Sequential Intercellular Delivery Nanosystem for Enhancing ROS

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Sequential Intercellular Delivery Nanosystem for Enhancing ROS-induced Anti-tumor Therapy binghua wang, huifang zhang, jingyi an, yiwen zhang, lulu sun, yajie jin, Jinjin Shi, mengjia li, hongling zhang, and zhenzhong zhang Nano Lett., Just Accepted Manuscript • Publication Date (Web): 29 Apr 2019 Downloaded from http://pubs.acs.org on April 29, 2019

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Sequential Intercellular Delivery Nanosystem for Enhancing ROS-induced Anti-tumor Therapy Binghua Wang1,2,3, Huifang Zhang1, Jingyi An1, Yiwen Zhang1, Lulu Sun1, Yajie Jin1, Jinjin Shi1, Mengjia Li4, Hongling Zhang1,2,3*, Zhenzhong Zhang1,2,3* 1 School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, China 2 Key Laboratory of Targeting Therapy and Diagnosis for Critical Diseases, Henan Province, China 3 Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou, China 4 School of Materials Science and Engineering, Zhengzhou University, Zhengzhou, China * Corresponding Author: Email address: [email protected] (Hongling Zhang), [email protected] (Zhenzhong Zhang); Tel.: +86 371 6778 1910. Fax: +86 371 6778 1908. Mailing address: No.100, Kexue Avenue, Zhengzhou 450001, China

ABSTRACT Despite recent advances in enhancing photodynamic therapy (PDT) efficacy, high-efficiency reactive oxygen species (ROS)-based therapy approach, especially in malignancy tumor treatment, remains challenging. Relieving the hypoxia of tumor tissue has been considered to be an attractive strategy for enhancing ROS-based treatment effect. Nevertheless, it is frequently neglected that the hypoxic regions are usually located deep in the tumors and therefore are usually inaccessible. To address these limitations, herein we constructed a sequential intercellular delivery system (MFLs/LAOOH@DOX) that consists of a membrane fusion liposome (MFLs) doped with Linoleic Acid Hydroperoxide (LAOOH) in the lipid bilayer, and anti-tumor doxorubicin (DOX) encapsulated inside. In this report, LAOOH, one of the primary products of lipid peroxidation in vivo, was selected as ROS-generated agent herein, which dependent Fe2+ rather than oxygen and other external stimuli to produce ROS. Upon the

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enhanced permeation and retention effect (EPR), MFLs/LAOOH@DOX firstly fused with tumor cell membranes in the perivascular region in synchrony with selective delivery of LAOOH into the plasma membrane and the on-demand intracellular release of DOX. By hitchhiking with extracellular vesicles, LAOOH, as cell membrane natural ingredient, spread gradually to neighboring cells and throughout the entire tumor eventually. Combining with subsequent administration of nano Fe3O4, ROS was specially generated on tumor cell membrane by LAOOH throughout the tumor tissues. This study offers a new method to enhance ROS-based anti-tumor treatment efficiency. KEYWORDS: Photodynamic Therapy; Reactive oxygen species; Linoleic Acid Hydroperoxide; Membrane Fusion Liposome; Nano Fe3O4; Extracellular Vesicles;

Introduction Reactive oxygen species (ROS), including singlet oxygen (1O2), superoxide radicals (O2•−), hydroxyl radicals (•OH), and peroxides (O22−), play imperative roles in the cell life cycle1-3. At lower concentrations, ROS serve as a key signaling molecule to modulate cell growth, proliferation, and differentiation4. However, aberrant generation of ROS in cell could cause oxidative damage to lipids, proteins, and DNA5. Normally, cells are capable of balancing the oxidative damage to protect cells from death. Whereas excessive production of ROS could overwhelm this homeostasis, resulting in irreversible oxidative damage to the cells and eventually cell apoptosis6-9. Breaking the threshold of ROS level to induced tumor cell death has been extensively applied to cancer treatment9-13. Photodynamic therapy (PDT), a representative approach of ROSbased cancer therapies, show tremendous potential to kill cancer cells with reduced side effects, negligible drug resistance, and low minimal toxicity14-17. Despite PDT have been approved by the U.S. Food and Drug Administration (FDA) as a clinical cancer treatment method, in most cases it remains difficult to generate sufficiently therapeutic efficiency for further application as a first-


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line cancer treatment option18. Hypoxia, a common pathophysiological characteristic within many types of solid tumors, has been widely considered to be mainly responsible for the compromise clinical outcomes of PDT in human cancer treatment19-22. To address this limitation, substantial efforts have been devoted to relieve the hypoxia of tumor tissue, such as, applying perfluorocarbon as oxygen selfenriching system23, catalysts25,

26.

24,

in situ generation of oxygen inside the tumor with

However, these strategies still dependent on complexity

photosensitive effect, in which oxygen is necessarily involved in the treatment process. To further magnify ROS generation, extensive research efforts have been directed towards the design of O2-independent ROS-generating systems to overcome hypoxia-induced PDT resistance6,

27-30.

For instance, Qu and

coauthors applied nanozymes to catalyze intracellular biochemical reactions to produce ROS and then induce intracellular oxidative damage against hypoxic tumors31. Although promising, these systems frequently neglect the fact that the hypoxic regions are usually located deep in the tumors and therefore are usually inaccessible to the intravenously injected drug carrier or drug32. Abnormal tumor microvessels and increased diffusion distances from cells to blood vessel induced insufficient oxygen supply while making drug or nanoparticles with little opportunity to reach the deep tumor cell33-35. Therefore, it is highly desired to develop a paradigm that not only can directly generate ROS inside tumor hypoxic microenvironment, but also effectively deliver ROSgenerated agent to tumor deeper. It is increasingly evident that extracellular vesicles (EVs) have a key role in intercellular communication by transferring proteins, lipids and nucleic acids3639.

Inspired by intercellular lipid transfer mediated by EVs, we propose a new

treatment modality to enhance ROS-based antitumor efficiency, in which an artificial membrane fusion liposome (MFLs) composed of DOPC/DOPE/SM/CH (35/30/15/20) was proposed as antitumor agent-carrier40,

41.

linoleic acid

hydroperoxide (LAOOH), one of the primary products of lipid peroxidation in


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vivo42,

43,

was loading within the phospholipid bilayer of MFLs, meanwhile,

clinically used doxorubicin (DOX) was selected as the model drug to be encapsulated into the hydrophilic core of MFLs, obtaining MFLs/LAOOH@DOX. Compared with conventional photosensitizer, LAOOH was used in this report as ROS-generated agent, which generated ROS catalyzed by metal ions (for example, Fe2+, Ce4+) rather than dependent on O2 and other external stimuli4446.

Firstly, MFLs/LAOOH@DOX selectively fused with tumor cell membranes in

the perivascular region via the enhanced permeation and retention effect (EPR). Followed the intercellular lipids exchange mediated by EVs, LAOOH, as cell membrane natural ingredient, spread gradually from the outer layer tumor cells to neighboring cells closer to the center of the tumor and distribute throughout the entire tumor eventually. With the assistance of subsequent administration of nano Fe3O4, which was dissociated to Fe2+ under tumor tissue or lysosome acidic pH environment, tumor membrane-locating LAOOH was catalyzed and produced abundant ROS through the Russell mechanism, resulting in tumor cells apoptosis and necrosis (Scheme 1). Furthermore, DOX be efficiently delivered into nucleus of tumor cells accompanied by membrane fusion between MFLs and tumor cells, thus decreasing DOX degradation in lysosomes (Scheme 2). This sequential intercellular delivery system mediated by EVs, which are not directional, like the random walk Brownian motion of molecules causes diffusion, offers a new ROS-based modality for treatment of malignant tumors.


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Scheme 1. Schematic design of MFLs-based anticancer drug delivery for treatment of malignant tumors. Upon membrane fusion effect between MFLs and tumor cells, ROSgenerated agent-LAOOH spread autonomously the entire tumor via EV-mediated intercellular transport like Brownian motion. With the assistance of subsequent administration of nano Fe3O4, substantial ROS was produced on tumor cells plasma membranes specifically. Combining with anti-tumor drug DOX, the final formulation (MFLs/LAOOH@DOX) inhibited tumor growth effectively.

Results and Discussion


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Figure 1 Characterization of MFLs formulation. A) Transmission electron microscopy images of MFLs (scale bar 100 nm). B, C) Size and zeta potential of the MFLs analyzed by DLS, respectively. D) Detection of ROS generated from LAOOH under different pH (7.4, 6.5, 5.5), respectively. E) Release profiles of DOX from different formulations in PBS with or without Fe2+. “+” represent adding Fe2+.Data are presented as mean ± SD from three independent experiments. Statistical significance was calculated by Student’s t-test, ***P < 0.001, **P < 0.01, *P < 0.05.

We firstly synthesized LAOOH through photooxidation of linoleic acid under 660 nm laser, using methylene blue as the photosensitizer. Liquid chromatography– mass spectrometry (LC-MS) was employed to confirm the successful production of LAOOH. As shown in Figure S1, the mass spectra of linoleic acid displayed a molecular ion [M-H]- at m/z 279, moreover, the mass spectrum of LAOOH exhibits an intense ion at m/z 311, corresponding to the hydroperoxide molecular ion [M-H]-. Additionally, the result of ultraviolet (UV) absorption spectrum also confirmed the successful synthesis of LAOOH (Figure S2). Next,


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we loaded the purified LAOOH and DOX in the hydrophobic lipid bilayer and hydrophilic inner core of MFLs, respectively. Transmission electron microscopy (Figure 1A) reveals that MFLs/LAOOH@DOX possess uniform shape with primary size of around 70 nm. By utilizing dynamic light scattering (DLS), the hydrodynamic diameter of MFLs/LAOOH@DOX was measured to be 144 nm (Figure 1B), which was larger than that of TEM result, mainly because of the formulation of hydration shell on the liposome surface. Additionally, the zeta potential (ζ) of MFLs/LAOOH@DOX was also measured by DLS and displayed a negative ζ potential (−15.2 mv) (Figure 1C). We hypothesized that LAOOH, one of the hyperoxide in mammalian cell, could be used as a potential ROS generating agent to circumvent complex photosensitizer effect, therefore the ability of LAOOH to generate singlet oxygen (1O2) was researched in detail. An increase in the fluorescent intensity (F.I.) of singlet oxygen sensor green (SOSG) indicated that the 1O2 was generated from LAOOH through catalysis of iron(II) (Figure S3). Next, we directly compared the generation rate of 1O2 by the LAOOH with the hermimether (HMME), a wildly applied photosensitizer in clinical (Figure S4). By measuring the F.I. of SOSG, we found that 1O2 generated by LAOOH and HMME was dependent on Fe2+ concentration and laser irradiation time, respectively. Simultaneously, we found that LAOOH could not cause evident 1O2 production in very light concentration of Fe2+ (LAOOH : Fe2+ > 100 : 1), implying that microelement of iron in body not affect the LAOOH stability. Together, these results indicated that LAOOH could act as a potential ROS-generated agent. It is well known that most tumors tend to develop an acidic microenvironment owing to abnormal tumor microvessels and poor perfusion. Next the profile of iron release from Fe3O4 was assessed under different pH values (that is, 6.5 and 5.5) to mimic the tumor environment and especially endosome (or lysosome). Firstly, TEM results indicated that the Fe3O4 possessed good monodispersity in water and diameter of about 10 nm (Figure S5). Additionally, Figure S6 demonstrated that the degree of ferric ions released


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from Fe3O4 is a pH-dependent manner. Under pH 7.4, the release rate of ferric ions was < 5% within 48 h incubation. Whereas the release rate relatively increased under acid condition, particularly, under pH 5.5 condition, the Fe3O4 showed 30% release of ferric ions for 48 h incubation. We next evaluated whether the iron ions production in acidic environment would be beneficial to the ROS generation from LAOOH. As revealed by Figure 1D, under pH 5.5, 1O2 was efficiently generated by LAOOH, whereas it was rarely detected in pH 7.4. This indicated that LAOOH could be catalyzed effectively by Fe3O4 under acidic environment, because of ferric iron (II) releasing. If ROS generated by LAOOH could damage tumor cells, theoretically it also could breakdown liposome and therefore triggered DOX burst release. To address the potential toxicity of LAOOH toward tumor cells, the release profile of the encapsulated DOX from different liposomes with or without ferric iron (II) was determined. As shown in Figure 1E, the result indicates that the release profile of DOX from MFLs/LAOOH@DOX was strongly correlated with ROS generation as a result of iron (II) addition. Additionally, the TEM image also confirmed that the morphology of MFLs/LAOOH was destroyed upon Fe2+ incubation (Figure S7). These results confirmed that the MFLs/LAOOH nanoparticles in the presence of iron (II) resulted in the generation of ROS.


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Figure 2. Investigation of the ability of MFLs/LAOOH to generate ROS in 4T1 cell in vitro. A) CLSM examination of ROS generation in 4T1 cells treated by MFLs/LAOOH with or without Fe3O4. The cells were co-stained by ROS probe (DCFH-DA) before image. Images were taken by confocal microscope (LEICA TCS SP8, Germany). “+” or “-” represent with or without Fe3O4, respectively. B) Flow cytometry analysis of the fluorescence intensity of tumor cells as shown in A.

Next, the ability of LAOOH to generate ROS in cell level was further examined using 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA) probe. Meanwhile, conventional liposomes (CLs) shielded with PEG chains on the surface were employed as a control. As confocal microscopy images shown (Figure 2A), the F.I. of DCFH-DA had negligible change after 4 h incubation with MFLs/LAOOH in different concentration, whereas further adding nano Fe3O4 into the medium led to an increase of F.I. in both MFLs/LAOOH and CLs/LAOOH group, which deepened

monotonically

with

increasing

LAOOH

concentration.

Simultaneously, we observed that the morphology of cells in MFLs/LAOOH plus Fe3O4 groups changed dramatically, which lost their original phenotype, whereas the cells treated with CLs/LAOOH did not show any distinct morphological change. These results implied that the LAOOH delivered by MFLs shown more cytotoxicity to tumor cells47. Additionally, flow cytometry showed

an

increased

level

of

ROS

for

cells

incubated

with

MFLs/LAOOH+Fe3O4, compared with those incubated with MFLs/LAOOH along (Figure 2B).


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Scheme 2. Schematic illustration of cellar internalization of MFLs and CLs, respectively.


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Figure 3. Cellar internalization and cytotoxicity assessment in vitro. A) CLSM images of 4T1 cells after cultured with CLs or MFLs. Images were taken by confocal microscope (LEICA TCS SP8, Germany). The merged image is the overlay of the individual images. B) Annexin V/PI staining assays of 4T1 cells after incubated with different formulation for 24h with or without Fe3O4. X-axis shows FITC-labeled Annexin V-positive cells and Y-axis shows PI-positive cells.

We further investigated the subcellular localization of MFLs in 4T1 (mouse breast carcinoma) cell lines. To track the distribution of liposomes in tumor cell, the 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (DII) were anchored into the lipid bilayer of liposomes. Confocal microscopy images revealed that MFLs delivered lipid mainly on the surface of tumor cells, and the fluorescent


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intensity increased over time (Figure S8). Thus, we guessed that the MFLs were internalized into tumor cells through the membrane fusing approach. In order to prove this idea more directly, we next examined the distribution of DOX and lipid of MFLs/LAOOH@DOX in tumor cells simultaneously, in which DOX was encapsulated in the hydrophilic core of liposomes. The lipid membrane of liposome was stained with 3,3′-dioctadecyloxacarbocyanine perchlorate (DIO). Theoretically, if MFLs were based on membrane fusing approach into tumor cell, DOX and lipid membrane of MFLs should show different subcellular distribution (as illustrated by Scheme 2). As expected, the Figure 3A results revealed that DOX (red fluorescence) and lipid membrane (green fluorescence) shown evidently different distribution in cells treated with MFLs/LAOOH@DOX. DOX mainly located in cell nucleus, whereas green fluorescence represented liposomes membranes distributed primarily into the plasma membranes of cancer cells. In contrast, the green fluorescence delivered intracellularly by CLs were observed as dots in the cytoplasm, indicating that most of CLs lipid were trapped in the endosomes/lysosomes. Unexpectedly, in CLs/LAOOH@DOX group, except to cell cytoplasm, cell nucleus shown obvious green fluorescence, which co-localized with DOX. This likely because that CLs were internalized through endocytosis pathway and subsequently localized in the endosomes. Under acid condition and enzymatic degradation environment of lysosomes, DIO was released from CLs lipid and then enter in nucleus. Additionally, considering the membrane permeable ability of DOX, next we applied propidium iodide (PI) instead of DOX to assess membrane fusion of MFLs48. As shown in Figure S9, PI also could be transported into cell nucleus by MFLs distinctly, and liposomes membranes distributed into the plasma membranes of cancer cells simultaneously. Collectively, these results demonstrate that MFLs enable efficient and selective delivery its lipid constituent into the plasma membrane of cancer cells. In this report, LAOOH, a lipid peroxidation, was encapsulated in the lipid bilayer of MFLs as the liposome lipid membrane ingredient. Based on the membrane-specific delivery of MFLs, LAOOH was


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delivered selectively to the plasma membrane of tumor cell. Next the ability of MFLs/LAOOH@DOX to induce tumor cells apoptosis was studied. We conducted the cytotoxicity study of these nano-formulations in 4T1 cell lines by Annexin V/PI co-staining and flow cytometry assay. As shown in Figure 3B, after 24 h incubation, MFLs/LAOOH@DOX induced obvious apoptosis with an apoptotic rate of 45.2%, significantly higher than CLs/LAOOH@DOX (30.5%). This phenomenon was ascribed to the different internalized pathway between MFLs and CLs by tumor cells probably. As previous study, we have confirmed that MFLs were internalized into tumor cells through membrane fusion, so DOX encapsulated in liposome hydrophilic core could directly enter in cell cytoplasm and therefore improved their translocation to the nucleus (Scheme 2). When incubated

with

Fe3O4

for

24h,

both

CLs/LAOOH@DOX

and

MFLs/LAOOH@DOX noticeably increased tumor cell apoptosis, which implied that the ROS was generated and could improve the therapeutic potential of these

formulations.

It

is

noteworthy

that

the

cell

apoptosis

in

MFLs/LAOOH@DOX group increased primarily on later apoptosis (23.9%51.0%), significantly higher than CLs/LAOOH@DOX sample (17.5%-30.4%). In line above mentioned experiment, concomitant with substantial membranespecific spreading of MFLs lipid, LAOOH was localized on tumor cell membranes selectively. Accordingly, the ROS was generated on cell membranes specifically, thus resulted in tumor cell membrane integrity losing. It is rational that the red luminescent PI entered into tumor cells readily because of increased permeability of damaged tumor cell membranes. Simultaneously, the

dose-dependent

cytotoxicity

of

DOX,

CLs/LAOOH@DOX

and

MFLs/LAOOH@DOX was characterized by MTT (Figure S10). In agree with cell apoptosis results, when incubation with Fe3O4, MFLs/LAOOH@DOX showed the most inhibition effect in cell proliferation. Taken together, these experiments demonstrate that the LAOOH can be localized efficiently on the plasma membrane of tumor cells via MFLs-mediated membrane fusion, and selectively generated ROS on the plasma membrane of tumor cells, thus


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increasing the antitumor efficiency of ROS-based cancer therapies.

Figure 4. Transwell experiments. (A) Schematic drawing showing the design of the transwell system experiment. 4T1 tumor cells were seeded in the upper chamber and cocultured with different formulation for 4 h. B) Confocal fluorescence images of 4T1 cells in the transwell lower chamber. The nuclei of 4T1 cells were stained with Hoechst 33342, and the liposome membrane were stained with DIO. Scale bar, 10 μm. (C) Examination of ROS generation in 4T1 cells in the transwell lower chamber.

It is well known that EVs play an important role in cell to cell communication. Through packaging cytosolic contents with the membrane of parental cells, EVs


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were secreted from the cells. Based on communication exchange, lipids in the membrane of parental cells be incorporated in the membrane of EVs and further be transferred to the membrane of neighboring and distant tumor cells. Herein, we loaded LAOOH, a lipid peroxidation that could generated ROS catalyzed by Fe2+, in the lipid bilayer of MFLs, expecting that LAOOH could be delivered to deep tumor cell surfaces via EV-mediated intercellular lipid transfer. Previously, we have verified that MFLs were internalized in tumor cells through membrane fusion. Next, we studied whether lipid of MFLs could be transferred on neighboring cell surfaces through a transwell experiment (As illustrated by Figure 4A). 4T1 cells in the upper filter with 400-nm pores were pre-incubated with different formulations for 4 h, then take away the formulation and coincubated with the fresh cells on the lower chamber for 8 h. By the confocal microscopy results (Figure 4B), the highest level of green fluorescence intensity was detected on the lower chamber cell when MFLs delivered lipids to the upper filter cells. Importantly, the fluorophore-lipid of MFLs were highly colocalized with the plasma membrane of 4T1 cells in the lower chamber. Contrarily, in CLs group, the lower chamber cell shown faint fluorescence, and the green fluorescence mainly localized into cell cytoplasm and nucleus rather than the surface of the lower chamber cells. These results demonstrate that the selective delivery of lipids into the plasma membrane significantly enhanced the lipid transfer of MFLs to neighboring tumor cells. Next, we doubted whether the ROS generated agent-LAOOH would be transferred into the neighboring tumor cells accompanied with the lipid transport of MFLs. We proceeded to assess the ROS-generation in lower chamber cells (Figure 4C). After incubation with Fe3O4, the generation of ROS was scarcely observed in the lower chamber cells when LAOOH were delivered to the upper filter cells by CLs. However, substantial ROS was generated in the neighboring cells on lower chamber of MFLs group. These results suggested that, based on the membrane fusion of MFLs, LAOOH was not only delivered selectively to the plasma membrane of tumor cells but also autonomously transferred to the plasma membrane of


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neighboring tumor cells. Taken together, these results demonstrated that the locally elevated LAOOH concentration at the cell membrane could accelerate its intercellular penetration.

Figure 5. The penetration of MFLs in vitro 3D multicellular tumor spheroids. A) Penetration of different formulations into the 3D 4T1 tumor spheroids after incubation for 8 h. CLSM images were obtained from the surface to the middle of the tumor spheroids in a Z-stack thickness of 40 µm. B) The 3D images of entire tumor spheroid. Green fluorescence represented DIO, red fluorescence represented DOX. Scale bar, 100 µm.


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Since intercellular spreading of MFLs was observed, we next evaluate the tumor-penetration

capability

of

MFLs-based

formulation

using

three-

dimensional (3D) multicellular tumor spheroids49, 50. In this experiment, the lipid bilayer of liposome was stained with DIO to visualize the permeability of the nanomedicines. After incubation with MFLs/LAOOH@DOX for 8 h, both DIO and DOX signals were monitored simultaneously in the middle of regions of tumor spheroid, even 120 μm from the surface towards the middle. Quantitative analysis demonstrates that lipid of MFLs are capable of permeating nearly 40% of a tumor spheroid with an average diameter of 400 µm. However, the CLs were not detectable except for trace amounts accumulated on the periphery of the tumor spheroid (Figure 5A). Considering the similar size and electric potential between MFLs and CLs, this difference seems to be caused by their different cell-internalized pathway. Indeed, from the 3D image of tumor spheroid, we clearly observed that DIO and DOX from the MFLs mostly localized on the cell plasma membrane and the cell nucleus, respectively. While a high degree of co-localization between the DOX and DIO signals was found in tumor spheroids treated with CLs/LAOOH@DOX (Figure 5B). These results suggesting that MFLs remain based on membrane fusion into tumor cells during the penetration process. Firstly, MFLs-membrane fused with the peripheral tumor cell membrane, then the locally elevated MFLs-lipid (such as LAOOH) transported autonomously to the neighboring tumor cells membrane via EVsmediated intercellular lipid transfer. Iterated exocytosis and fusion of EVs, which are not directional, accelerate the transport of MFLs-lipid into the tumor tissue deeper in a manner similar to that the random walk Brownian motion of molecules causes diffusion. Therefore, it is comprehensible that MFLs was capable of penetrate more deeply into tumor tissue.


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Figure 6. The penetrated-capacity of MFLs-based nanomedicines in zebrafish embryos. A) Lateral views of the whole embryos treated with different formulation. Green fluorescence represented DIO, red fluorescence represented DOX. B) Assessment of intranuclear delivery using liposomes loaded with PI.

In this study, we used the zebrafish embryos as a vertebrate screening model to evaluate the penetrated-capacity of MFLs-based nanomedicines directly in vivo, thus bridging the gap between in vitro cell-based models and in vivo mammalian models. As the first step toward clinical application, the zebrafish possess unique advantages including high reproducibility, high level of genetic homology to humans and most importantly optical transparency51, 52. Embryos of zebrafish were stripped off the egg sheath 48 hpf. During the embryonic stages, the zebrafish skin is composed of a layer of ridged, mucus-covered enveloping layer cells (EVLs). Due to mucus covering or membrane ridging, EVLs was inaccessible. Therefore, it is necessary to across the EVLs if someone want to reach cells within the underlying epidermal basal layer (EBL). We exposed 48-h-old zebrafish embryos to different formulation in embryo medium for 1 h. As shown in Figure 6A, an enhancement of both DIO and DOX


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fluorescence signals occurred in MFLs treated zebrafish embryos as compared to CLs. CLs incubated zebrafish shown a very slight fluorescence and appeared to be at the periphery of the larvae sac. It is ascribed that the pegylated surface of the conventional liposome limited the penetrate in EVLs. However, the DIO signals of MFLs were observed randomly throughout the sac of larvae. The distribution of the MFLs-lipid can be partially explained by the membrane fusion of MFLs which allows liposomes diffuse to neighboring cells. Additionally, we further confirmed intracellular delivery using liposomes loaded with PI, which becomes highly fluorescent only after interaction with cellular DNA or RNA. As shown in Figure 6B, obvious PI fluorescence signals was observed in zebrafish embryos as dots, indicated that MFLs could deliver efficiently therapeutic agent into cell nucleus.

Figure 7. In vivo animal experiment. A) Therapeutic effect of systemic administration of different therapeutic formulations against 4T1 tumor xenograft. B) Panorama scanning analysis of hearts in 4T1 xenograft mice treated with various formulations. The black arrows represent ventricular wall. Scale bar, 1000 μm. C) Fluorescence images of 4T1


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tumor sections after intravenous injection of CLs or MFLs. Scale bar, 1000 μm. n= 6 in each group. Data are presented as mean ± SD from three independent experiments. Statistical significance was calculated by Student’s t-test, ***P < 0.001, **P < 0.01, *P < 0.05.

To investigate the therapy efficacy of MFLs-based formulation, BALB-c micebearing 4T1 tumors were intravenously injected with saline, DOX (5 mg/kg), MFLs/LAOOH, CLs/LAOOH@DOX, MFLs/LAOOH@DOX with or without Fe3O4 (10 mg/kg). To optimize the therapeutic schedule, the pharmacokinetics of MFLs/LAOOH@DOX and Fe3O4 were evaluated to elucidate their behaviors in vivo. We firstly researched the in vivo distribution of MFLs/LAOOH@DOX and nano Fe3O4. DIR (1,1'-dioctadecyl-3,3,3',3'-tetramethyl indotricarbocyanine Iodide) labeled MFLs/LAOOH@DOX was intravenously injected into tumor xenografts models and the fluorescent was monitored by Two-dimensional fluorescence reflectance imaging (2D FRI). As Figure S11 shown, the tumor accumulation of MFLs/LAOOH@DOX reached maximum at 1 h after i.v. injection and remain at the higher level until 48 h. Furthermore, the time of the maximum enrichment of nano Fe3O4 was at 4 h after i.v. injection. Thus, we decided that Fe3O4 following MFLs/LAOOH@DOX injection be administered at 1 h. Simultaneously, the blood circulation time of MFLs/LAOOH@DOX and Fe3O4 was assessed by High Performance Liquid Chromatography (HPLC) and Inductively Coupled Plasma Mass Spectrometry (IPC-MS) respectively. According to blood circulation curve (Figure S12), the blood circulating half-life of MFLs/LAOOH@DOX and Fe3O4 was 35.8 h and 23.3 h respectively. Therefore, the final therapeutic schedule was decided: MFLs/LAOOH@DOX was injected every 36 h, and after 1 h, the Fe3O4 be administered. As presented in Figure 7A, MFLs/LAOOH plus Fe3O4 exhibited certain therapeutic outcome, but failed to inhibit the growth of tumors and weaker than DOX. Comparatively, the inhibitory effect of MFLs/LAOOH@DOX is much better than the free drug DOX applied under the same concentration, which mainly contributed that the


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EPR effect of tumor promoted the penetration of nano MFLs/LAOOH@DOX in tumor tissue. Evidenced by Two-dimensional fluorescence reflectance imaging (2D FRI) results (Figure S11), the MFLs indeed enhanced the tumor specific accumulation and retention compared with sole IR783. Furthermore, MFLs/LAOOH@DOX exhibits more prominent antitumor efficacy upon Fe3O4 administration than the sole MFLs/LAOOH@DOX, which decreases the tumor volume by 78% compared with the control group. This result illustrated that Fe3O4 plus MFLs/LAOOH@DOX inhibit tumor in a combinatorial way. More importantly, compared with CLs/LAOOH@DOX+Fe3O4 (47% tumor volume of saline), MFLs/LAOOH@DOX+Fe3O4 produced the more effective tumor inhibition at day 13. Consistent with the results of tumor growth curves, DOX and CLs/LAOOH@DOX+Fe3O4 induced moderate amounts of apoptosis as compared with the saline control group. However, a large number of apoptotic cells were detected in tumor of mice received MFLs/LAOOH@DOX+Fe3O4 (Figure S13). Taken together, these results indicated that MFLs have a direct effect on the excellent therapeutic efficacy. To visualize the difference between MFLs and CLs in antitumor efficiency, we next studied the penetrated degree of these liposomes in tumor. As Figure 7C results revealed, compared with injection with conventional liposomes, the MFLs-based nanosystem exhibited superior accumulation in both peripheral and interior regions. Accordingly, we also observed that ROS was produced substantially in the tumor of mice treated with MFLs/LAOOH+Fe3O4. Whereas, in CLs/LAOOH+Fe3O4 treated mice, we only detected ROS in a small area of tumor tissue (Figure S14). Taken together, these observations demonstrate that tumor cell membrane-selective delivery mediated by MFLs could enhanced tumor deeper penetration of therapeutic agents and thus improve the antitumor treatment efficiency. Although nano Fe3O4 were internalized into cells by endocytosis that is same with CLs, Fe2+ as the degradation product of Fe3O4 more easily penetrate into tumor deep than conventional antitumor drug or photosensitizer, because these antitumor therapeutic agents may be degraded in tumor edge prior to reach to tumor


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center. Additionally, from Figure S15, we observed that, except to DOX, all of the drug-treated mice do not show obvious body weight loss, implying that MFLs/LAOOH@DOX has no obvious system toxicity under the in vivo experimental conditions. To further investigate the systemic toxicity in details, after the therapy studies, the hearts of mice were harvested for immunohistochemical analysis. As shown in Figure 7B, abnormal thinning of the ventricular wall was observed for the free DOX treatment, but it is normal for the MFLs/LAOOH@DOX treatment, suggesting that the nanomedicine we developed could decrease drug retention in heart. Furthermore, images of hematoxylin

and

eosin

(H&E)

stain

confirmed

that

nanomedicine

MFLs/LAOOH@DOX had little toxicity to various major organs (liver, spleen, lung,

kidney)

of

mice

(Figure

S16).

Since

the

potential

risks

of

MFLs/LAOOH@DOX in inducing inflammatory responses would be reflected in hematological factors, the standard haemotology markers including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), mean corpuslar hemoglobin (MCH), mean corpuslar volume (MCV) and mean corpuslar hemoglobin concentration (MCHC) were also measured. No significant difference between MFLs/LAOOH@DOX-treated groups and control group was observed at all blood indexes, elucidating that no significant infection and inflammation were arose by MFLs/LAOOH@DOX (Figure S17). These results indicate that the MFLs/LAOOH@DOX nanosystem has excellent therapeutic capability without noticeable toxicity, which favors their further safe bioapplications.

Conclusion For effective photodynamic therapy, it is necessary to generate sufficiently ROS to all of the cells within tumor tissues to exert a therapeutic effect. This has been challenging since meticulous coordination between light, photosensitizer (PS), and oxygen confers the application of PDT in cancer treatment with complexity and difficulty. In this report, we applied linoleic acid hydroperoxide (LAOOH) as


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ROS-generated agent. In contrast to conventional PS, LAOOH generated ROS is catalyzed by Fe2+, which independent of oxygen or other external stimuli. Considering the fact that the hypoxic regions are usually located deep in the tumors and inaccessible, we developed a membrane fusion liposome-based nanosystem, in which LAOOH was loaded within the phospholipid bilayer and DOX

was

encapsulated

into

the

hydrophilic

core

of

MFLs

(MFLs/LAOOH@DOX). The obtained MFLs/LAOOH@DOX was able to deliver selectively LAOOH into the plasma membrane of tumor cells accompanied by the on-demand intracellular release of DOX to induce the cell death. Based on intercellular lipids exchange mediated by EVs, LAOOH gradually distribute throughout the entire tumor eventually. We believe that this sequential intercellular delivery system will provide opportunities to enhance ROS-induced antitumor efficacy through improving ROS productivity and tumor distribution simultaneously.

Statistical analysis Data were presented as mean ± standard deviation (SD). The oneway analysis of variance (ANOVA) was performed in statistical evaluation. A p-value below 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001) was considered to be statistically significant.

ACKNOWLEDGEMENTS The work is supported by grants from the National Natural Science Foundation of China (Nos.81573364, 81572991), Science and Technology Project of Henan Province (162102310510) and Modern Analysis and Computer Center of Zhengzhou University.

COMPETING FINANCIAL Interests The authors declare no competing financial interests.


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Experimental section Materials Doxorubicin (DOX, purity > 98%) was gotten from Beijing Yi-He Biotech Co. Ltd.

1,

2-dioleoyl-sn-glycero-3-phosphocholine

(DOPC),

Dioleoyl

Phosphatidylethanolamine (DOPE) were purchased from Switzerland Solarbio Co. Ltd. Sphingomyelin (SM) was purchased from Sigma-Aldrich. Linoleic acid (LA, purity > 98%) was purchased from Shanghai Yuanyebios. RPMI 1640 cell culture medium, penicillin, streptomycin, fetal bovine serum (FBS), and heparin sodium were bought from Gibco Invitrogen. Other reagents were acquired from China National Medicine Corporation Ltd. Cell culture The mice breast carcinoma 4T1 used in this study was from the China Center for Type Culture Collection at Wuhan University (Wuhan, Hubei, China). 4T1 cells were cultured in RPMI 1640 medium with 10% fatal bovine serum (FBS) and 1% penicillin-streptomycin solution. All cells were maintained at 37 °C in an atmosphere with 5% CO2 and cells in all experiments were performed at the stage of cell index growth. Animals Female BALB-C mice (18-22 g), aged 5-6 weeks, were feed at the condition of 25 °C and 55% of humidity in Experimental Animal Center of Zhengzhou University. All animal experiments were performed according to the guidelines approved by Henan laboratory animal center. Synthesis of Linoleic Acid Hydroperoxide (LAOOH) LAOOH was synthesized by photooxidation of linoleic acid using methylene blue as the photosensitizer. Briefly, 500 mg of linoleic acid was dissolved in 25 ml of chloroform, then 50 μl of methylene blue solution (100 mM in methanol) was added. Thereafter, the whole system was connected to an O2 gas cylinder. The mixture was irradiated with 660 nm laser irradiation for 5 h and stirred continuously. The LAOOH was purified by column chromatography and was stored in chloroform at -20 °C. The LAOOH concentration was determined by


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absorbance at 235 nm. Preparation of MFLs/LAOOH@DOX Lipids mixtures at appropriate molar ratios (DOPC/DOPE/SM/CH: 35/30/15/20) in chloroform (5 ml) were dried under nitrogen. The dried mixed lipids were dissolved in 1 ml of cyclohexane containing a small aliquot of methanol and 2 mg LAOOH. The mixed lipid was lyophilized under high vacuum overnight. The dried lipid powders were suspended in 1 ml DOX buffers (2 mg/ml) for 2 hours at room temperature. Then, the solution was dialyzed against deionized water for 48 h to remove unencapsulated DOX (MWCO 3000). The resulting MFLs/LAOOH@DOX nanosuspension was stored at 4 °C until use. Preparation of Conventional Liposome (CL/LAOOH@DOX) The conventional liposomes were fabricated by a solvent evaporation method as our previous work. Brieﬂy, DPPC (40 mg), cholesterol (5mg) and DSPE-PEG (5 mg) were dissolved in chloroform (10 ml) containing 2 mg LAOOH, after evaporation to remove the chloroform, the product was dispersed in DOX solution (4 ml, 5 mg/ml). After incubation at 40°C for 1h, the solution was dialyzed against deionized water for 2 days to remove unencapsulated DOX (MWCO 3000). The resulting CLs/DOX nanosuspension was stored at 4°C in the dark until use. Preparation of Fe3O4 Nanoparticles Brieﬂy, polymer poly(acrylic acid) (PAA, 5.56 mmol) was dissolved in water (50 mL), and the solution was then heated up to 80 °C in nitrogen atmosphere under refluxing condition. Meanwhile, the mixture (2 mL) of FeCl2⋅4H2O (0.54 mmol) and FeCl3⋅6H2O (0.279 mmol) were quickly injected into the hot polymer solution under vigorous stirring in a nitrogen atmosphere. Then, 25% NH4OH was added to the reaction mixture to adjust the pH value to 9–10. After another 4 h, the mixture was cooled to room temperature. The obtained precipitates were then separated from the supernatant solution by using a permanent magnet and thoroughly rinsed with water. Characterization of MFLs/LAOOH@DOX


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To identify the morphology of MFLs/LAOOH@DOX, a TEM (Tecnai G2 20, FEI) operated at 100 kV was employed. DLS (Zetasizer Nano ZS-90, Malvern, UK) was applied to examine the average size and zeta potential of particles. Each measurement was performed in triplicate, and a mean value was reported. LAOOH Singlet oxygen detection A fluorescence singlet oxygen sensor green (SOSG) was employed to evaluate the singlet oxygen generation of LAOOH. Briefly, a 20 μl of ethanol containing linoleic acid hydroperoxides (5 mM) was mixed with 1 ml PBS (pH 7.4) containing 3% SOSG. The fluorescence of the mixture solution was measured under excitation/emission (~ 490/525) nm as the background for comparison purpose. In the same way, a 20 μl FeSO4 solution with different concentration (0.05, 0.5, 5, 50, 500 μM) freshly prepared was added to the mixture solution. After incubation for 30 min under 37 °C, the fluorescence was measured and the singlet oxygen generation of LAOOH was detected by comparing the SOSG fluorescence enhancement with the control samples. Detection of ROS generated by HMME The method was the same as LAOOH Singlet oxygen detection. Briefly, a 20 μl of ethanol containing linoleic acid hydroperoxides (5 mM) was mixed with 1 ml PBS containing 3% SOSG. The fluorescence of the mixture solution was measured under excitation/emission (~ 490/525) nm as the background for comparison purpose. The mixture solution was exposure under 532 nm (1 W/cm2) laser with different time (0.5, 1, 2, 5, 10 min) and the fluorescence was then measured. The singlet oxygen generation of linoleic acid hydroperoxides was quantified by comparing the SOSG fluorescence enhancement with the background or control samples. Iron ions Release Studies Fe3O4 nanoparticles were incubated with different pH values of Tris/HCl buffer solutions (7.4, 6.5 and 5.5) for different durations. At the given time points, the supernatants were obtained through centrifugation and measured by ICP-MS. Then the precipitation at each tube was redispersed by the original pH buffer


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solutions after each centrifugation. Detection of ROS generated by LAOOH under different pH Firstly, Fe3O4 nanoparticles were incubated with different pH values of Tris/HCl buffer solutions (7.4, 6.5 and 5.5) for 1 h (1 mg/ml). Then 20 μl supernatant was added into 1 ml PBS containing 3% SOSG and 20 μl of ethanol containing linoleic acid hydroperoxides (5 mM). After incubation for 30 min under 37 °C, the fluorescence was measured. Release profiles of DOX from different formulations in PBS with or without Fe2+ MFLs/DOX, MFLs/DOX+Fe2+, MFLs/LAOOH@DOX, MFLs/LAOOH@DOX+ Fe2+ (DOX: 1 mg/ml, LAOOH: 1 mg/ml, Fe2+: 0.01 mg/ml) release were measured using dialysis membranes (MW cutoff 12-14 kDa; Spectrapor) in phosphate buffered saline at 37 °C with 10% FBS added to each dialysis tube. Dialysis samples were collected periodically and quantified the DOX by HPLC (1100; Aglient Technologies Santa Clara, CA, USA). Assessment of the ability of MFLs/LAOOH to generate ROS in cell level A ROS probe (DCFH-DA) were employed to evaluate the ROS generation in cell level. 4T1 cells were seeded in a 6-well plate at a density of 1×105 cells per well and incubated at 37 °C for 24 h. Then, medium was removed and cells were

incubated

respectively

with

MFLs/LAOOH,

CLs/LAOOH

(the

concentration of LAOOH: 0, 10, 30 μg/ml), and added 1 μl DCFH-DA for 4 h. Thereafter, Fe3O4 nanoparticles (10 μg) were added in medium. The ROS generation of samples were monitored using a Fluorescence Microscope (Zeiss LSM 510) and were quantified by flow cytometry (BD FACSCalibur). Research the distribution of MFLs in tumor cell 4T1 cells were seeded into a 6-well plate. After 24 h of incubation, MFLs loading with DII (DII concentration: 1 μg/ml) was added, and then cells were cultured for 1h or 2h continuously. Thereafter, cell nucleus was stained by Hoechst 33,342 (10 μg/ml) solution. Ten minutes later, residual Hoechst 33,342 was removed with PBS buffer washing, and samples were imaged directly via a


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confocal microscope (LEICA TCS SP8, Germany). Cellar internalization and Membrane Fusogenic Property of MFLs 4T1 cells were seeded into 6-well plates (1×105 cells per well) for 24 h of incubation

and

then

treated

with

DIO

labeled

CLs/LAOOH@DOX,

MFLs/LAOOH@DOX (with the same DOX concentration: 10 μg/ml) for 4h. Thereafter, cell nucleus was stained by Hoechst 33,342 (10 μg/ml) solution. Ten minutes later, residual Hoechst 33,342 was removed, and then the samples were imaged directly by a confocal microscope (LEICA TCS SP8, Germany). Cytotoxicity assessment in vitro 4T1 cells were seeded in 6-well plates at a density of 2×105 cells per well in 2 ml of complete RPMI 1640 medium and incubated for 24 h. The groups of control, CLs/LAOOH@DOX, MFLs/LAOOH@DOX with or without Fe3O4 nanoparticles (with the same DOX concentration: 10 μg/ml, Fe3O4 concentration: 20 μg/ml) were added to each well and the plate was incubated for another 24 h. Subsequently, the cells were collected and stained with annexin V-FITC/PI and then analyzed by flow cytometry (BD FACSCalibur). Transwell experiment 4T1 cells were seeded at 1 × 105 cells per well in upper chambers of twelvewell plates. After culturing for 24 h, DIO labeled MFLs/LAOOH, CL/LAOOH were added to upper chambers at an equivalent LAOOH does (20 µg/ml), respectively. Then the treated cells on the transwell filter were co-incubated with the lower chamber for 8 h. Thereafter they were removed and washing well with pbs. Finally, the samples were imaged directly via a confocal microscope (LEICA TCS SP8, Germany). Subsequently, we use the same way to evaluate the ROS generation of lower chamber 4T1 cells with a ROS probe (DCFH-DA). Three-dimensional (3D) tumor spheroids formation The vitro 3D tumor spheroids of 4T1 cells were developed by a liquid-covering method. Each well of 96-well plates was added with 100 μl of sterile agarosebased pbs (1%, w:v). Subsequently, 4T1 cells were seeded into each well at a


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density of 6×103 cells per well in 100 μl of complete RPMI 1640 medium and then the 96-well plates were gently shaken for 10 min. The tumor spheroids were allowed to grow up to appropriate diameter for some days at 37 °C and 5% CO2. The formation of 3D tumor spheroids was monitored using a Fluorescence Microscope (Zeiss LSM 510). The penetration of MFLs in vitro 3D multicellular tumor spheroids The tumor spheroids were incubated with the MFLs/LAOOH@DOX and CLs/LAOOH@DOX (20 ug/ml DOX) for 8 h respectively. Then the tumor spheroids

were

washed

thrice

with

ice-cold

PBS

and

fixed

with

paraformaldehyde (4%, w:v) for 30 min, and placed in glass bottom cell culture dish. The images of the tumor spheroids were acquired by tumor scan using Zstack imaging with 40 μm intervals from the top of the spheroid to the middle by a confocal microscope (LEICA TCS SP8, Germany). Zebrafish Embryo Assay Zebrafish (Danio rerio) were treated according to the local animal protection regulations and maintained according to standard protocols. In order to prevent pigment formation, the embryos were treated with 0.16 mM 1-phenyl-2-thiourea from 24 h post fertilization (hpf). Then, embryos were treated for 30 min with MFLs/LAOOH@PI, MFLs/LAOOH@DOX, CLs/LAOOH@DOX at 48 hpf. Thereafter 3× washing in embryo medium, embryos were anesthetized in 0.02% tricaine methane sulfonate, mounted in glass bottom cell culture dish, and imaged by a confocal microscope (LEICA TCS SP8, Germany). In vivo distribution DIR and DIR labeled MFLs/LAOOH@DOX were injected to 4T1 tumor-bearing mice (1 mg/kg DIR), respectively. Whole body fluorescence images were acquired by two-dimensional fluorescence reflectance imaging (2D FRI) at desired time. In Vivo Tumor Inhibition Study The antitumor activity was conducted using 4T1 tumor-bearing BALB-c mice. When the tumor volume reached about 100 mm3, 4T1 tumor models were


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randomly divided into 5 groups (six mice per group) and treated with saline (0.2 ml),

free

DOX,

MFLs/LAOOH@DOX,

CLs/LAOOH@DOX+Fe3O4,

and

MFLs/LAOOH@DOX+Fe3O4 (5 mg/kg DOX, 10 mg/kg LAOOH). Fe3O4 treatment was performed at predetermined time. Afterward, the tumor sizes and mice weight were monitored and recorded every 2 d over a period of 15 d after the first administration. The tumor sizes were measured by a vernier caliper and calculated as the volume = (tumor length) × (tumor width)2 / 2. All the animal experiments were performed in accordance with the protocol approved by the Chinese Academy of Medical Science and Peking Union Medical College and adhered to the Guiding Principles in the Care and Use of Animals of the American Physiological Society. Histological analyses After treatment for 15 days, the mice were sacrificed and tumor sections were detected with the In Situ Cell Death Detection Kit for in situ terminal deoxynucleotidyltransferase mediated UTP end labeling (TUNEL) assay. Additionally, after treatment, the hearts of mice treated with saline, free DOX, MFLs/LAOOH@DOX+Fe3O4 were harvested for panorama scanning analysis. Morphological changes were observed under microscope (Zeiss LSM 510). In vivo tumor penetration depth evaluation 200 μl CLs/DIO and MFLs/DIO (0.5 mg/kg DIO) were respectively injected to 4T1 tumor-bearing mice. After 24 h, the mice were sacrificed and tumor sections images were analyzed by a Fluorescence Microscope (Zeiss LSM 510).

ASSOCIATED CONTENT Supporting Information Available: Mass spectra of LAOOH, ROS detection, TEM image of nano Fe3O4, the cumulative release profiles of iron ions, internalized pathway assessment, in vitro cytotoxicity assay, biodistribution studies, ROS assessment in vivo, body weight assessments, H&E-stained major organs samples, complete blood analysis.


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Nano Letters

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281x176mm (300 x 300 DPI)


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Scheme 1 281x176mm (300 x 300 DPI)


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Scheme 2 221x138mm (300 x 300 DPI)


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Figure 2 458x259mm (300 x 300 DPI)


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Figure 3 282x302mm (300 x 300 DPI)


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Figure 4 240x241mm (300 x 300 DPI)


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Figure 6 270x173mm (300 x 300 DPI)


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Nano Letters


Sequential Intercellular Delivery Nanosystem for Enhancing ROS

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