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Amplification of Tumor Oxidative Stresses with Liposomal Fenton Catalyst and Glutathione Inhibitor for Enhanced Cancer Chemotherapy and Radiotherapy Ziliang Dong, Liangzhu Feng, Yu Chao, Yu Hao, Muchao Chen, Fei Gong, Xiao Han, Rui Zhang, Liang Cheng, and Zhuang Liu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b03905 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

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Amplification of Tumor Oxidative Stresses with Liposomal Fenton Catalyst and Glutathione Inhibitor for Enhanced Cancer Chemotherapy and Radiotherapy Ziliang Dong, Liangzhu Feng*, Yu Chao, Yu Hao, Muchao Chen, Fei Gong, Xiao Han, Rui Zhang, Liang Cheng, Zhuang Liu* Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, 199 Ren'ai Road, Suzhou, 215123, Jiangsu, PR China *E-mail: [email protected], [email protected]

ABSTRACT

Amplification of intracellular oxidative stress has found to be an effective strategy to induce cancer cell death. To this end, we prepare a unique type of ultrasmall gallic acid-ferrous (GA-Fe(II)) nanocomplexes as the catalyst of Fenton reaction to enable persistent conversion of H2O2 to highly cytotoxic hydroxyl radicals (•OH). Then, both GA-Fe(II) and L-buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, are co-encapsulated within a stealth liposomal nano-carrier. Interestingly, the obtained BSO/GA-Fe(II)@liposome is able to efficiently amplify intracellular oxidative stress via increasing •OH generation and reducing GSH biosynthesis. After chelating with 99mTc4+ radioisotope, such BSO/GA-Fe(II)@liposome could be tracked under in vivo single-photon-emission-computed-tomography

(SPECT) 1

imaging,

ACS Paragon Plus Environment

which

illustrates

the

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time-dependent tumor homing of such liposomal nanoparticles after intravenous injection. With GA-Fe(II)-mediated •OH production and BSO-mediated GSH depletion, treatment with such BSO/GA-Fe(II)@liposome would lead to dramatically enhanced intra-tumoral oxidative stresses, which then result in remarkably improved therapeutic efficacies of concurrently applied chemotherapy or radiotherapy. This work thus presents the concise fabrication of biocompatible BSO/GA-Fe(II)@liposome as an effective adjuvant nanomedicine to promote clinically used conventional cancer chemotherapy and radiotherapy, by greatly amplifying the intra-tumoral oxidative stress.

KEYWORDS: GA-Fe(II) nanocomplexes, Fenton reaction, GSH depletion, disruption of redox homeostasis, cancer combination therapy

INTRODUCTION It has been found that the amplified intracellular oxidative stress is involved in radiotherapy and various types of chemotherapies to directly or indirectly kill cancer cells.1-4 As the main cause of intracellular oxidative stress, reactive oxygen species (ROS), a group of highly reactive chemical species containing oxygen such as hydroxyl radical (•OH), hydrogen peroxide (H2O2) and superoxide radical (O2-), have been found to be produced at an elevated basal concentration inside the cancerous cells as the result of their altered metabolism.5, 6 To adapt to such a high level of intracellular ROS, cancer cells normally would have developed a high level of adaptive antioxidants including different types of enzymes (e.g. superoxide dismutase, catalase and glutathione peroxidase) and reductants (e.g. glutathione (GSH)) to maintain the balance of redox homeostasis 2


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for cell survival.7 Therefore, it has been recognized that disruption of intracellular redox homeostasis by promoting ROS production and / or reducing ROS scavenging should be able to enable efficient amplification of intracellular oxidative stress, so as to enable more effective cancer treatment.8, 9. Fenton reaction, which utilizes ferrous ions as the catalyst to in situ convert H2O2 to highly oxidative •OH, has been extensively explored to remove organic contaminates in water during the past decades.10-13 Motivated by the strongest oxidative capacity of •OH among those common ROS (e.g. E(•OH/H2O) = 2.80 V, E(1O2/H2O) = 2.17 V, E(H2O2/H2O) = 1.78 V), Fenton reaction has shown to be an innovative strategy to induce cancer cell elimination by utilizing the high level of endogenous H2O2 within most types of solid tumors.14-17 To date, several different types of iron containing formulations such as ferrocence, iron oxide nanoparticles and iron nanoparticles have been utilized as catalysts of Fenton reaction to induce cell death singly or in combination with other types of cancer therapies.18-20 However, considering the limited catalytic activity and / or stability of those previously developed catalysts in biological environments, it is necessary to develop innovative formulations with efficient and stainable catalytic activity in converting H2O2 to •OH to enable effective cancer treatment. Moreover, as a powerful type of ROS scavenger, GSH overexpressed within cancer cells could adaptively maintain intracellular redox homeostasis to counteract the cell killing effect of ROS generated by Fenton reaction.21-23 Therefore, it would be interesting to realize GSH depletion within the tumor, so as to promote the therapeutic effect of Fenton reaction for cancer killing. As -glutamylcysteine synthetase (-GCS) is responsible for intracellular GSH synthesis, the inhibitor of -GCS, L-buthionine sulfoximine (BSO), has been found to be able to induce efficient intracellular GSH depletion.24-26 Therefore, in this study, we design a liposome-based nanomedicine 3


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system that is able to co-deliver the catalyst of Fenton reaction together with -GCS inhibitor BSO into tumors, to achieve enhanced oxidative stress for tumor cells. In our design, a biocompatible Fenton catalyst was prepared by mixing gallic acid (GA) with Fe2+ ions, obtaining ultrasmall GA-Fe(II) nanocomplexes with sustainable catalytic activity in converting H2O2 to highly oxidative •OH, as well as greatly improved catalytic stability over free Fe2+ owing to the GA-mediated Fe3+ to Fe2+ conversion. Upon co-encapsulation of both GA-Fe(II) and BSO within stealth liposomes, the obtained BSO/GA-Fe(II)@liposome could collectively amplify the intracellular oxidative stress by efficient ROS production and simultaneous GSH depletion to endow more efficient cancer cell killing. Utilizing the metal ion chelating ability of GA, BSO/GA-Fe(II)@liposome could be easily labeled by a radioisotope 99mTc4+ to enable convenient in vivo tracking. Upon intravenous injection, our BSO/GA-(99mTc)Fe(II)@liposome showed remarkably prolonged blood circulation and improved tumor homing compared to GA-(99mTc)Fe(II) without liposome encapsulation, as observed under SPECT imaging. Interestingly, the intra-tumoral oxidative stress was dramatically amplified after treatment with BSO/GA-Fe(II)@liposome, which could not only suppress the tumor growth on its own, but also greatly improve the therapeutic efficacies of clinically applied chemotherapy and radiotherapy, without imposing additional side effects to the treated animals. This work thus presents an innovative strategy to enhance conventional cancer therapies by effectively amplifying the tumor oxidative stress.

RESULTS AND DISSCUSSION To develop the sustainable and efficient ROS-generating nanoplatform for tumor therapy, ultrasmall metal-polyphenol networks GA-Fe(II) nanocomplexes were fabricated by utilizing the strong coordination interactions between the polyphenol groups of GA and ferrous ions (Fe(II)) 4


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(Figure 1a). Upon dropping GA solution into the aqueous mixture of ferrous chloride (FeCl2) and polyvinyl pyrrolidone (PVP) at the optimized molar feeding ratio of 1: 2: 7 (GA : Fe : PVP) under stirring based on preliminary screening (Figure S1), the solution color instantly changed from colorless to dark purple (Figure 1b). The Fourier transform infrared (FTIR) spectrum of GA-Fe(II) showed a band at 1250 cm-1 (OH-C stretching band), which was much lower than that of free GA molecules, indicating the efficient coordination interaction between -OH groups in GA and Fe2+ (Figure S2). Under the transmission electron microscopy (TEM), the as-prepared GA-Fe(II) nanocomplexes showed uniform size distribution with an average diameter at 2.16 ± 0.31 nm (Figure 1c), which was slightly smaller than their hydrodynamic diameter of 4.85 nm as measured by dynamic light scattering (DLS) (Figure 1d). Moreover, the mass ratio of Fe2+ in such GA-Fe(II) nanocomplexes was determined to 5.6~6.7% by thermogravimetric analysis (TGA) and inductively coupled plasma mass spectrometry (ICP-MS) (Figure S3). In addition, under the measurement of X-ray photoelectron spectroscopy (XPS), the Fe in the obtained GA-Fe(II) nanocomplexes was confirmed to be ferrous ions as indicated by the two strong binding energy peaks appeared at 711 eV and 724 eV, responsible for the appearances of Fe2p3/2 and Fe2p1/2 according to the standard binding energy (BE) Lookup Table (Figure 1e). Taken together, those results indicate that the success formation of GA-Fe(II) nanocomplexes. To investigate the potential of GA-Fe(II) as a Fenton agent, the electron spin resonance (ESR) spectroscopy was firstly used to identify the •OH generation with the assistance of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a typical capture-agent for short-lived hydroxyl radicals.27 It was found that efficient generation of •OH, presented as the emergence of the characteristic 1 : 2 : 2 : 1 signals in the ESR spectrum, was only observed for the GA-Fe(II) sample incubated with H2O2 (1 mM), while not detected in the samples of bare GA-Fe(II) or H2O2 alone 5


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(Figure 1f). In addition, upon being incubated with GA-Fe(II) in the presence of H2O2 (1 mM), the color of methylene blue (MB) solution, a widely employed indicator of •OH generation, gradually changed from blue color to colorlessness with a time-dependent degradation manner, indirectly indicating the generation of •OH (Figure 1g). Besides MB, two other widely used free-radical-capturing agents, 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) and 1,2-diaminobenzene (OPDA), were also employed to detect the generation of ROS (Figure S4a-d). Consistently, the efficient catalytic activity of GA-Fe(II) nanocomplexes in converting H2O2 to •OH was also confirmed in those experiments. Then, the catalytic activity of such GA-Fe(II) in converting H2O2 to •OH were compared in parallel with previously reported catalysts including Fe3O4 nanoparticles, hemin and aminoferrocene. Based on the MB degradation experiment, we found that GA-Fe(II) triggered the highest level of ROS generation in the presence of H2O2, compared to the other tested Fenton reaction catalysts including Hemin, Fe3O4 nanoparticles and aminoferrocene at the same molar concentration of Fe (Figure 1h). Moreover, unlike free Fe2+, which could be easily oxidized by the dissolved oxygen and thus lose its catalytic activity, our GA-Fe(II) showed largely maintained Fenton catalytic capacity without being obviously disturbed for up to 30 days (Figure 1i). Such super stable Fenton-like catalytic activity of GA-Fe(II) should be ascribed to the fact that the excess phenol groups within GA-Fe(II) nanocomplexes would enable instant reduction of the less active Fe3+ ions generated during the Fenton reaction or oxygen oxidization, to highly active Fe2+ ions. This hypothesis was confirmed by the finding that the Fenton-like catalytic capacity of Fe3+ ions was significantly improved in the presence of GA (Figure S5), consistent with those previous reports.28,

29

Most importantly, unlike free Fe2+ ions whose Fenton catalytic activity would be

significantly suppressed in the presence of serum, such GA-Fe(II) with excellent stability in 6


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physiological conditions (Figure S6) showed comparable Fenton-like catalytic activity in serum to that measured in pure water (Figure 1j). Therefore, GA-Fe(II) nanocomplexes are efficient Fenton catalysts with superior catalytic activity and stability in physiological environments, promising for further biological applications. Given that small molecules and ultra-small nanoparticles (< 10 nm) would be rapidly excreted via the renal clearance pathway and thus show limited tumor accumulation, stealth liposome with versatile molecular loading capacity, efficient passive tumor homing capacity as well as excellent biocompatibility was selected in this work for encapsulation of GA-Fe(II).30-32 Therefore, we encapsulated as-prepared GA-Fe(II) and BSO with liposomes by hydrating the pre-prepared lipid film composed of 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethyleneglycol)-5000 (DSPE-PEG) at a molar ratio of 6 : 4 : 0.5 with the phosphate buffered saline (PBS) solution of GA-Fe(II) nanocomplexes and BSO (Figure 2a). As observed under TEM imaging, the obtained BSO/GA-Fe(II)@liposome showed spherical morphology with multiple smaller dark dots within one particle, indicating the successful encapsulation of GA-Fe(II) (Figure 2b, Figure S7). By utilizing

the

dynamic

light

scattering

(DLS)

measurement,

the

as-prepared

BSO/GA-Fe(II)@liposome showed mono-dispersed size distribution profile with mean diameter determined to be ~100 nm, which was comparable to that of BSO@liposome and GA-Fe(II)@liposome prepared by the same procedure (Figure S8). Encouraged by the efficient Fenton-like catalytic activity of GA-Fe(II) in •OH generation and the well-known capacity of BSO in the depletion of intracellular GSH, we then carefully evaluated the impact of such BSO/GA-Fe(II)@liposome on the intracellular oxidative stress using the commercial Ellman’s reagents. As showed in Figure 2c, the intracellular GSH was found to be 7


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dramatically

decreased

in

4T1

murine

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breast

cancer

cells

incubated

with

BSO/GA-Fe(II)@liposome, to the level much lower than that observed in cells incubated with GA-Fe(II)@liposome alone or BSO@liposome alone. Afterwards, the intracellular oxidative stress of those 4T1 cells after various treatment were carefully studied by utilizing the confocal laser scanning microscopy (CLSM) imaging with 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) as the intracellular ROS probe. It was found that the cells treated with BSO/GA-Fe(II)@liposome (BSO = 200 M, GA-Fe(II) = 1.1 mM) displayed strongest intracellular green fluorescence, while those cells treated with GA-Fe(II)@liposome or BSO@liposome only showed moderate intracellular green fluorescence (Figure 2d). In addition, the intracellular oxidative stress of 4T1 cells after different treatments was quantitatively analyzed by flow cytometry, which revealed consistent trend to that observed under CLSM (Figure S9). Taken together, these results indicate that BSO/GA-Fe(II)@liposome could significantly amplify the intracellular oxidative stress and disrupt intracellular redox homeostasis through GA-Fe(II)-enhanced ROS generation and BSO-mediated GSH depletion. Then, the effect of amplified intracellular oxidative stress on the cell viability was analyzed using the standard cell viability assay. It was found that BSO/GA-Fe(II)@liposome would induce more severe cytotoxicity to 4T1 cells compared to BSO@liposome and GA-Fe(II)@liposome (Figure 2e). Only 21.2% cells kept alive after being incubated with BSO/GA-Fe(II)@liposome (BSO = 100 M, Fe2+ = 550 M) for 24 h, while there were 91.0% or 67.2% of viable cells for those treated with BSO@liposome or GA-Fe(II)@liposome, respectively. Consistently, we found that BSO/GA-Fe(II)@liposome could induce a much higher level of cell apoptosis compared to GA-Fe(II)@liposome

or

BSO@liposome

(Figure

S10),

demonstrating

that

such

BSO/GA-Fe(II)@liposome could offer excellent cell growth inhibition effect via the apoptosis 8


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pathway as the result of amplified intracellular oxidative stress (Figure 2f). Moreover, although BSO/GA-Fe(II)@liposome treatment showed severe cytotoxicity to 4T1 murine breast cancer cells, it only showed limited cytotoxicities to normal cell lines including RAW264.7 macrophages, human embryonic kidney 293 (HEK293) cell line and NIH 3T3 mouse embryo fibroblast cell line, as determined by using the standard cell viability assay (Figure S11). Such phenomenon might be ascribed to lower production of H2O2 inside those cells. SPECT imaging that can provide accurate information for quantitative analysis of diseases has been widely used in the clinic with the help of suitable imaging probes.33-35 By means of the strong coordination interactions between the -OH groups and metals ions (e.g.

64Cu2+,

Fe3+), various

nanostructures with abundant -OH groups have been exploited as potentials imaging probes after metal ion binding.36, 37 As expected, it was found that

99mTc4+,

a widely used radioactive tracer for

clinical SPECT imaging, could also be efficiently labeled onto the as-prepared GA-Fe(II) nanocomplexes at a high radiolabeling efficiency and stability (Figure 3a&b, Figure S12). More surprisingly, we found that GA-Fe(II)@liposome could also be labeled with

99mTc4+

at a

comparable labeling efficiency to that of GA-Fe(II) nanocomplexes upon simple mixing, without the need of any other agents to assist this process (Figure S12). The ability of

99mTc4+

to cross the

lipid bilayer in our system is somewhat unexpected. Although the detailed mechanism is still not very clear, similar findings have also been reported in a number of previous reports for radiolabeling of liposomes (e.g. with 64Cu2+). 38-40 Moreover, we found that 89.3% of radioactivity was remained on such oC

99mTc4+

labelled GA-Fe(II)@liposome after being incubated in serum at 37

for up to 24 h (Figure 3b), indicating such labeling would be quite stable. Therefore, this

incredible finding, to some extent, broadened the feasibility and applications of chelator-assisted radiolabeling of liposomes. 9


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Then, the in vivo behaviors of such

99mTc4+

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labeled GA-Fe(II) nanocomplexes and

BSO/GA-Fe(II)@liposome were studied under a SPECT imaging system (MILabs, Utrecht, the Netherlands). It was found that mice with i.v. injection of BSO/GA-(99mTc)Fe(II)@liposome (dose: 500 μCi per mouse) showed gradually increased SPECT signals in tumors, while the tumor signals in mice with i.v. injection of GA-(99mTc)Fe(II) appeared to be rather weak (Figure 3c&e). The tumor uptake of radioactivities was found to be ~5.99% ID g-1 (percentage of injection dose per gram) and ~2.53% ID g-1 at 24 h post injection (p.i.), for mice injected with of BSO/GA-(99mTc)Fe(II)@liposome and free GA-(99mTc)Fe(II), respectively (Figure 3d&f). In addition, the tumor-to-muscle (T/M) ratios of BSO/GA-(99mTc)Fe(II)@liposome showed gradual enhancement over time, higher than that in GA-(99mTc)Fe(II) treated mice at each corresponding time point (Figure S13). Meanwhile,

the

detailed

pharmacokinetic

profiles

of

GA-(99mTc)Fe(II)

and

BSO/GA-(99mTc)Fe(II)@liposome were quantitatively studied by gamma counter reading. As expected, BSO/GA-(99mTc)Fe(II)@liposome showed significantly prolonged blood circulation time compared to that of GA-(99mTc)Fe(II), with half-life times estimated to be t1/2() = 1.19 ± 0.032 h and t1/2() = 10.16 ± 0.88 h for BSO/GA-(99mTc)Fe(II)@liposome (Figure 3g). Besides, the ex vivo bio-distribution analysis revealed that the tumor accumulation of BSO/GA-(99mTc)Fe(II)@liposome at 24 h p.i. was quantified to be ~5.63% ID/g, much higher than 2.68% for GA-(99mTc)Fe(II), comparable to those quantified by the in vivo SPECT imaging data (Figure 3h). Those results demonstrate that the liposome encapsulation method could confer ultrasmall GA-Fe(II) remarkably prolonged blood circulation time and increased tumor accumulation via the via the enhanced permeability and retention (EPR) effect.

10


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After that, the effect of BSO/GA-Fe(II)@liposome treatment on the intra-tumoral oxidative stress was carefully studied with DCFH-DA as the ROS probe. A total of 15 tumor-bearing mice were randomly divided into 5 groups (n = 3) and i.v. injected with PBS, GA-Fe(II)@liposome, BSO@liposome, GA-Fe(II) plus BSO, or BSO/GA-Fe(II)@liposome. The doses for BSO and Fe were 10 mg kg-1 and 6 mg kg-1, respectively. At 24 h post various injection, those mice were sacrificed with tumors collected for cyro-section and stained by DCFH-DA for CLSM observation. It was intriguingly uncovered that those tumor slices from the mice with i.v. injection of BSO/GA-Fe(II)@liposome exhibited the strongest DCF fluorescence signals, while moderate DCF florescence was observed for mice with injection of GA-Fe(II) plus BSO (Figure 4a). However, minimal fluorescence signals were observed in tumors on those mice with injection of GA-Fe(II)@liposome or BSO@liposome alone under the tested dose. These results therefore demonstrate that BSO/GA-Fe(II)@liposome upon i.v. injection could significantly amplify the intra-tumoral oxidative stress. Afterwards, the in vivo therapeutic efficacy of BSO/GA-Fe(II)@liposome was evaluated by utilizing mice bearing 4T1 tumors. A total of 25 tumor-bearing mice with tumor volumes of ∼100 mm3 were randomly divided into 5 groups (n = 5) as follows: Group I with injection of PBS, Group II with injection of GA-Fe(II)@liposome, Group III with injection of BSO@liposome, Group IV with simultaneous injection of free GA-Fe(II) + BSO, and Group V with injection of BSO/GA-Fe(II)@liposome. At day 0, 4, 8, various agents were i.v. injected into corresponding groups of mice at equivalent doses of BSO (10 mg kg-1) and Fe (6 mg kg-1) (Figure 4b). From day 0, the tumor size and body weight were recorded. It was found that the treatment of BSO/GA-Fe(II)@liposome could significantly inhibit the tumor growth, while the other treatments in group II, III and IV showed negligible inhibitory effect on tumor growth (Figure 4c&d). Notably, 11


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it was also found that the average body weights during various treatments showed negligible variation (Figure S14). Moreover, the therapeutic effects of various treatments were studied by evaluating the histological damages and apoptosis levels of those tumor slices collected at 24 h post injection of various agents through the hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively. We found that the tumor slices collected from the mice of Group V showed severe histological damages, while no obvious damages were observed on tumor slices collected from the other three groups of mice (Figure 4e). Moreover, the TUNEL staining assay showed consistent trend to those abovementioned H&E staining

results,

further

evidencing

the

superior

therapeutic

efficacy

of

our

BSO/GA-Fe(II)@liposome (Figure 4e). Considering that conventional radiotherapy and many types of chemotherapies would induce cell death with excess ROS generation involved, we therefore hypothesized that the ability of BSO/GA-Fe(II)@liposome to disrupt intra-tumoral redox hemostasis might be helpful to enhance the therapeutic performance of conventional cancer therapeutics. By utilizing the standard in vitro cell viability assay, we found that the concurrent treatment of BSO/GA-Fe(II)@liposome and chemo-drugs (e.g. cisplatin, Doxorubicin, oxaliplatin) showed greatly increased cell killing ability in comparison with the treatment of BSO/GA-Fe(II)@liposome or chemo-drugs alone (Figure S15a-c). Based on such interesting results, we then selected oxaliplatin as the model chemo-drug to explore the enhancement effect of BSO/GA-Fe(II)@liposome on the therapeutic efficacy of oxaliplatin to 4T1 tumors grown on mice (Figure 5a). We found that concurrent systemic administration of BSO/GA-Fe(II)@liposome and oxaliplatin showed the most potent inhibitory effect on tumor growth in comparison to administration of BSO/GA-Fe(II)@liposome alone or 12


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oxaliplatin alone (Figure 5b). The body weight data also showed negligible variation during treatments (Figure S16). Furthermore, utilizing the H&E staining, it was found that the tumor slices collected from mice with concurrent systemic administration of BSO/GA-Fe(II)@liposome and oxaliplatin showed the most severe histological damages, which appeared to more significant than those treated with BSO/GA-Fe(II)@liposome or oxaliplatin alone (Figure 5c). Moreover, we also investigated the possibility of using BSO/GA-Fe(II)@liposome to enhance conventional radiotherapy (Figure 5d). Consistently, it was found that 4T1 tumors grown on the mice with sequential treatment of BSO/GA-Fe(II)@liposome administration and X-ray exposure were significantly suppressed, while tumors on mice with treatment of BSO/GA-Fe(II)@liposome alone or X-ray exposure alone were only partially inhibited (Figure 5e, Figure S17a&b). Similarly, no significant body weight drop was noticed for various groups of mice (Figure S18). Consistently, micrographs of H&E stained tumor slices also verified that the combinational treatment of BSO/GA-Fe(II)@liposome plus X-ray exposure resulted in the most significant histological damages compared to the respectively mono-therapies (Figure 5f). Collectively, our results demonstrate that the increased tumor oxidative stresses and disruption of intra-tumoral redox hemostasis by BSO/GA-Fe(II)@liposome could effectively enhance the therapeutic responses of both oxaliplatin-based chemotherapy and X-ray-induced radiotherapy (Figure 5g). At last, the potential in vivo toxicity of our BSO/GA-Fe(II)@liposome was carefully investigated through the routine blood assay and serum biochemistry indexes for healthy mice with i.v. injection of BSO/GA-Fe(II)@liposome. We found that the hepatic and renal function markers of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and blood urea nitrogen (BUN) within 14 days post injection of BSO/GA-Fe(II)@liposome kept consistent compared to those of control mice (Figure S19). Moreover, the levels of red blood cells (RBC), 13


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platelet (PLT), hemoglobin (HGB), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBC), mean corpuscular hemoglobin (MCH), and mean corpuscular volume (MCV) were all found to be normal after i.v. injection of BSO/GA-Fe(II)@liposome. Moreover, the main organs including liver, spleen, kidney, heart and lung of those 4T1 tumor bearing mice post treatments of BSO/GA-Fe(II)@liposome alone, BSO/GA-Fe(II)@liposome plus oxaliplatin injection, and BSO/GA-Fe(II)@liposome plus X-ray exposure were collected for H&E staining at 14 days post treatments. No obvious histological damage was observed for the examined organs in those mice with various treatments (Figure S20). Our results demonstrated that such BSO/GA-Fe(II)@liposome while acting as an adjuvant nanomedicine to significantly promote the antitumor efficacy of conventional chemotherapy and radiotherapy, would not impose additional side effects to the treated animals.

CONCLUSIONS In

this

study,

we

have

designed

a

biocompatible

liposomal

nano-formulation

BSO/GA-Fe(II)@liposome to enable disruption of redox hemostasis within the tumor,

of

conferring

efficient cancer combination therapy. Taking advantage of the highly stable Fenton catalytic activity of GA-Fe(II) in physiological environments and the function of BSO in inhibiting GSH synthesis, our BSO/GA-Fe(II)@liposome could significantly amplify the intracellular oxidative stress and thus induce severe cell death. Based on in vivo SPECT imaging, it was found that 99mTc-labeled

BSO/GA-Fe(II)@liposome upon systemic administration showed obviously

increased tumor accumulation compared to bare GA-Fe(II). Owing to the combinational effect of GA-Fe(II)-triggered ROS production and BSO-mediated GSH disruption, i.v. injection of BSO/GA-Fe(II)@liposome could elicit a high level of intra-tumoral oxidative stress. As the results, 14


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BSO/GA-Fe(II)@liposome treatment could not only directly suppress the tumor growth, but also effectively enhance the treatment outcomes of conventional chemotherapy and radiotherapy. Therefore, our work highlights a new avenue to enhance the therapeutic responses of clinically used cancer therapies, by amplifying the tumor oxidative stress through targeting high levels of H2O2 and GSH within the tumor microenvironment. With high biocompatibility and no additional side effect, BSO/GA-Fe(II)@liposome could be a promising type of adjuvant nanomedicine for future clinical translation.

EXPERIMENTAL SECTION Materials. Gallic acid, L-buthionine sulfoximine (BSO), DCFH-DA, ABTS and PVP were obtained from Sigma-Aldrich. Iron dichloride tetrahydrate (FeCl2·4H2O), cholesterol, OPDA and methylene blue trihydrate (MB) were purchased from J&K Chemical Co. DPPC and DSPE-PEG were purchased from Avanti Lipids Polar, Inc. Other chemicals were purchased from Sinopharm Chemical Reagent CO, Ltd., China.

Synthesis of GA-Fe(II) nanocomplexes. GA-Fe(II) nanocomplexes were synthesized following the previously reported method.41 Briefly, FeCl2·4H2O (23 mg) and PVP (80 mg) were added into 4 mL degassed deionized (DI) water and vigorously stirred at room temperature for 5 min. Later, GA (1 mL, 10 mg mL-1 in degassed DI water) was dropwise added into above solution and stirred in the nitrogen atmosphere for 24 h. Then, the obtained GA-Fe(II) nanocomplexes in purple color were condensed and purified by using a ultra-filtration filters with a molecular weight cut off (MWCO) at 10 kDa. The obtained nanocomplexes were stored at 4 oC for further use.

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Preparation of GA-Fe(II)@liposome, BSO@liposome and BSO/GA-Fe(II)@liposome. The dried lipid film was prepared by following the previously developed protocol.42 Afterwards, GA-Fe(II)@liposome, BSO@liposome and BSO/GA-Fe(II)@liposome were obtained by hydrating the dried lipid films with aqueous solutions of GA-Fe(II), BSO or BSO + GA-Fe(II), respectively, extruded through a 200 nm polycarbonate membrane, and purified by running sephadex G-100 columns by following our previously developed method.43 The obtained liposomes were condensed and stored at 4 oC for further use.

Characterization. The morphologies of GA-Fe(II) and BSO/GA-Fe(II)@liposome were observed via a TEM (Tecnai F20, FEI). The DLS and UV-vis-NIR spectra of GA-Fe(II)@liposome and BSO/GA-Fe(II)@liposome were measured by a Malvern Zetasizer (ZEN3690, Malvern) and spectrophotometer (Lambda750, PerkinElmer), respectively. The loading capacity of Fe2+ and BSO were quantified with the ICP-MS (Jena, PlasmaQuant MS) and high performance liquid chromatography (HPLC) (Agilent 1260), respectively.

Evaluation of •OH producing. To detect the generation of •OH, a classical colorimetric method was used based on the degradation of MB under the oxidative environment.44 In brief, GA-Fe(II)@liposome was added into an aqueous solution containing H2O2 (1 mM) and MB (15 g mL-1). After incubation at 37 °C for various time intervals, the absorption of above solutions at 665 nm was measured to record the degradation of MB. In addition, ESR spectroscopy was used to evaluate the •OH production via a commonly used radical trap agent, DMPO.45

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Cell Experiments. The 4T1 murine breast cancer cells were cultured at 37 °C under 5% CO2. To evaluate the efficient generation of intracellular ROS, 4T1 cells were incubated with BSO@liposome, GA-Fe(II)@liposome or BSO/GA-Fe(II)@liposome for 4 h at the BSO or Fe concentrations of 200 M or 1.1 mM, respectively. The cells were then washed with fresh PBS and further incubated with fresh 1640 medium containing DCFH-DA (20 μM) for 30 min. The intracellular ROS was then examined by recording the fluorescence of DCF (λex = 488 nm, λem = 525 nm) under a confocal microscope (Leica SP5II). Meanwhile, flow cytometer was used to further quantitatively evaluate the intracellular ROS generation. The intracellular GSH was detected using Ellman’s reagents.46 In Brief, 4T1 cells were cultured with BSO@liposome, GA-Fe(II)@liposome or BSO/GA-Fe(II)@liposome for 12 h (BSO = 200 M, Fe = 1.1 mM). The treated cells were lysed by repeated cycle of freezing and thawing and then centrifuged to collect the supernatants for the measurement of GSH based on the standard protocol.47 For cytotoxicity evaluation, 4T1 cells were seeded in 96-wells culture plates and then cultured with

BSO@liposome,

GA-Fe(II)@liposome

or

BSO/GA-Fe(II)@liposome

at

varying

concentrations and incubated for 24 h. Later, the cell viability was measured by a standard methyl thiazolyl tetrazolium (MTT) assay. The apoptosis levels of the cells with different treatments were also evaluated by using the commercial Annexin V-FITC&PI dual staining Kit following the vendor’s protocol (Abcam).

Tumor model. All animal experiments were performed following the protocols approved by the Soochow University Laboratory Animal Center. The 4T1 tumors were generated by subcutaneous injection of 106 cells in 50 L PBS into the back of each female Balb/c mouse. 17


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In vivo SPECT imaging. GA-Fe(II) or BSO/GA-Fe(II)@liposome were labelled with

99mTC,

purchased from Shanghai GMS Pharmaceutical Co., Ltd, and their labeling yields and stability were measured by adopting our previously used methods.34 For SPECT imaging, tumor-bearing mice (n = 3 each group) were i.v. injected with

99mTc-labeled

GA-Fe(II) nanocomplexes or

BSO/GA-Fe(II)@liposome nanoparticles, and then imaged by in vivo animal SPECT imaging system (MILabs, Utrecht, the Netherlands) at different time post injection (p.i.). The signal intensities of liver, heart, tumor and muscle at each time intervals were also recorded by following our previously developed methods.48

In vivo pharmacokinetic study. = 6 mg kg-1,

99mTc4+

99mTc-labeled

GA-Fe(II) or BSO/GA-Fe(II)@liposome (dose: Fe

= 500 μCi per mouse) were i.v. injected into tumor-bearing mice (n = 3 per

group) with their blood circulation and tissue bio-distribution profiles measured by according to our previously used procedure.48

In vivo cancer therapy. When tumor sizes reached ∼100 mm3, those tumor bearing mice were randomly divided into five groups (n = 6 each group): (1) PBS, (2) BSO@liposome, (3) GA-Fe(II)@liposome, (4) free BSO + GA-Fe(II), and (5) BSO/GA-Fe(II)@liposome. At day 0, 4 8, mice were i.v. injected with 200 μL of different agents (BSO = 10 mg kg-1, Fe = 6 mg kg-1). From day 0, the tumor sizes and body weights of each mouse were monitored every two days. The tumor sizes were defined as ‘volume = length x width2/2’. At day 1, one mouse from each group was sacrificed, and their tumors were collected, and sliced for H&E and TUNEL staining to further evaluate the therapeutic effects of each group. 18


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For combination therapy of BSO/GA-Fe(II)@liposome treatment and chemotherapy, 200 L of various agents, including (1) PBS, (2) oxaliplatin, (3) BSO/GA-Fe(II)@liposome, (4) BSO/GA-Fe(II)@liposome + oxaliplatin were i.v. injected into tumor bearing mice at day 0, 4, 8 (dose: BSO = 10 mg kg-1, Fe = 6 mg kg-1, oxaliplatin = 4 mg kg-1). The tumor size and body weight of each group mice were recorded following the aforementioned procedure. At day 1, one mouse from each group was sacrificed to collect tumor for H&E staining. For combination therapy of BSO/GA-Fe(II)@liposome treatment and radiotherapy, mice were randomly divided into four groups (n = 6 each group): (1) PBS, (2) X-ray, (3) BSO/GA-Fe(II)@liposome, (4) BSO/GA-Fe(II)@liposome + X-ray. At day 0, 200 μL of various agents were i.v. injected into each mouse. At 24 h p.i., the mice of group (2) and (4) were exposed to X-ray radiation at a dose of 8 Gy. The doses of BSO and Fe were 10 mg kg-1, 6 mg kg-1, respectively.

Blood

biochemistry

analysis.

Healthy

Balb/c

mice

were

i.v.

injected

with

BSO/GA-Fe(II)@liposome (BSO = 10 mg kg-1, Fe = 6 mg kg-1, n = 5 each group). The untreated healthy Balb/c mice were used as the control. At day 1, 7 and 14 p.i., the blood samples were collected and measured in South Shanghai Research Center for Biomodel Organism for blood biochemistry and hematology analysis.

ASSOCIATED CONTENT Supporting Information Figures S1-S20 can be found in the supporting information.

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AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected], *E-mail: [email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENTS This work was partially supported by the National Research Programs from Ministry of Science and Technology (MOST) of China (2016YFA0201200), the National Natural Science Foundation of China (51525203, 51761145041, 51802209), the Natural Science Foundation of Jiangsu Province

(BK20180848),

the

China

Post-doctoral

Science

Foundation

(2017M610348,

2018T110545), Collaborative Innovation Center of Suzhou Nano Science and Technology, the 111 Program from the Ministry of Education of China, and a Project Funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

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Figure 1. Synthesis and characterizations of GA-Fe(II) nanocomplexes. (a) Schematic illustration of the synthesis and functions of GA-Fe(II) nanocomplexes. (b) UV-Vis-NIR spectra of free GA (1), FeCl2 (2) and GA-Fe(II) (3) solutions. Inset: photos of those solutions. (c) A TEM image and TEM-measured size distribution (inset) of GA-Fe(II) nanocomplexes. (d) DLS data of GA-Fe(II) in the aqueous solution. (e) XPS spectra of Fe 2p oribit for GA-Fe(II) nanocomplexes. (f) ESR spectra of different agents with DMPO as the radical trap. (g) Time-dependent UV-Vis-NIR spectra of MB degradation triggered by Fenton reaction. Inset: the photos of MB solutions at various time points. (h) Time-dependent degradation of MB triggered by Fe3O4, hemin, aminoferrocene, or GA-Fe(II). (i) Comparisons of the relative catalytic activity of free Fe2+ and GA-Fe(II) within one month. (j) Relative catalytic activity of free Fe2+ and GA-Fe(II) in water and serum.

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Figure 2. (a) In vitro oxidative stress-mediated anticancer effect. (a) A scheme showing the preparation of BSO/GA-Fe(II)@liposome. (b) A TEM image of BSO/GA-Fe(II) @liposome. (c) Intracellular GSH levels of 4T1 cells treated with BSO@liposome, GA-Fe(II)@liposome or BSO/GA-Fe(II)@liposome. The GSH concentration was detected using Ellman’s reagents. (d) Fluorescence images of 4T1 cells treated with various agents and stained with a ROS probe DCFH-DA (green) for intracellular ROS evaluation. (e) Relative viabilities of 4T1 cells after being treated with BSO@liposome, GA-Fe(II)@liposome or BSO/GA-Fe(II)@liposome. (f) Schematic illustration of the amplified oxidative stress via the effective •OH production and GSH depletion to induce cell death. P values in (e) were calculated by Tukey’s post-test (***p < 0.001, **p < 0.01, or *p < 0.05).

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Figure 3. In vivo SPECT imaging and pharmacokinetic study. (a) A scheme showing 99mTc-labeled nanoparticles. (b) Radiolabeling stability of GA-Fe(II) and BSO/GA-Fe(II)@liposome. (c&e) In vivo SPECT images of 4T1 tumor-bearing mice after i.v. injection of 99mTc-labeled BSO/GA-Fe(II)@liposome (c) or GA-Fe(II) (e). (d&f) Quantitative analysis of SPECT signals in the liver, kidney, tumor, and muscle at different time points after i.v. injection with 99mTc-labeled BSO/GA-Fe(II)@liposome (d) or GA-Fe(II) (f). (g) Blood circulation of 99mTc-labeled GA-Fe(II) or BSO/GA-Fe(II)@liposome after i.v. injection. (h) Biodistribution of 99mTc-labeled GA-Fe(II) or BSO/GA-Fe(II)@liposome at 24 h post i.v. injection.

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Figure 4. In vivo anticancer effect of BSO/GA-Fe(II)@liposome. (a) Fluorescence images of tumor slices after DCFH-DA staining (green). The nuclei were stained with DAPI (blue). (b) A scheme showing the experiment design. (c)Tumor growth curves after i.v. injection with various agents (5 mice per group). (d) Tumor weights of different groups collected at day 14 after various treatments. (e) Micrographs of tumor slices after H&E (upper) or TUNEL (bottom) staining collected at one day after various treatments indicated. P values in (c&e) were calculated by Tukey’s post-test (***p < 0.001, **p < 0.01, or *p < 0.05).

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f

Figure 5. ROS-mediated chemo-/radiotherapy sensitization. (a) A scheme showing experiment design for BSO/GA-Fe(II)@liposome enhanced oxaliplatin-based chemotherapy. (b) Tumor growth curves of different groups of mice after various treatments (5 mice per group). (c) H&E stained images of tumors slices collected from each group at 24 h post injection of nanoparticles and drug. (d) A scheme showing experiment design for BSO/GA-Fe(II)@liposome enhanced X-ray radiotherapy. (e) Tumor growth curves of different groups of mice after various treatments (5 mice per group). (f) H&E stained images of tumors slices collected 2 days after X-ray radiation. (g) Schematic illustration of the efficient disruption of redox homeostasis in the tumor by BSO/GA-Fe(II)@liposome for enhanced chemo-/radiotherapy. 27


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Table of Content

The efficient disruption of redox homeostasis by BSO/GA-Fe(II)@liposome for enhanced chemo-/radiotherapy

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