNCPs covers four steps. (i) Hydrophobic Co@Mn MNPs were firstly synthesized using a seeds-meditated growth method under a high thermal environment. 19,20 (ii) PMA contains poly(isobutylene-alt-maleic anhydride) and dodecylamine was reacted together following the published protocol. 21,22 (iii) PMA(Co@Mn + Ce6) MNPs were then assembled by using an oil-in water emulsion method modified from the previous studies. 23,24 (iv) At last, 5 mg of EDC and 10 mg of FA dissolved in 1 mL of SBB 9.0 buffer (50 mM sodium borate, pH 9.0) were gradually added into the above sample and kept at 37 °C for 3 h (cf. Scheme S1). 25 The obtained MNCPs were washed twice with Milli-Q water to remove the excess FA with centrifugation (5000 rpm, 10 min). Details were shown in Supporting Information.

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Characterization

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The morphology and sizes of Co@Mn MNPs and MNCPs were recorded by transmission electron microscopy (TEM), high-resolution TEM (HR-TEM, JEM-2100f, Japan) and

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Cell culture

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Human gastric cell line (MGC-803) was purchased from Chinese Academy of Sciences, China. The obtained cells were incubated at 37 °C (5% CO2) in Dulbecco's modified Eagle's medium (HyClone) supplemented with 0.1 mg/mL streptomycin, 100 U/mL penicillin and 10% fetal bovine serum (Gibco).

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Cell viability study and cellular uptake assay

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Cell viability studies with co-incubation of MNCPs in the presence/absence of laser irradiation were evaluated by MTT assay and apoptosis assay, respectively. Cellular uptake assay was investigated using a Leica TCS SP8 confocal laser scanning microscope (CLSM, Leica, Germany) and Flow cytometer (FCM, BD Biosciences, USA), respectively. Details were provided in Supporting Information.

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scanning electron microscopy (SEM, Zeiss, Germany), respectively. Zeta (ζ) potential value and hydrodynamic diameters (DLS) were measured by a Zetasizer Nano (Malvern Instruments Ltd., U.K.). UV–Vis spectra were measured via an UV–vis spectrophotometer (Varian Inc., Palo Alto, CA) and fluorescence spectra were recorded using a fluorescence spectrophotometer (Hitachi FL-4600, Japan). Fourier transform infrared (FTIR) spectroscopy was accomplished by an FTIR spectrometer (IR/ Nicolet 6700) in 4000-500 cm −1 region KBr disc IR spectrum calibrated using polystyrene film. 26,27 The XRD pattern was measured by 3/*D8ADVANCE DA Vinci.

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advanced gastric cancer, resulting in high mortality. 11,12 Thus, early diagnosis and accurate treatment technique development are of great importance. 13,14 In order to significantly enhance therapeutic and diagnostic efficacy, more progressive diagnostic and therapeutic modalities based on nanotechnology have been emerging in biomedical applications, owing to their unique physico-chemical properties, such as large specific surface areas, easy surface modification, biocompatibility and long-term blood circulation. 15 Since the U.S. Food and Drug Administration first applied ferumoxytol agents as an iron supplement for magnetic resonance imaging (MRI) and treatment of iron deficiency, 16 more medical agents based on magnetic nanoparticles (MNPs) have been developed for clinical applications, including targeted drug delivery, MRI contrast agent, magnetic fluid hyperthermia, among others. 17,18 Herein, we have designed novel co-loaded magnetic nanocapsules (MNCPs) using a microemulsion method. MNCPs were fabricated by co-loading with hydrophobic Co@ Mn MNPs and chlorin e6 (Ce6) into the matrix of an amphiphilic polymer (PMA); the exposed carboxyl groups of PMA can be further covalently coupled with gastric-targeted molecules (folic acid, FA) via EDC chemistry. In this system, the embedded Co@Mn MNPs exhibited an enhanced MRI due to their enrichment effect. Ce6 did not only act as an in vivo nearinfrared fluorescence molecule for real-time imaging, but also produced singlet oxygen (O2−·) for PDT therapy under 633 nm laser irradiation. Notably, FA molecules covalently modified on the surface of PMA can specifically target to gastric cancer cells MGC-803 with folate receptors to enhance the targeting efficiency of gastric tumors. Therefore, these MNCPs could achieve enhanced MRI and PDT efficacy, which may have significant benefit for future cancer diagnosis and treatment strategies (shown in Figure 1).

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Animal model

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Female nude mice were purchased from Shanghai LAC Laboratory Animal Co. Ltd., Shanghai, China. The average body weights (mean ± SD) are 20 ± 3 g, and ages are 5-6 weeks. Mice were housed in the animal facility at Shanghai Jiao Tong University. All animal experiments were performed under the direction of the “Guidelines for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH). The gastric tumor-bearing mice were established by injection MGC-803 cells into the right thigh of female nude mice until the tumor volume reached to ~100 mm 3.

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MRI evaluation

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MRI evaluation with r2 values were performed using lowfield nuclear magnetic resonance (NMR, 1.5 T, Niumag, Shanghai, China). Free Co@Mn MNPs with polymer coating were used as control (Figure S1). Details were provided in Supporting Information.

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Tumor-targeting evaluation and PDT therapy

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The biodistribution study for MNCPs in tumor-bearing mice was conducted by collecting real-time fluorescence of Ce6 in MNCPs (λEx: 630 nm, λEm: 700 nm) using the Bruker In Vivo FPRO imaging system (Billerica, MA, USA). For PDT therapy, the tumor-bearing mice were randomly divided into three groups: treated with MNCPs, free Ce6, and control group (100 μL of PBS). After injection for 24 h, half of them were treated with 633 nm laser at 50 mW/cm 2 for 30 min, and no

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Figure 1. Schematic illustration of MNCPs for enhancing targeted MR imaging and in vivo photodynamic therapy. 3

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Results

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Synthesis and characterization

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TEM, SEM and HR-TEM micrographs of Co@Mn MNPs in Figure 2, A-C showed that the obtained nanoparticles exhibit spherical structure, uniform size distribution and excellent atomic arrangement. The size histogram by counting the particles from TEM image showed that the average diameter size was 14.3 ± 2.1 nm (Figure 2, D). The EDS and XRD spectra further verified their structures (cf. Figure S2-3). TEM and SEM images of MNCPs in Figure 2, E-G exhibited the monodisperse spherical capsules with highly uniform sizes, and the Co@Mn MNPs were well wrapped into the capsules; the collected average diameter size was about around 151 ± 35 nm (Figure 2, H), which the system then verified by FTIR spectra (cf. Figure S4). UV–Vis spectra of free Ce6, FA, Co@Mn MNPs, PMA (Co@Mn + Ce6) and MNCPs were shown in Figure 3, A. The absorption peaks of Ce6 and FA were at 665 nm and 283 nm, which also appeared in the spectra of MNCPs, indicating the successful fabrication of MNCPs. Figure 3, B exhibited that there were evident fluorescence peaks of free Ce6 and MNCPs at 669 and 663 nm, respectively. Moreover, the Ce6 release in various pH conditions over time was investigated by fluorescence spectroscopy in Figure 3, C. The result demonstrated that Ce6 was gradually released within 24 h, and fluorescence intensity in pH 4.6 had significantly enhanced over time,

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meaning the MNCPs were acidic-sensitive system. DLS value of all the samples (Co@Mn, PMA (Co@Mn), PMA(Co@ Mn + Ce6) and MNCPs) were 16.1 ± 2.3 nm, 146.75 ± 5.07 nm, 162.16 ± 9.05 nm and 154.31 ± 7.83 nm, respectively (Figure 3, D). Meanwhile, their ζ-potential values were 0, −25.3 ± 3.05, −23.5 ± 3.4 and −34.9 ± 5.8 mV, respectively (Figure 3, E). In addition, analysis of the hydrodynamic diameter of MNCPs over time showed no obvious changes in Figure 3, F, indicating the commendable long-term stability. Moreover, based on the calibration curve of Ce6 (cf. Figure S5), the encapsulation efficiency (EE%) and Ce6 loading% in Table 1 approximately were up to 97% and 3.3%, illustrating that MNCPs have the ability to accommodate a large number of Ce6.

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Cellular uptake assay

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Cellular uptake and quantification analysis of MNCPs in MGC-803 cells were investigated using FCM and CLSM, respectively. As shown in Figure 4, A, the red fluorescence signal excited from Ce6 was significantly enhanced from 1 to 8 h, demonstrating that the uptake efficiency of MNCPs increased over time. Their relevant fluorescence signals using FCM collected from the FLH-3 channel gradually increased with incubation time, with the value increasing from 45 to 105. By contrast, the cellular uptake of no FA targeted MNCPs had lower red signal, indicating the targeting effect of FA enhanced the cell endocytosis (cf. Figure S6). Besides, as shown in the cell-TEM image (Figure 4, B), many MNCPs with a complete spherical structures were well distributed in lysosomes (red arrow), and meanwhile plenty of monodisperse Co@Mn MNPs sustainably released from the ruptured MNCPs were also clearly represented (blue arrow).

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laser treated mice were considered as control group. Then the tumor growth, weight changes, survival rate, and hematoxylin and eosin (H&E) staining were recorded to investigate the PDT efficacy. Details were shown in Supporting Information.

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Figure 2. Synthesis and characterization. (A) TEM, (B) SEM and (C) HR-TEM micrographs of Co@Mn MNPs evaporated from chloroform. (D) Size histogram of Co@Mn MNP counted by Image J, with a diameter of 14.3 ± 2.1 nm. (E-G) TEM and SEM micrographs of MNCPs evaporated from H2O. (H) Size distribution of MNCPs obtained from TEM measurements using Image J software, with a diameter of 151 ± 35 nm.

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Figure 3. Characterization of MNCPs. (A) UV–Vis spectra of Co@Mn MNPs, Co@Mn-PMA-Ce6 MNPs, free FA, free Ce6 and MNCPs. (B) The fluorescent spectra of free Ce6 and MNCPs. (C) The Ce6 release in different pH conditions over time. (D) DLS and (E) ζ-potential value of Co@Mn MNPs (in chloroform), PMA (Co@Mn) MNPs, PMA (Co@Mn + Ce6) MNPs and MNCPs, respectively. (F) The stability study of MNCPs at room temperature from H2O according to analyze of the hydrodynamic diameter of MNCPs over time.

Table 1 Encapsulation efficiency (EE%) and % loading of Ce6 for different batches of MNCPs.

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Cell viability studies of MNCPs were performed using MTT and apoptosis assays, respectively. According to the MTT assay in the dark (Figure 5, A), both free Ce6 and MNCPs exhibited good cell viability at a dose of 2.5 μg/mL; However, the cell viability with free Ce6 decreased to 30% with a concentration of 20 μg/mL, while that incubated with MNCPs remained above 75% at 80 μg/mL, indicating that MNCPs have better biocompatibility than free Ce6. In comparison, after co-exposure with MNCPs, free Ce6, or mechanical mixture of free Ce6 and no Ce6 loaded nanocapsules (in Figure S7) in the presence of laser irradiation, the cell viability was markedly decreased and inversely proportional to their dosages, particularly the one with MNCPs that were drastically decreased below 30% at 10 μg/mL. This observation indicated that MNCPs had excellent photocytotoxicity properties, thus presenting great potential for PDT treatment. To study cell death induced by MNCPs under laser irradiation, the apoptosis assay was further investigated using flow cytometry. As shown in Figure 5, B, all the cells remained almost consistently in a living state in the dark; but a

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significantly decreased ratio of cells' viabilities occurred when treated with free Ce6 and MNCPs under laser irradiation; particularly, maximum necrosis and apoptosis were measured in the cells incubated with MNCPs under 633 nm laser irradiation, with a value of N90%. This result indicated the MNCPs had good biocompatibility as well as high PDT treatment potential for cancer cells. Moreover, a singlet oxygen detection study in Figure S8 demonstrated that MNCPs had the ability to produce singlet oxygen as free Ce6, which was consistent with the photodamage study results (Figure 5, C).

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MRI evaluation

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T2-weighted MRI images in vitro and in vivo were respectively investigated with their corresponding r2 values for free Co@Mn MNPs and MNCPs. As shown in Figure 6, A-B, the white circles as T2-weighted MRI images gradually appeared darker with the increase in Fe content. Due to the enrichment effect, the MNCPs exhibited a higher r2 value than that of free Co@Mn MNPs, with values of 323.2 mM −1 s −1 and 248.7 mM −1 s −1, respectively. Furthermore, equal amounts of

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Figure 4. Cellular uptake assay. (A) Laser confocal images and FCM (FLH-3 channel) images of MGC-803 cells incubated with MNCPs (5 μg/mL) for 1, 4 and 8 h respectively. (B) Cell-TEM images of MGC-803 cells co-exposure with MNCPs (80 μg/mL) for 24 h. The MNCPs with whole spherical structures in lysosomes (red arrows) and some Co@Mn MNPs released from the ruptured MNCPs (blue arrows). Scale bar: 10 μm.

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Tumor-targeting evaluation

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In the presence of 633 nm laser irradiation, Ce6 exhibit NIR fluorescence to apply for the real-time position detection of the injected MNCPs in MGC-803 tumor-bearing mice in vivo. The free Ce6 and MNCPs with consistent dose were respectively tail vein injected into the MGC-803 tumor-bearing mice, and their time dependent distribution over time was monitored. As shown in Figure 7, NIR fluorescence could be detected all over the body of the mice after post-injection of free Ce6 at 2 h, and rapidly metabolized at 8 h, to the point when almost no fluorescent signal was left at 3 days. In comparison, significant fluorescence

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MNCPs and free Co@Mn MNPs were intravenously injected into the MGC-803 tumor-bearing mice and the MRI images were recorded (time points: pre-injection, 2, 4, 6, 8 and 12 h) for in vivo MRI evaluation. Notably, both MNCPs and free Co@Mn MNPs treated mice have the significant MRI signal intensity changes at the tumor site, while the value decreased into the minimum at 4 h post-injection (Figure 6, C-D).

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intensity of MNCPs treated tumor-bearing mice was observed at 2 h post-injection, demonstrating that MNCPs exhibited relatively high tumor accumulation efficiency. Over time, a decrease in the fluorescence intensity in the areas surrounding the tumor tissue was accompanied by an increased fluorescence intensity at the tumor site. This fluorescence signal maintained its intensity without obvious change over the course of 72 h. To further investigate the MNCPs tumor-targeting efficiency and to estimate the metabolic pathways in vivo, all the mice were anesthetized 72 h post-injection to extract tumor tissue and major organs (lungs, heart, liver, kidneys, and spleen) and recorded using small animal fluorescence imaging. As anticipated, the fluorescence signal in nude mice treated with MNCPs was adequately accumulated at the tumor site, with few nanocapsules being distributed throughout the lung, heart, liver, spleen, and kidney tissue. However, the fluorescence signal in samples treated with free Ce6 was not located at the tumor site. These results demonstrated excellent MNCP tumortargeting efficiency and further confirmed that MNCPs had less hepatotoxicity than that of free Ce6.

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Figure 5. In vitro cellular toxicity study. (A) The MGC-803 viability evaluated by MTT assay after co-incubation with MNCPs and free Ce6 (Ce6 content: 2.580 μg/mL) in the absence or presence of laser irradiation. (B) MGC-803 cell death evaluated by FCM induced by PDT of MNCPs and free Ce6. The photocytotoxicity was carried by 633 nm laser irradiation; the value was acquired via apoptosis assay with Annexin V-FITC and propidium iodide (PI) double staining. (C) Photodamage detection of MGC-803 cells after incubation with PBS, free Ce6 and MNCPs recorded using fluorescence microscopy (double staining with PI and calcein-M). Live cells: green fluorescence of calcein-AM, dead cells: red fluorescence of PI. The data are shown as mean ± SD (n = 3). Scale bar: 500 μm.

In vivo PDT study

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The Ce6 delivered into tumor sites by MNCPs increased the curative effect of drugs, causing tumor cell death under laser irradiation. The tumor volumes in tumor-bearing nude mice were monitored using Vernier calipers over the course of 15 days. As shown in Figure 8, A-B, D, the final PDT efficiency was arrayed as follows: free Ce6 ≈ MNCPs ≈ PBS b free Ce6 (with laser irradiation) b MNCPs (with laser irradiation). The tumor growth of MNCPs treated mice with laser irradiation was significantly inhibited as compared to that treated with PBS, free Ce6, and MNCPs samples (no laser). Interestingly, the mice treated with free Ce6 under laser irradiation also exhibited some inhibition, yet showed a trend of tumor growth at 15 days. As the potential toxicity in vivo is always a major concern in the circulation process of the delivery system, H&E staining of

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main organs was performed to detect potential in vivo toxicity of MNCPs after treatment. As shown in Figure 8, F, the major organs exhibited no apparent abnormalities or lesions 15 days after laser irradiation of MNCPs. To further confirm the toxicity levels in vivo after PDT, body weight over time and survival rate from different treatments were recorded in Figure 8, C and E, respectively. The result showed that there was no evident weight loss in all the groups, and the MNCPs treated mice kept a long survival rate compared to other groups.

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Discussion

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In this study, we have successfully designed novel multifunctional MNCPs using a microemulsion method, which consists of hydrophobic Co@Mn MNPs and photosensitizer

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Figure 6. MRI evaluation. (A) T2-weighted MR images and (B) r2 values (1/T2) of Co@Mn MNPs and MNCPs in H2O with varying of Fe concentration at 25 °C. (C) MR images with (D) their corresponding signal intensities of tumor bearing mice with pre- and post-injection of free Co@Mn MNPs and MNCPs at a time range from 2 to 12 h under 1.5 T magnetic field. The data are shown as mean ± SD (n = 3).

Figure 7. (A) The fluorescence images and biodistribution of MNCPs in vivo and ex vivo investigated by MGC-803 tumor-bearing mice. (B, C) The quantitative analysis of average fluorescence intensity in the tumor sites of mice and the signals of major organs and tumor after intravenous injection MNCPs by real-time observation for 72 h. The data are shown as mean ± SD (n = 3). 313 314 315

Ce6 into the matrix of an amphiphilic polymer, with further covalent coupling with gastric-targeted molecules FA. The collected MNCPs have a mean ± SD diameter of about 151 ±

35 nm, with a smooth spherical shape and highly uniform sizes. Compared to the previous studies, 28,29 the Ce6 molecules wrapped in the shielding layer of MNCPs maintained their

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monomolecular structures without aggregation, and sustained release in acidic tumor environment, to carry out the high PDT efficacy under 633 nm laser irradiation. In the meantime, the MNCPs showed a more favorable r2 value as a T2 contrast agent in MRI than free Co@Mn MNPs due to the enrichment effect. This effect might be attributed to the interparticle interactions between each other in a nanocapsule less than 200 nm, that the original superparamagnetic cores became more effective at dephasing the spins of water protons. 30 Moreover, the MNCPs have much longer retention and targeted accumulation in tumor site than free Ce6, owing to the MGC-803 cells targeting effect of FA molecules. In addition, the MNCPs have an excellent stability in aqueous solution at room temperature and faster elimination rate from in vivo. For in vitro cell uptake studies, red fluorescent signals excited from Ce6 gradually increased after incubation MGC-803 cells with MNCPs, which was associated with the enrichment of

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Figure 8. In vivo PDT efficacy study. (A) Images of all the mice with different treatments before and after 633 nm laser irradiation. (B) The tumor volume curves and (C) body weight growth curves of tumor-bearing mice after different treatments over time. (D) The final tumor weights, (E) the survival rate, and (F) the slices of major organs with H&E staining of tumor-bearing mice after PDT treatment for 15 days (scale bar: 100 μm). The data are shown as mean ± SD (n = 5).

MNCPs in lysosomes by cell endocytosis. Upon the acid environment of lysosome, MNCPs gradually degraded, and the wrapped Ce6 was slowly released to produce singlet oxygen for apoptosis under 633 nm laser irradiation. The MGC-803 cells co-incubated with MNCPs showed a high survival rate without laser irradiation, indicating the good biocompatibility of MNCPs; conversely, the photocytotoxicity induced by MNCPs exhibited higher efficacy than that of free Ce6 at a low dosage of 10 μg/mL, which demonstrated the high PDT treatment potential of MNCPs for cancer cells. Furthermore, in vivo bio-distribution study has illustrated that the MNCPs play an important role in fluorescence/magneto imaging and specific targeting of tumor sites. These results of the fluorescence images demonstrated that the MNCPs could prolong circulation time and accumulation ability at the tumor site. In contrast, the fluorescence of free Ce6 rapidly disappeared in mice body. The phenomenon was attributed to a specific

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Supplementary data to this article can be found online at https://doi.org/10.1016/j.nano.2019.102047.

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Appendix A. Supplementary data

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metabolism characteristic of small dye molecules. The results of MRI experimentation demonstrated that MNCPs could gradually accumulate at the tumor sites via the intravenous injection for MRI enhancement. Meanwhile, the signal intensity value of MNCPs treated mice was lower than that of free Co@Mn MNPs treated mice; this can be ascribed not only to the enhanced permeability and retention (EPR) effect of MNCPs at the tumor site during blood circulation over time, but also to the fact that FA molecules could significantly improve specific targeting of MNCPs to tumor tissue. For in vivo PDT evaluation, MNCPs exhibited the most superior PDT performance to significantly inhibit tumor growth. The reason might be that Ce6 could be continually released from MNCPs to produce singlet oxygen under 633 nm laser irradiation, which induced the tumor tissue lesions and tumor cell apoptosis. In contrast, rapid metabolism in the tumor-bearing nude mice treated with free Ce6 was not sufficient to provide sufficient singlet oxygen for tumor suppression. Moreover, the main metabolism pathways in vivo include kidney metabolism and liver metabolism. The toxicity of the drug can be judged by evaluating the degree of damage to organ in body. The H&E staining of MNCPs presented no apparent abnormalities or lesions, suggesting that the MNCPs side effects were comparatively lower than other groups. Overall, the MNCPs demonstrated good blood circulation, biocompatibility, anti-degradation, low cytotoxicity, high selectivity targeting and pH response, efficient tumor accumulation, and enhancement of MRI signals in MGC-803 tumor-bearing mice model, which has achieved dual modal imaging and PDT treatment for accurate diagnosis and treatment of gastric cancer. Therefore, the outstanding properties of synthesized nanocapsules in this study could provide new ideas for simultaneous tumor imaging and drug delivery in the near future.

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