Self-Assembled Responsive Bilayered Vesicles with Adjustable

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Self-Assembled Responsive Bilayered Vesicles with Adjustable Oxidative Stress for Enhanced Cancer Imaging and Therapy Jibin Song, Lisen Lin, Zhen Yang, Rong Zhu, Zijian Zhou, Zhan-Wei Li, Feng Wang, Jingyi Chen, Huang-Hao Yang, and Xiaoyuan Chen J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13902 • Publication Date (Web): 03 May 2019 Downloaded from http://pubs.acs.org on May 4, 2019

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Journal of the American Chemical Society

Self-Assembled Responsive Bilayered Vesicles with Adjustable Oxidative Stress for Enhanced Cancer Imaging and Therapy

Jibin Song,† Lisen Lin,‡ Zhen Yang,‡ Rong Zhu,† Zijian Zhou,‡,* Zhan-Wei Li,§,* Feng Wang,‖ Jingyi Chen,‖ Huanghao Yang, †,* Xiaoyuan Chen‡,*

†

MOE Key Laboratory for Analytical Science of Food Safety and Biology, College of

Chemistry, Fuzhou University, Fuzhou 350116, China ‡

Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of

Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland 20892, United States

ACS Paragon Plus Environment

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Journal of the American Chemical Society 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

§ State

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

Chemistry, Chinese Academy of Sciences, Changchun 130022, China ‖

Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville,

Arkansas 72701, United States

ABSTRACT In the present study, we report the development of magnetic-plasmonic bilayer vesicles assembled from iron oxide-gold Janus nanoparticles (Fe3O4-Au JNPs) for reactive oxygen species (ROS) enhanced chemotherapy. The amphiphilic Fe3O4-Au JNPs were grafted with poly(ethylene glycol) (PEG) on Au surface and ROS generating poly(lipid hydroperoxide) (PLHP) on Fe3O4 surface, respectively, which were then assembled into vesicles containing two Fe3O4-Au NPs closely attached layers in opposite directions. The self-assembly mechanism of the bilayered vesicles was elucidated by performing a series of numerical simulations. The enhanced optical properties of the bilayered vesicles were verified by the calculated results and experimental data. The vesicles exhibited enhanced T2 relaxivity and photoacoustic properties over single JNPs due to the interparticle magnetic dipole interaction and plasmonic coupling. In particular, the vesicles are pH responsive and disassemble into single JNPs in the acidic tumor environment, activating an intracellular biochemical reaction between the


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grafted PLHP and released ferrous ions (Fe2+) from Fe3O4 NPs, resulting in highly efficient local ROS generation and increased intracellular oxidative stress. In combination with the release of doxorubicin (DOX), the vesicles combine ROS mediated cytotoxicity and DOX induced chemotherapy, leading to greatly improved therapeutic efficacy than monotherapies. High tumor accumulation efficiency and fast vesicle clearance from the body were also confirmed by positron emission tomography (PET) imaging of radioisotope 64Cu-labeled vesicles.

KEYWORDS: amphiphilic polymer, Janus nanoparticle, cancer imaging, cancer therapy, reactive oxygen species

INTRODUCTION


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Recently, exploiting altered redox status and increased reactive oxygen species (ROS) levels in cancer cells has become a new approach to fight against cancer by enhancing therapeutic efficacy and improving cancer specific cytotoxicity over normal cells.1-3 Although moderate ROS increase can lead to faster cell proliferation and increase differentiation rates of cancer cells, oxidative insults are generally more toxic to cancer cells than to normal cells.4-6 Furthermore, additional ROS stress generated by exogenous agents can disrupt the redox homeostasis and overwhelm the relatively vulnerable balance between ROS generation and elimination of cancer cells, leading to selective cancer cell toxicity.6-10 Meanwhile, therapeutic drugs that specifically generate ROS in cancer cells have been extensively studied. For example, radiation sensitizer motexafin gadolinium is now in phase III clinical trials for tumor therapy, because of its preferred accumulation in cancer cells and intracellular ROS generation.11-14 Another widely explored ROS production system is based on Fenton reaction, which involves the reaction between reductive metal ions and hydrogen peroxide (H2O2) to generate hydroxyl radicals.15


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With the recent advancements in nanomedicine, a plethora of tumor environment responsive nanoplatforms have been developed to produce ROS with high efficiency.16,17 For example, superparamagnetic iron oxide nanoparticles can be metabolized and degraded into the cell endocytic organelles and the released iron ions (Fe2+) are transported through the endosomal/lysosomal membranes and finally moved into cytosol.18 It is noticeable that the released Fe2+ may dramatically disrupt the intracellular redox reactions and further affect ROS homeostasis inside cells. Thus, intracellular ROS production efficiency is highly dependent on H2O2 concentration. Therefore, it is of utmost importance to develop a ROS generation system, which is able to adjust ROS production in a controlled manner for effective cancer therapy.15,19

Doxorubicin (DOX) is a highly effective drug to treat a broad spectrum of cancer types.5,6 However, its efficacy in treating cancer is limited by a cumulative dosedependent cardiotoxicity, which can cause irreversible cardiomyopathy and heart failure.5 Meanwhile, chemotherapy based on a single drug, such as DOX, is often insufficient for cancer therapy due to the drug resistance and side effects.20 Therefore, combination


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therapies based on several treatment methods rather than one treatment approach with enhanced and synergistic therapeutic effects are being explored.20-23


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Figure 1. (a) Schematic representation of the self-assembly of amphiphilic Janus NPs into dimer, micelle, chain and bilayered vesicles and their corresponding simulation model. (b) Schematic illustration of the anticancer effect of doxorubicin (DOX) loaded bilayered magnetic-plasmonic vesicles. The disassembly into monodisperse Janus F3O4-Au NPs in acidic environment results in ROS generation, increases oxidative stress and releases DOX, leading to combined ROS and chemotherapy of cancer guided by PET, magnetic resonance and photoacoustic imaging.

Herein, we report the development of a magnetic-plasmonic bilayer vesicular formula that generates ROS and releases DOX. The double layered vesicles were fabricated by self-assembly of amphiphilic iron oxide-gold Janus nanoparticles (Fe3O4-Au JNPs) grafted with ROS active agent poly(lipid hydroperoxide)-co-poly(4-vinyl pyrene) (PLHPVP) on Fe3O4 and hydrophilic poly(ethylene glycol) (PEG) on Au. The vesicular shell was composed of two layers of JNPs with Fe3O4 face-to-face localized in the inner side and Au extended to the outer side (Figure 1a). Lipid hydroperoxide (LHP) is a lipid


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peroxidation product that exists in several diseases, which can produce 1O2 radical through reaction with redox-active metal ions, such as Fe2+ by the Russell mechanism (Figure 1b).24 In an acidic environment, the vesicles are dissociated into single JNPs driven by pH responsive change of PLHPVP from being hydrophobic to hydrophilic, allowing H+ diffusion and contact with Fe3O4 NPs to release Fe2+.25 Thus, under acidic condition, the released Fe2+ can react with LHP to generate ROS, leading to oxidative stress induced apoptosis. The combination of ROS and DOX is expected to have improved anticancer effect than DOX or ROS alone. Furthermore, due to the magnetic dipole interaction and strong plasmonic-coupling of the Janus Fe3O4-Au NPs in the vesicular shell, the vesicles exhibit greatly enhanced magnetic and optical properties, which can be used for magnetic resonance (MR) and photoacoustic (PA) imaging to guide and track cancer therapy.26 In vivo positron emission tomography (PET) imaging of radio metal [64Cu] labeled vesicles was further used to calculate the tumor accumulation efficiency and evaluate the in vivo biodistribution of the vesicles.27


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RESULTS AND DISCUSSION

Preparation of the Bilayered Magnetic-Plasmonic Vesicles

Figure 2. Characterization and physical properties of the bilayered vesicles. (a) TEM image of iron oxide-gold Janus nanoparticles (Fe3O4-Au JNPs). TEM (b) and SEM (c)


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images of the double-layered vesicles. (d) High magnification SEM image of a collapsed vesicle caused by high vacuum pressure during SEM test. (e) Dark-field (DF) TEM and element mapping (Fe and Au elements) of a single vesicle. Hydrodynamic diameter distribution (f), T2-weighted spin-echo MR images and r2 values (g), UV-vis spectra (h) and photoacoustic images and intensity (i) of the JNP and as-prepared JNP vesicles dispersed in water.

To prepare the bilayered magnetic-plasmonic vesicles, Fe3O4-Au Janus NPs were first stepwise synthesized using small Au NPs as seeds to grow Fe3O4 NPs (the synthetic procedure is available in the Supporting Information).28 The size of the Fe3O4-Au Janus particles was approximately 11 nm, where the size of Fe3O4 and Au NPs were around 6 nm and 5 nm, respectively, based on TEM images (Figure 2a). By taking advantage of the specific surface conjugation abilities of Fe3O4 to phosphate group and Au NPs to thiol groups,29 pH responsive phosphorylated poly (lipid hydroperoxide80–co-4-vinylpyridine20) (PLHPVP) (MW= 28 kDa) (Scheme S1, Figure S1 and S2) was grafted onto Fe3O4 and


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hydrophilic thiolated PEG (SH-PEG) (MW= 5 kDa) was grafted onto Au. The pH responsive amphiphilic Janus NPs were denoted as Fe3O4@PLHPVP-Au@PEG NPs (Scheme 1).29,30 The weight fraction of the organic part of Fe3O4@PLHPVP-Au@PEG NPs was ~54.5% based on thermogravimetric analysis results (Figure S3), indicating high density grafting of polymers on JNP surface.25 Owing to the Janus distribution of the hydrophobic PLHPVP and hydrophilic PEG on JNP surface, these amphiphilic JNPs selfassembled into different nanostructures of dimer, micelle, chain and bilayered hybrid vesicles (Figure 1a, Figure 2b,c, and Figure S4). As shown in TEM images of the asprepared vesicles (Figure 2b), hydrophilic Au@PEG was placed at the outer side of the vesicular shell, while hydrophobic Fe3O4@PLHPVP was face-to-face localized at the inner side. The vesicular shell thickness was ~27 nm measured from the high magnification SEM images of the collapsed vesicles (Figure 2d), which was about the total size of two JNPs. As shown by element mapping results of the vesicle (Figure 2e), the signal map of Au possessed a larger size and was surrounded by Fe, further confirming that Au was localized at the outer side of the shell. The vesicle size was approximately 92 nm as determined by the dynamic light scattering measurement (Figure


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2f). The vesicles exhibited good water dispersibility and high stability, presumably due to the presence of high-density PEG at the outer side of the vesicles.

Clustering magnetic nanoparticles with decreased interparticle distance can significantly increase the transverse relaxation, and thus, nanoparticle assembly has been extensively used to improve the relaxivity.31-33 At the same concentration of Fe, the double layered vesicles showed much stronger T2 contrast enhancement than JNPs (Figure 2g). Furthermore, the double layered vesicles exhibited approximately 6 times higher r2 value (~445 mM-1 s-1) than JNPs (~76 mM-1 s-1), due to the strong magnetic field coupling of Fe3O4 NPs in the vesicular shell. Also of note is that the r2 value of double layered vesicles was 2.5 times higher than that of the single layered vesicles assembled from Fe3O4-Au coated with mixed polymer brushes of PLHPVP and PEG (Figure S5). Therefore, the JNP@PLHPVP-PEG vesicles could be used as an excellent T2 contrast agent for MR imaging.

The maximum absorption peak of the vesicles was in the near-infrared (NIR) region, which exhibited large red-shift and color (blue) change compared with JNPs (violet), due


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to the plasmonic coupling of Au NPs in the vesicular shell (Figure 2h).26,27 Plasmonic coupling of gold nanocrystals can also lead to enhanced photothermal conversion efficiency, which is associated with its PA performance.34,35 Therefore, we next investigated PA property of vesicles of different optical densities at 808 nm (OD808) illuminated with an 808-nm laser. The vesicles showed greatly enhanced PA signal over JNP under the same OD808 value (Figure 2i). The strong vesicle PA performance was ascribed to the high optical absorption coefficient and enhanced electromagnetic filed induced by the strong plasmonic coupling of the AuNPs in the vesicular shell.35 The multifunctional hybrid double-layered vesicles with excellent stability and enhanced physical properties may be used as simultaneous MRI and PA dual modality imaging agents.34,36-38

Formation Mechanism of the Bilayered Vesicles of Janus NP


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Figure 3. Self-assembly and formation mechanism of bilayered magnetic-plasmonic vesicles. (a) Schematic illustration of coarse-grained Janus particle model used in the simulations. (b-d) SEM images (left) and corresponding simulation snapshots (right) of aggregate structures assembled by Janus F3O4-Au NPs at different concentrations: (b) small micellar clusters (50 nM and Φ = 0.03%), (c) wormlike micelles (70nM and Φ = 0.05%), and (d) bilayered vesicles (120nM and Φ = 0.3%). (d1-d5) Simulation snapshots of the formation process of the bilayered vesicles in (d) at different simulation times: 10000 (d1), 20000 (d2), 30000 (d3), 60000 (d4), 100000 (d5) time units. For the sake of visualization, solvents are not shown in (d1-d5).


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To better understand the self-assembly mechanism of the bilayered vesicles of Janus NPs, we simulated the self-assembly process of amphiphilic polymer-grafted Janus Fe3O4-Au NPs into bilayered vesicles with the aid of the general mesoscale soft patchy particle model,39-41 using GALAMOST software package.42,43 To achieve high computational efficiency, each Janus F3O4-Au NP was coarse-grained into one singlesite Janus particle, as shown in Figure 3a. Please see the details of the model and simulation in the SI. Consistent with our experimental results, the small micellar clusters (Figure 3b), wormlike micelles (Figure 3c), and bilayered vesicles (Figure 3d) were observed in the simulations with increasing concentrations of Janus NPs. The simulations were further performed to investigate the formation mechanism of the bilayered vesicles in Figure 3d, which are formed by gradually reducing the solvent quality (i.e. adding water into the solution in a stepwise fashion) in experiments. As shown in Figure 3d1-d5, upon reducing the solvent quality, the small micellar clusters formed at the early stage (Figure 3d1) will fuse into larger wormlike micelles (Figure 3d2), and then the wormlike micelles will further assemble and realign into the bilayered vesicles (Figure 3d3-d5). Therefore, the major kinetic mechanism governing the formation of the bilayered vesicles is the


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formation of wormlike micelles from the fusion of small micellar clusters and the subsequent assembly of wormlike micelles into bilayered vesicles.

Theoretical Simulation of Electromagnetic Coupling of the AuNPs in the Vesicular Shell

Figure 4. Theoretical calculation of the absorption, extinction and scattering spectra of the JNPs (a), dimer of JNPS (b), chain (c) and vesicle (d).


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The optical properties of the bilayer vesicle and their building blocks were simulated using the discrete dipole approximation (DDA) using the DDSCAT 7.3 program.44,45 Figure 4a displays the optical spectra of the building block Fe3O4-Au JNP and the comparison Au NP. The Fe3O4-Au JNP was built by attaching a Fe3O4 half shell on an Au nanosphere that is 7 nm in diameter. Assuming the Fe3O4 is spherical, the diameter of the hypothetical sphere is 7 nm and the center of this hypothetical sphere is 7 nm away from the center of the Au sphere (see the inset of Figure 4a). The extinction spectrum of the Janus dumbbell exhibits a peak at 535 nm slightly redshifted from the 7 nm Au extinction peak at 520 nm. Compared to the Au sphere, the optical efficiency of the Janus dumbbell slightly decreases due to the damping effect from the adjacent Fe3O4 component. The optical spectra of a dimer and a chain of dimers assembled from the Fe3O4-Au JNPs are shown in Figure 4b and 4c, respectively. Interestingly, the LSPR of the chain of their dimers at 550 nm slightly blue shifted from that of the dimer at 570 nm. This observation can be attributed to the difference in the aspect ratios of the assemblies, which is 4:1 for dimer while 4:3 for the chain of dimer pairs. In order to calculate the optical spectra of the vesicle shell, we built two bilayer vesicles with a diameter of 100 nm using Au-JNP dimers as building blocks. One of them is assembled by facing the Fe3O4 component of the JNP toward each other at a distance of 1 nm (Figure 4d). The other one was assembled by facing the Au component toward each other dimer with Au component facing at a separation of 1 nm (Figure 4f). The peak positions of the vesicles are 570 nm and 550 nm for the former and the latter, respectively. Irrespective of the slight difference in the peak positions, both vesicles show dramatically improved the optical efficiency of the LSPR peaks when compared to the Fe3O4-Au JNP dimer, increasing from ~0.7 for the dimer to ~2.5 for the vesicle. In addition, the peak width is broadened for the vesicle when compared to the Fe3O4-Au


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JNP dimer building block. This observation can be accounted for by an increase of both absorption and scattering cross-section as the size of the particles increase. For both vesicles, the light absorption dominates despite a slight increase in the ratio of scattering to absorption when compared to a pair of dimers.

ROS Generation and Drug Release of the pH Responsive Bilayered Vesicles


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Figure 5. Representative TEM images of the double layered vesicles (Ve) after incubation in acidic environment (pH 5.4) for 30 (a), 60 (b) and 90 min (c). (d) Fe2+ release from the JNPs and double layered vesicles in acidic environment. (e) Fluorescence detection of 1O 2

generation by singlet oxygen sensor green (SOSG). (f) UV detection of 1O2 production

by 1O2 scavenger reacting with JNPs and JNP vesicles. (g) UV-vis spectra of DOX, JNP Ve and DOX loaded JNP Ve. (h) Fluorescence spectra of DOX and DOX loaded JNP Ve incubated at pH 5.4 or 7.4. (i) DOX release from DOX loaded JNP Ve at pH 7.4 and 5.4.

We next investigated the pH responsiveness of the nanovesicles. Because the pKa value of 4-vinylpyridine (4VP) is 5.4, it is protonated under acidic condition, turning the hydrophobic PLHPVP into hydrophilic polymer.25 The change of hydrophilicity of PLHPVP is expected to change the integrity and morphology of the vesicles. As shown in TEM images, the vesicles were disrupted after incubation at pH 5.4 buffer solution for 30 min, which were further disassembled into small clusters and even single JNPs after incubation for 60 and 90 min, respectively (Figure 5a-c). Absorbance blue shift was also


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observed with increasing incubation time, due to the decrease of interparticle plasmonic coupling caused by increasing interparticle distance (Figure S6).46-48 Sample hydrodynamic size also changed from ~92 nm to ~19 nm after incubation for 90 min, indicating dispersion of single JNPs in the solution (Figure S7).

The disassembly of vesicles into single JNPs under acidic condition allows H+ to react with JNPs and release Fe2+ from IO NPs. Because most invasive tumors have an acidic environment, we further tested the Fe2+ release behaviors in buffer solution at different pH values mimicking tumor and healthy tissue biological environments.49 The results showed that ~1% Fe2+ was released from JNP vesicles within the first 3 h and this value increased to 3.4% after 10 h incubation in pH 5.4 solution (Figure 5d). Fe2+ release at pH 6.7 was also observed, which reached 0.4% and 1.4% at 3 h and 10 h, respectively.

1O 2

production by the biochemical reaction of released Fe2+ and PLHP was detected

using both singlet oxygen sensor green (SOSG) and UV-based singlet oxygen scavenger 9,10-diphenylanthracene (DPA).50 Significant increase in the fluorescence intensity of SOSG (Figure 5e) and clear drop in DPA absorption peak (Figure 5f) demonstrated the


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high 1O2 production efficiency of the vesicles. Negligible amount of 1O2 generation was observed from the doubled vesicle in neutral buffer (pH 7.4), as no functional Fe2+ was released to react with PLHP. 1O2 was also not detected from the control sample of gold nanovesicles assembled from gold nanoparticles (12 nm) coated with PLHPVP and PEG (Au@PLHPVP-PEG Ve) in pH 5.4 buffer (Figure S8). Thus, 1O2 production from the vesicles occurred only in the presence of released Fe2+ in acidic environment, suggesting that 1O2 generation could be controlled at tumor site and adjusted by tuning the number of vesicles injected.

Vesicular nanostructures with large hollow cavity and stimuli responsive shell have been widely used as drug carriers.51-53 Herein, DOX, a widely used anticancer drug, was loaded into the JNP vesicle during its formation process. The characteristic UV-vis absorption peak of DOX at 489 nm was observed in the UV-vis absorption spectra of the DOX loaded vesicles, indicating that DOX was successfully encapsulated into the vesicle (JNP@PLHPVP-PEG Ve-DOX) (Figure 5g). Due to the nanometal surface energy transfer, the DOX fluorescence signal in the JNP Ve-DOX was significantly quenched as


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compared with free DOX of the same concentration.20 Noting that the fluorescence signal of the encapsulated DOX was almost fully recovered after JNP Ve-DOX was incubated in acidic solution (pH 5.4) for 90 min (Figure 5h). DOX release experiment also confirmed effective release of DOX from the vesicle (Figure 5i).

In Vitro ROS Formation, Drug Release and ROS Mediated Therapy Combined with Chemotherapy by DOX Loaded Hybrid Vesicles


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Figure 6. In vitro ROS generation, drug delivery and synergistic cytotoxicity studies. (a) Confocal images of U87MG cancer cells showing ROS generation and DOX release after incubation with JNP@PLHPVP-PEG Ve-DOX. (b) Quantification of DOX fluorescence signal (mean fluorescence intensity/cell). (c) H2DCFA was used to measure ROS, whose


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fluorescence intensity increased with increasing ROS level. (d) Confocal images of the cells treated with different compounds for 24 h followed by TUNEL-FITC (green color) and Hoechst 33342 (blue color) staining. Sectional TEM images of cancer cells treated with PBS (e) and JNP@PLHPVP-PEG vesicle for 24 h (f). (g) Flow cytometry analysis of cell apoptosis induced by different samples for 24 h using Annexin V-FITC/PI staining. (h) Cancer cell viability after incubation with different compounds for 24 h.

Intracellular ROS generation and anti-cancer drug release were evaluated in U87MG tumor cells. After 3 h incubation, large amount of JNP@PLHPVP-PEG Ve was taken up by cancer cells based on the inductively coupled plasma mass spectrometry (ICP-MS) results, showing a concentration of 3.6 × 105 JNPs/cell (Figure S9).52 However, very weak DOX fluorescence signal was observed in the cells at 0.5 h post-incubation (Figure 6a3), due to the quenching of DOX fluorescence by Au. At 1.5 h post-incubation, strong DOX red signal was observed in the cytoplasm, indicating that DOX molecules were released from the vesicles (Figure 6a7). Interestingly, most of the DOX finally entered the nucleus


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at 2 h and 4 h post-incubation (Figure 6a11, a15), the fluorescence intensity of the cells treated with JNP@PLHPVP-PEG Ve was approximately 2.5-fold higher than free DOX, indicating high drug delivery efficiency (Figure 3b). Furthermore, little fluorescence signal was detected in the cells treated with a pH non-responsive JNP@PLHP-PEG vesicles, indicating no DOX leakage from these control vesicles (Figure S10).

To further verify JNP@PLHPVP-PEG Ve-mediated ROS generation in cancer cells, we treated cells with the vesicles and ROS probe DPA simultaneously to analyze ROS production.50 Cells treated with pH responsive vesicles exhibited a continuous increase in ROS generation (green color) over time (Figure 6a6, 10, 14), due to the reaction of released Fe2+ with PLHPVP. Quantitative analysis of DPA fluorescence intensity further confirmed time-dependent of increase in ROS generation (Figure S11). Nearly no ROS was found in the controls, such as cells treated with PLHPVP or Au@PLHPVP (Figure S12). Overall, intracellular disassembly of the vesicles and biochemical reaction between the released Fe2+ and LHP are responsible for the simultaneous DOX release and ROS production.


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The cytotoxicity of the JNP@PLHPVP-PEG Ve was also studied. Round shrunken shape and condensed nuclei were found after JNP@PLHPVP-PEG Ve treatment for 24 h as shown by Hoechst 33342 staining (Figure 6d). Cell DNA fragmentation analyzed using deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) suggested that cells underwent apoptosis probably due to the 1O2 production. Cells treated with JNP@PLHPPEG Ve for 24 h exhibited 38.6% of cells in early apoptosis, as shown by flow cytometry after Annexin V-FITC and PI staining (Figure 6g). The percentages of apoptotic cells were much lower after being treated with PBS, PLHP or Au@PLHP-PEG for 24 h (Figure 6g, Figure S13, 14). In comparison with the control cells treated with PBS (Figure 6e), the cancer cells treated with JNP@PLHPVP-PEG vesicles for 24 h showed surface blebbing, membrane

disruption,

cytoplasmic

vacuolation,

and

chromatin

margination,

condensation, and fragmentation as shown by the sectional TEM images (Figure 6 f), further confirming the ROS induced cell apoptosis.

To further investigate cancer cell death exerted by DOX loaded in the JNP@PLHPVPPEG Ve, viability of U87MG cells treated with Au@PLHPVP-PEG Ve-DOX,


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JNP@PLHPVP-PEG Ve loaded with or without DOX were tested and the IC50 values were calculated. As expected, JNP@PLHPVP-PEG Ve-DOX with combined therapeutic effects induced more effective cell death than Au@PLHPVP-PEG Ve-DOX and JNP@PLHPVP-PEG-Ve without DOX. JNP@PLHPVP-PEG Ve exhibited significant anticancer activity due to cell apoptosis induced by the increased oxidative stress mediated by ROS. Thus, JNP@PLHPVP-PEG Ve-DOX carrying ROS-active PLHP and encapsulated DOX showed obvious advantages over single therapy agent-loaded nanoplatforms. The vesicles in the cells overlaid well with the endosome after incubation (Figure S15), further indicating that the acidic environment of the endosome could induce the dis-assembly of the vesicles and drug release. However, negligible cell cytotoxicity was observed when the endosome acidic condition was blocked by endosomal acidification inhibitor chloroquine, because nearly no DOX and Fe2+ could be released this way (Figure S16).

In Vivo Multi-Modes of Optical and MR Imaging of Bilayered Vesicles


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Figure 7. In vivo imaging and biodistribution studies of the hybrid vesicles. (a) PET imaging of U87MG tumor-bearing mice at different time points after intravenous injection of [64Cu] radiolabeled JNP vesicles. (b) PET region of interest (ROI) analysis of tumor, muscle and liver at 1, 5, 25 and 40 h post-injection. (c) Direct tissue sampling biodistribution in different organs at day 1 and 12 post-injection analyzed by ICP-MS. (d)

In vivo PA images and (e) average PA intensity in the tumor after intravenous injection of


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the hybrid vesicles. (f) T2-weighted MR images and (g) the relative T2 values of the tumor before and after intravenous injection of vesicles at different times (tumors highlighted by yellow circles). Scale bar: 5 mm. (h) In vivo fluorescence imaging and (i) tumor signal intensity after injection of IRDy800CW loaded vesicles.

To quantitatively evaluate the in vivo biodistribution and pharmacokinetics of the hybrid vesicles, radiometal [64Cu] labeling on Au of the JNPs was used for PET imaging.45 [64Cu] labeling experiment was performed at room temperature and purity was checked by using instant thin layer chromatography (ITLC) (Figure S17).45 Whole-body PET imaging of U87MG tumor-bearing mice was performed after intravenous injection of [64Cu]-labeled vesicles (150 µCi/mice) (Figure 7a). Tumor uptake of vesicles was 0.69 ± 0.36, 2.4 ± 1.2, 11.5 ± 1.6, and 8.9 ± 1.9 %ID/g at 2, 5, 25, and 40 h post-injection, respectively (Figure 7b). The biodistribution of vesicles was also evaluated using ICPMS, which showed similar results as PET imaging (Figure 7c). The DOX content in the blood is very little, suggesting negligible DOX was released during vesicle circulate in the


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blood (Figure S18). Furthermore, clearance from the liver was observed over time (less than 1 %ID/g at day 12), suggesting that vesicles could be dissociated and cleared from the body (Figure 7c), greatly reducing the toxicity to the liver.

PA imaging is a hybrid imaging approach, which can provide higher spatial resolution and deeper tissue penetration than traditional optical imaging techniques.34,54,55 JNP@PLHPVP-PEG with strong PA property was employed as a PA agent to investigate the in vivo imaging behavior.56 Tumor PA images were obtained after intravenous injection of the proper contrast agent into the U87MG tumor mice. PA signal in the tumor continuously

increased

over

time,

suggesting

a

continuous

accumulation

of

JNP@PLHPVP-PEG in the tumor area (Figure 7d,e, Figure S19). Tumor PA images at 25 h post-injection showed clear tumor characteristics, including size, position, and morphology. Furthermore, the PA spectrum of the contrast agent in the tumor blue shifted at 40 h post-injection, indicating disassembly of JNP@PLHPVP-PEG caused by the acidic tumor environment (Figure S20).


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MR imaging with high spatiotemporal resolution, nonionizing radiation and excellent soft issue contrast has been widely applied in clinical imaging and diagnosis.57 However, MR imaging is impaired by relatively low sensitivity.58 Thus, new contrast agents are usually required to increase the accuracy of differentiating tumor from normal tissue. Herein, we employed the as-prepared vesicles with high r2 value as a T2 contrast agent for tumor imaging (Figure 7f). Significantly enhanced T2 contrast in the tumor region was observed after vesicle injection (Figure 7g). Strong MR signal in the tumor region was ascribed to the tumor accumulation and enhanced relaxivity of the vesicles. In contrast, the tumor showed weaker PA and lower T2 signals at 12 h and 25 h post-injection of the JNPs (Figure S21).

NIR

fluorescence

imaging

was

further

employed

to

investigate

in vivo

pharmacokinetics of the pH responsive vesicles, especially their disassembly behavior in the tumor. NIR dye IRCW800 was encapsulated into the vesicle using a film-rehydration method.36 The NIR fluorescence was recorded over the whole mouse body soon after intravenous injection of the dye loaded vesicles. Almost no fluorescence signal was found


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in the body during 0-5 h post-injection (Figure 7h), due to the fact that the dye molecules were still loaded in the vesicles, thus the fluorescence signal was quenched by Au. The fluorescence signal in the tumor region slowly appeared and enhanced over time (Figure 7h). Tumor fluorescence signal was significantly higher than that in the surrounding healthy tissue (Figure 7i), suggesting that vesicles were localized in the tumor and released the content locally.

In Vivo Synergistic ROS Mediated Chemo-Therapy


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Figure 8. In vivo cancer therapy effect of JNP vesicles. (a) Tumor growth profiles in mice after different treatments (* P < 0.05; ** P < 0.01). (b) Tumor MR images before and 18 days after treatment with different formulations (Yellow circle: Tumor). (c) Representative photographs of the dissected tumors in different treatment groups. (d)TUNEL-labeled tumor sections treated with different formulations (Green color: positive TUNEL staining,


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blue color: nuclei stained by Hoechst 33342) and histological analysis of tumor sections after staining with H&E in different treatment groups.

The excellent physical properties and stability of the vesicles encouraged us to further investigate their in vivo behavior and anticancer effect (Figure S22). The vesicles were injected into the U87MG tumor-bearing mice via tail vein when the tumor volume reached ~60 mm3. To demonstrate the effect of combined ROS and DOX therapy, Au@PLHPVPPEG Ve-DOX and JNP@PLHPVP-PEG Ve were also tested as controls. The other two control groups were PBS and free DOX groups. Tumor volumes were then recorded every two days for 18 days. A nearly complete tumor ablation was found in the mice treated with JNP@PLHPVP-PEG Ve-DOX (Figure 8a). However, Au@PLHPVP-PEG Ve-DOX or JNP@PLHPVP-PEG Ve treatment resulted in continuous tumor growth although somewhat slower than the PBS group. Moreover, the tumor growth was not completely inhibited after being treated with the mixture of DOX JNP@PLHPVP-PEG Ve, further indicating the encapsulation of DOX in the vesicle is necessary to improve the antitumor efficacy.


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Tumor therapy effect was also monitored using MR imaging, which showed a clear difference in tumor volume between different treatment groups (Figure 8b). Ex vivo tumor size measurement was consistent with that obtained from digital caliper and MR imaging (Figure 8c). Consistent with the tumor ablation results, mice treated with JNP@PLHPVPPEG Ve-DOX lived for more than two months, while those treated with Au@PLHP-PEG Ve-DOX or JNP@PLHPVP-PEG Ve had to be euthanized within 20 days after the treatment due to excessive tumor growth (Figure S23). The body weight of the mice treated with JNP@PLHPVP-PEG Ve-DOX was unchanged or slighted increased, indicating that simultaneous delivery of ROS and DOX by the hybrid vesicles did not induce systemic toxicity (Figure S24). To assess the toxicity of the vesicles in vivo, we measured the liver enzyme levels such as ALT, AST, ALP, and GGT in the blood and liver in the vesicle treated tumor and normal mice (Figure S25). Before and after treatment, these parameters were still within the normal range, indicating biocompatibility of the vesicles. For the in vivo cancer therapy, the dosage of the vesicle was as low as 0.01 mg/g of animal body weight, which led to negligible depletion of kupffer cells (Figure S25, 27).59,60 To detect the ROS production in


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vivo, a luminol derivative (L-012) was injected into the peritoneal cavity of the tumor bearing mice after administration of the vesicles, ROS generation at the tumor site was found whereas liver showed nearly no ROS production (Figure S28).61 To further determine the synergistic antitumor effect by ROS and DOX, apoptosis was evaluated by TUNEL assay in tumor sections of mice treated with different samples. Mice treated with PBS showed no TUNEL signal (Figure 8d1-4). Au@PLHPVP Ve-DOX or JNP@PLHPVP-PEG Ve treated mice showed diffuse TUNEL signals indicating apoptosis (Figure 8d5-7, d9-11). JNP@PLHPVP-PEG Ve-DOX treated mice showed much stronger TUNEL signal (Figure 5d13-15) than that of Au@PLHPVP Ve-DOX or JNP@PLHPVPPEG Ve. Tumor tissue hematoxylin and eosin (H&E) staining after JNP@PLHPVP-PEG Ve-DOX treatment showed more condensed cell nuclei compared with those of control groups (Figure 8d). H&E staining of the different organs of the mice treated with the vesicles showed negligible damage, suggesting little systemic toxicity of the samples (Figure S29).62 Taken together, ROS (1O2) generation and DOX release from the vesicles induces a synergetic combination of ROS mediated therapy and chemotherapy.


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CONCLUSION

In conclusion, our results demonstrated the use of double-layered magneticplasmonic vesicles made from Janus Fe3O4-Au NPs for synergistic ROS mediated therapy and chemotherapy, guided by PET, MR and PA imaging. The amphiphilic JNPs can be self-assembled into dimer, micelles and vesicles by changing the parameters of the concentration of the JNPs. Moreover, the simulated results show that the major kinetic mechanism governing the formation of the bilayered vesicles is the formation of wormlike micelles from the fusion of small micellar clusters and the subsequent assembly of wormlike micelles into bilayered vesicles. Benefiting from the assembly of the JNPs in the bilayer, the vesicles exhibited enhanced T2 relaxivity and photoacoustic properties than single JNPs due to efficient interparticle magnetic and plasmonic coupling, which was verified by the calculated results. Indeed, tumor acidic environment generated abundant singlet oxygen through the reaction of released Fe2+ and PLPHVP in the vesicles. In combination with the loaded DOX and the high tumor accumulation through EPR effect,


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these vesicles showed significantly better inhibition of tumor growth over each single therapy approaches. The ROS active vesicles modulate the cellular redox environment within cancer cells and increase oxidative stress, resulting in apoptosis for the treatment of cancer. Multi-model imaging, combined therapy, and stimuli responsiveness make this new hybrid vesicle structure a promising cancer theranostic platform.

EXPERIMENTAL SECTION

Preparation

of

the

ROS

Active

Bilayered

Magnetic-Plasmonic

Vesicles

of

Fe3O4@PLHPVP-Au@PEG

For the preparation of bilayer vesicle, amphiphilic Janus Fe3O4@PLHPVP-Au@PEG was dispersed in the solution of DMF/THF with the concentration of 0.1 mg/mL, followed by adding the mixture of water and THF with predetermined ratio drop-by-drop. After that, the mixed solution was dialyzed by using dialysis membrane tubing (with 3000–5000 molecular-weight-cutoff) for 24 h to remove the DMF and THF. Similarly, the DOX loaded


38

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vesicle was prepared using the same method by dissolving amount of DOX in THF containing JNP@PLHPVP-PEG.

Simulation Model and Calculation Details of the Self-assembly Process of the JNPs To achieve high computational efficiency, we adopt the simple and general soft patchy particle mode to simulate the self-assembly of polymer-grafted Janus F3O4-Au NPs. In this model, each Janus F3O4-Au NPs is coarse-grained into one single-site Janus particle, as shown in Figure 1a and Figure 3. The interaction between Janus particles is described by a single-site anisotropic attractive potential.

𝑈𝑖𝑗 =

{

𝛼𝑅𝑖𝑗𝑑𝑖𝑗 2

(

1―

𝑟𝑖𝑗 𝑑𝑖𝑗

)

2

―

𝑀𝑖

𝑀𝑗

𝜅=1

𝜆=1

∑ ∑

(

𝑣

𝑓

𝒏𝜅𝑖,𝒏𝜆𝑗,

𝒓𝑖𝑗)

[ ( )]

𝛼𝐴𝑖𝑗𝑑𝑖𝑗 𝑟𝑖𝑗 2

𝑑𝑖𝑗

―

𝑟𝑖𝑗

2

𝑑𝑖𝑗

0,

𝑟𝑖𝑗 ≤ 𝑑𝑖𝑗 𝑟𝑖𝑗 > 𝑑𝑖𝑗 (1)

where 𝑓(𝒏𝑘𝑖,𝒏𝜆𝑗, 𝒓𝑖𝑗) =

{

𝜋𝜃𝜅𝑖

𝜋𝜃𝜆𝑗

cos 2𝜃𝜅 cos 2𝜃𝜆 , 𝑖𝑓cos 𝜃𝜅𝑖 ≥ cos 𝜃𝜅𝑚 𝑎𝑛𝑑 cos 𝜃𝜆𝑗 ≥ cos 𝜃𝜆𝑚. 𝑚 𝑚 0, 𝑜𝑡ℎ𝑒𝑟𝑤𝑖𝑠𝑒 (2)


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Here, the magnitude of 𝛼𝑅𝑖𝑗 controls the strength of repulsion, and 𝛼𝐴𝑖𝑗 controls the strength of attraction between the attractive patches. 𝑑𝑖𝑗 = (𝑑𝑖 + 𝑑𝑗)/2, where 𝑑𝑖 and 𝑑𝑗 are the diameters of particle i and particle j. In this study, all the particles have the same diameter, 𝑑𝑖𝑗 = 𝑑𝑖 = 𝑑𝑗 ≡ 1.0. 𝑀𝑖 and 𝑀𝑗 are the number of attractive patches on the surfaces of particle i and j, which also equal to 1. As shown in Figure 1a, the unit vectors 𝒏𝜅𝑖 and 𝒏𝜆𝑗 are the directions of the orange patches of particle i and particle j. The sizes of the orange attractive patches κ and λ are described by 𝜃𝜅𝑚 and 𝜃𝜆𝑚, which are half of the opening angle of the attractive patches. The fraction of surface of particle i covered by the orange attractive patch, χ, is related to 𝜃𝜅𝑚 by the relation χ = sin2

𝜃𝜅𝑚 2

[4-6].

In the simulations, all the variables and parameters are given in the reduced units, 𝑑𝑖 is chosen as the unit of length, 𝑘𝐵𝑇 is used as the unit of energy, and thus the unit of time 𝜏 = 𝑚𝑖𝑑2𝑖𝑗/𝑘𝐵𝑇. As given in Refs.[4-6], the simulation parameter 𝛼𝑅𝑖𝑗 is related to the linear elastic modulus of the particle by 𝛼𝑅𝑖𝑗 = 𝜋𝐸𝑑2𝑒𝑓𝑓/6, where 𝑑𝑒𝑓𝑓 is the effective diameter of Janus particle. 𝛼𝐴𝑖𝑗 is related to the adhesion energy between Janus particles by 𝐺 = 𝛼𝐴𝑖𝑗 (1 ― 𝑑𝑒𝑓𝑓)/4. 𝛼𝑅𝑖𝑗 is kept at 396 as in Refs. [4-6], and the corresponding elastic modulus E of


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Janus particles is approximately equal to 4.0 MPa, which ensures that the polymergrafted Janus particles are soft and compressible as compared to the usual hard-sphere particles with modulus about 2.0 GPa. In the simulations, the strength of attraction 𝛼𝐴𝑖𝑗 (i.e. the adhesion energy G) is stepwise increased from 44 (G ≈ 0.6) to 176 (G ≈ 6.8) at slow enough rate of Δ𝛼𝐴𝑖𝑗 = 5.5 per 2.0 × 106 time steps, which corresponds to reducing the solvent quality by gradual addition of water in experiments. The fraction of the orange attractive patch χ estimated by experiment is about 70%, and thus we choose 𝜃𝜅𝑚 = 115 ∘ .

The simulations are performed in the NVT ensemble. The Nose-Hoover (NH) thermostat is used to control the temperature at target value[4,5]. The coarse-grained solvent particles are explicitly considered in the simulations. The systems of 1.92 × 105 particles are simulated in a 40 × 40 × 40 cubic box with periodic boundary conditions. Φ is used to describe the concentration of Janus particles in solution. Simulations in larger systems (6.48 × 105 particles in a 60 × 60 × 60 cubic box) and from different initial configurations are also performed to check the size effect and the reproducibility of simulation results. Time step δt = 0.002τ is used. All the simulations are performed with


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the aid of GALAMOST software package, a GPU-accelerated large-scale molecular simulation toolkit.

MR Relaxivity Measurements of the Bilayer Vesicles

T2 relaxation time of the JNP@PLHPVP-PEG vesicle was measured using a Bruker 7T 347 scanner (Pharmascan) equipped with a small animal-specific body coil. Samples of the double layered vesicles containing 1% of agarose gel were prepared in tubes with different concentrations of 0.5, 0.25, 0.15, 0.1 and 0.05 mM with respect to iron mass. The T2 MR images were obtained using 350 spin echo sequence with the following experimental condition: repetition time = 3000 ms, echo time = 10, 20, 30, 40, 351 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160 ms, slice thickness = 3.00 mm matrix = 256 × 256, field of view (FOV) = 352 40 × 40 mm2. The T2 relaxation time of the samples was calculated by fitting these 353 multiple spin echo images.


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In Vitro ROS Generation and DOX Release of JNP@PLHPVP-PEG Ve-DOX

The cancer cells (5×104) were seeded in 12 well confocal glass slides and incubated for 24 h under cell culture condition. To monitor the intracellular ROS generation and DOX release by fluorescence imaging, the cells were first treated with freshly prepared carboxy-H2DCFDA (2 µM) for 20 min under cell culture condition. After removing the carboxy-H2DCFDA solution and washing with PBS, JNP@PLHPVP-PEG Ve-DOX, JNP@PLHPVP-PEG Ve, PLHPVP, Au@PLHPVP-PEG Ve, and PBS were added into the well, respectively. The cell nuclei of U87 MG cells were counterstained with Hoechst 33342. The red fluorescence signal of DOX, generated green fluorescence signal of carboxy-H2DCFDA and blue fluorescence signal of Hoechst 33342 were detected at different time intervals.

In Vitro Cytotoxicity of JNP@PLHPVP-PEG Ve-DOX

U87MG cells (1×104 cells/well) were seeded in a 96-well plate. After incubation at 37 °C for 24 h, the double layered vesicles with a final concentration of 0.05, 0.1, 0.2, 0.4, 0.8,


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1.6 μg/mL of DOX were incubated with cells for 24 h, respectively. The cell viability was examined by the Cell Counting Kit-8 (CCK-8) assay. The relative cell viability was determined with the calculated ratio of absorbance of treated cell to that of untreated cell. All in vitro cytotoxicity experiments of the samples were triplicated and results were averaged.

In Vitro Apoptosis Assay

Apoptosis was studied using the Annexin-FITC/PI Apoptosis Detection Kit (Thermal Fisher Scientific). Briefly, the cancer cells were grown in 12-well plates at a density of 5×104 cells per well for 24 h. The PBS, PLHPVP, Au@PLHPVP-PEG Ve and JNP@PLHPVP-PEG Ve were added in the well, respectively. After 3 h incubation, the solution was removed and replaced with fresh cell culture medium. After additional 24 h incubation, the Annexin-FITC detection was processed using flow cytometry following the manufacturer’s protocol. The data was analyzed using FlowJo.

PET Imaging of Tumor-bearing Mice Treated with [64Cu] labeled Hybrid Vesicles


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For in vivo PET imaging, [64Cu] labeled JNP@PLHPVP-PEG vesicle was prepared according our previously reported method. When the tumor volume reach about 60 mm3, 200 µCi [64Cu] labeled JNP@PLHPVP-PEG vesicles in PBS was intravenously injected into the tumor-bearing mice. Whole-body PET imaging of the mice was processed using an Inveon micro PET scanner (Siemens Medical Solutions).

MR Imaging and T2 Contrast Test of the Tumor For in vivo MR imaging, tumor-bearing mice were anesthetized by isoflurane (1.5~2.0%) in oxygen and placed in an animal-specific body coil. T2-weighted images of the tumor were obtained at before and after intravenous injection of the bilayer vesicle on axial planes focusing region of interest (ROI) of tumor. Multi-slice multi-echo sequence was employed to obtain MR images using the following parameters: echo time = 30 ms, repetition time = 2000 ms, flip angle = 180, slices = 16, slice thickness = 1 mm, matrix size = 256 × 256, field of view (FOV) = 40 × 40 mm2. We employed MR compatible small


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animal respiratory gating device to reduce the artifacts caused by respiration. We employed Image J, a NIH supported software, to analyze the MR images.

In Vivo Photoacoustic Imaging The bilayer vesicle in PBS (200 μL) was injected into the tumor-bearing mice through tail vein when the tumor volume was approximately 60 mm3. Afterwards, the whole tumor region was scanned using a VisualSonic Vevo 2100 LAZR system equipped with a 40 MHz and 256-element linear array transducer at predetermined time points.

In Vivo Anticancer Efficacy

DOX loaded hybrid vesicle was intravenously injected into the mice when the tumor volume reached ~60 mm3. The injection of vesicles (200 µL, 1 mg/mL of vesicle, and about 2 mg equivalent DOX/kg of the mouse body weight) was repeated every two days for three times. The tumor volume was measured every two days to evaluate the cancer therapy effects. The tumor volume was calculated using the following equation: V=Lⅹ W2/2, where L and W is the length and width of the tumor, respectively. The tumor volume


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is normalized against to the original volume before injection of the samples. Other groups were done using the same protocol. The mice weight was recorded every two days after injection of the samples.

ASSOCIATED CONTENT

Supporting Information. Supporting data includes GPC, TGA, TEM, ILTC, and DLS measurements and properties analysis of the bilayered vesicles. The model and simulation details, in vitro/vivo fluorescence, photoacoustic and MR imaging of the bilayered vesicle are also presented. This material is available free of charge via the internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Zijian Zhou: [email protected]


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Zhan-Wei Li: [email protected] Huanghao Yang: [email protected] Xiaoyuan Chen: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Acknowledgments

This work was supported by the intramural research program of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH). This research was supported by the National Natural Science Foundation of China (Nos. U1505221, 21475026, 21874024, 21674116, 21474110) the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT15R11), and the Youth Innovation Promotion Association CAS (2018257).


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Notes The authors declare no competing financial interest.

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