O2-Economizer for Inhibiting Cell Respiration to Combat Hypoxia

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O2-Economizer for Inhibiting Cell Respiration to Combat Hypoxia Obstacle in Tumor Treatments Wuyang Yu, Tao Liu, Mingkang Zhang, Zixu Wang, Jingjie Ye, ChuXin Li, Wenlong Liu, Runqing Li, Jun Feng, and Xian-Zheng Zhang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b07852 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

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O2-Economizer for Inhibiting Cell Respiration to Combat Hypoxia Obstacle in Tumor Treatments

Wuyang Yu,† Tao Liu,† Mingkang Zhang, Zixu Wang, Jingjie Ye, Chu-Xin Li, Wenlong Liu, Runqing Li, Jun Feng* and Xian-Zheng Zhang

Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China

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ABSTRACT: Hypoxia, a ubiquitously aberrant phenomenon implicated in tumor growth, causes severe tumor resistance to therapeutic interventions. Instead of the currently prevalent solution through intratumoral oxygen supply, we put forward an “O2-economizer” concept by inhibiting O2 consumption of cell respiration to spare endogenous O2 to overcome the hypoxia barrier. A nitric oxide (NO) donor responsible for respiration inhibition and a photosensitizer for photodynamic therapy (PDT) are co-loaded into PLGA nanovesicles to provide a PDT-specific O2 economizer. Once accumulating in tumors and subsequently responding to the locally reductive environment, the carried NO donor undergoes breakdown to produce NO for inhibiting cellular respiration, allowing more O2 in tumor cells to support profound enhancement of PDT. Depending on biochemical re-allocation of cellular oxygen resource, this “O2-economizer” concept offers a way to address the important issue of hypoxia-induced tumor resistance to therapeutic interventions, including but not limited to PDT.

KEYWORDS: tumor treatment, hypoxic tumor, nitric oxide, respiration inhibition, photodynamic therapy


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Tumor environment is pathologically featured with extreme hypoxia. This robust hallmark has been acknowledged as one of the major hurdles accounting for the tumor resistance to many therapeutic interventions, such as radiotherapy, chemotherapy and photodynamic therapy (PDT).1,2 To overcome this challenge, great efforts have been made to provide extra oxygen (O2) supply toward tumors.

For

instance,

catalase,3,4

manganese

dioxide,5,6

and

gold

nanoclusters7 are transported into tumors to decompose H2O2 for O2 supply. However, the outcomes are limited by the locally low H2O2 level.8 Meanwhile, red blood cell,9,10 hemoglobin,11,12 perfluorocarbon vesicles13,14 have been utilized to directly carry O2 to hypoxic tumors. This approach faces the problems including poor O2 loading, rapid O2 leakage and difficult co-delivery with desired therapeutics. Recently, carbon nitride has been proposed for photo-splitting water to generate O2 inside tumors, which suffers from the low O2-generation efficiency and the safety concern arising from the nondegradable nature of carbon nitride.8 In addition to the difficult access to intratumoral oxygen supply, the correlation between promoting oxygen supply and tumor proliferation remains ambiguous and argued. It is generally thought that sufficient oxygen supply disfavors tumor proliferation, but a part of animal experiments and clinical trials offered the contrary outcomes.15-17 Johnson and Lauchlan postulated that hyperbaric oxygen may even facilitate malignant tumor growth because wound healing would benefit from hyperbaric oxygen.18 To combat the hypoxia obstacle in tumor treatments, this study put forward


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an “O2-economizer” solution instead of the currently prevalent oxygen-supply strategy. Respiration is the major way of living cells for O2 consumption.19 Though cancer cells acquire energy mainly through aerobic glycolysis (as termed “Warburg effect”),20 mitochondrial respiration still plays fundamental roles in the development and progression of diverse tumors.21,22 It has been reported that a significant number of tumor cell lines exhibit elevated rates of respiration for tumorigenesis and tumor survival.23,24 Hypoxia occurs in tumors because the oxygen requirements of tumors outstrip the ability of the local vasculature to supply oxygen.25,26 We thus intend to inhibit physiological O2 consumption of tumor cells to spare endogenous O2 for therapy purpose, thus making tumors more sensitive to hypoxia-resistant treatments without exogenous O2 supply. We note that nitric oxide (NO) can inhibit cell respiration and disturb cell metabolism because of its competition with O2 by binding to the oxygen-binding site of mitochondrion.27-29 In this regard, NO may be suitable as the starter of O2-economizer for cell respiration inhibition. PDT has been an effective modality for tumor treatments with minimal invasive damages to normal tissues.30-32 In principle, a photosensitizer delivered at tumor site would be photo-activated to convert O2 to cytotoxic reactive oxygen species (ROS), consequently killing tumor cells.33 Apparently, tumorous hypoxia plays a predominant role in limiting PDT efficacy. To validate our idea, a PDT-specific O2-economizer is specifically fabricated on the basis of polymeric nanovesicles (PVs) composed of biocompatible poly(D,L-lactide-


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co-glycolide) (PLGA, Mw=38-54 Kda). PLGA was chosen as the carrier for its superior biocompatibility and biodegradability, which facilitated sustained drug delivery with high biosafety.34,35 The PVs are co-loaded with sodium nitroprusside (SNP), a hydrophilic NO donor, in the aqueous cavity and tetraphenylporphyrin (TPP), a hydrophobic photosensitizer, in the outer polymeric layer, respectively (Scheme 1). SNP is a medication used to lower blood pressure, relying on the breakdown to NO upon the contact with vascular epithelial cells.36,37 On one hand, the vesicular structure can protect SNP from the capture by vascular epithelial cells during the period of blood circulation. On the other hand, the cellular glutathione (GSH) concentration (0.5-10 mM) is much higher than that in the plasma (ca. 2-20 μM), and the tumor cells are rich in thiol-bearing compounds (GSH and cysteine). For instance, GSH in the tumor cells is more than 4-fold higher than that in normal cells.38,39 Once the SNP and TPP co-loaded PV (PV-TS) accumulates in tumors due to the enhanced permeation and retention (EPR) effect of tumors,40 the locally released SNP would undergo the reaction with the overexpressed thiol compounds in tumor cell followed by the formation of S-nitrosothiol (RSNO, e.g. S-nitroso-cysteine (CSNO) and S-nitroso-glutathione (GSNO)).41 Thereafter, the formed S-nitrosated intermediates produce NO spontaneously or under the catalysis of cellular enzymes,42-44 enabling efficient inhibition of cellular respiration and in turn facilitating the PDT induced by the concurrently delivered TPP.


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RESULTS AND DISCUSSION PV-TS was prepared by the double emulsion method in darkness.45 The observations by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) clearly reflected the hollow spherical nanostructure of PV-TS (Figure 1A, B). By dynamic light scattering (DLS) analysis, the hydrodynamic diameter was measured to be around 200 nm, which is very close to the particle size determined by TEM (Figure 1A), indicating the well dispersion of PV-TS in an aqueous medium. Furthermore, the higher contrast in the vesicle cavity reflected the loading of water-soluble SNP. The stability of the nanoparticles was further investigated in phosphate buffer solution (PBS) and the serum-containing medium, respectively (Figure S1). All the nanoparticles exhibited high stability in the two media within 5 days. X-ray photoelectron spectroscopy (XPS) further illustrated the success of SNP loading (Figure S2, Supporting Information). Typically, the drug content reached 2.6% for SNP, as determined by inductively coupled plasma atomic emission spectrometry (ICP-AES), and 5.4% for TPP by ultraviolet-visible spectroscopy (UV-Vis), respectively (Figure S3A, Supporting Information). Confocal laser scanning microscopy (CLSM) observation over the microscaled vesicles manifested that the hydrophilic molecules (e.g. SNP) would be loaded in the vesicular cavity and the hydrophobic molecules (e.g. TPP) were merely located in the hydrophobic shell (Figure S4, Supporting Information). The SNPfree and TPP-free nanovesicles (termed as PV-T and PV-S, respectively) were


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prepared in the similar manner for comparison purpose (Figure S5, Supporting Information). We investigated the interaction of the nanovesicles with cells. As shown in Figure S6, red fluorescence representing TPP appeared in 4T1 cells after 2 h incubation and overlapped well with the green fluorescence used to label lysosome, indicating the cellular uptake was mainly via endocytosis pathway. As prolonging incubation time, the fluorescence tended to pervade cytoplasm with evidently enhanced intensity. The result suggested that PV-TS was readily internalized by cells owing to their nanoscale dimension. PV-TS showed similar release profiles of either SNP or TPP with PV-S and PV-T in PBS and the serum containing medium, in consistence with their good stability in two media (Figure S7A, and S7B). After irradiation treatments, the morphology of PV-TS was almost unchanged, indicating the excellent photostability of PV vesicles (Figure S8). The capability of PV-TS to generate NO was verified by the Griess assay.46 As aforementioned, NO liberation from SNP can be activated by intracellular thiol-bearing compounds, such as GSH and cysteine.41 Upon the addition of GSH or cysteine into the PV-TS contained solution, NO was rapidly generated in contrast to the minimal variation of NO content detected in the negative control (Figure 1C, Figure S9, Supporting Information). Without the addition of thiol compounds, the percent release of NO is calculated to be about 6% during the initial 12 hours, indicating the low level of burst release. Thiol concentration in tumor cells is dramatically higher than that in normal cells and extracellular milieu.38,39 Therefore, the thiol-


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dependent characteristic of NO generation is favorable for PV-TS to impose better efficacy of respiration inhibition toward tumor cells. The intracellular NO release was strongly verified by flow cytometry analysis using a NO probe of 4amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FM DA) (Figure 1D, Figure S10, Supporting Information).47 The observation indicated that NO was readily produced in the 4T1 cells treated with PV-TS, which would affect cellular respiration. Tumor hypoxia occurs because the requirement of oxygen outstrips the supply.25,26 To mimic the process of hypoxia formation, the oxygen supply of cells were cut off by sealing the culture medium with liquid paraffin. As measured by oxygen consumption assay kit, the partial pressure of oxygen (pO2) in the sealed cells was sharply decreased within 100 min to around 1.1% in the sample-free control and the PV-T treated group, due to the cellular oxygen consumption (Figure S11, Supporting Information). In comparison, pO2 after PV-TS treatment remained at about 4 folds as that measured at normal condition and PV-T treated group. The result indicated that PV-TS treatment could indeed inhibit oxygen consumption and thus potentially spare oxygen for PDT purpose. According to the reported method using an oxygen electrode, the oxygen content in the culture media was also measured to study the cellular respiration.48,49 In the dark, 4T1 cells were first incubated with PV-TS or PV-T for 10 hours in an open chamber. After that, the oxygen contents in the culture media were monitored in the sealed chamber. Under the condition, it is evident that the lower the O2 content was, the more the O2 consumption and the faster


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the respiration rate. In the absence of PV-T and PV-TS, cell respiration led to a decline of O2 content by 42% in 25 min (Figure 2A). The introduction of PV-T into the cell-containing medium did not affect cellular O2 consumption significantly. In comparison, the oxygen content was reduced by merely about 20% when the cells were exposed to PV-TS. The result reconfirmed that the SNP contained in PV-TS did suppress cellular O2 consumption. Since blocking the respiration chain would cause both the reduction of adenosine triphosphate (ATP) synthesis and the depolarization of mitochondrial membrane with a reduction of mitochondrial membrane potential (ΔΨm) value,50-52 the variations of MMP and ATP content were examined after different treatments to explore whether oxygen consumption suppression was due to the respiration inhibition at mitochondria. The treatment with PV-TS led to apparently lower ATP content inside cells compared with the PV-T treatment (Figure 2B). Based on CLSM using

a

probe

of

5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolo-

carbocyanine iodide (JC-1), ΔΨm alterations were examined. JC-1 forms aggregates at high ΔΨm while existing as monomer at low ΔΨm. The result manifested that mitochondrial membrane was depolarized in the PV-TS treated cells, in contrast to minimal changes detected in the PV-T treated cells (Figure 2C, Figure S12, Supporting Information), suggesting that PV-TS rather than PV-T induced the mitochondria dysfunction. Oxygen consumption in cells is closely related to cytochrome c oxidase (CcO), which converts oxygen to water in the respiration chain. Studies have shown that NO would bind to CcO and


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inhibit its activity,53,54 thus the CcO activity was here examined using the CcO activity assay kit. As shown in Figure 2D, the CcO activity was decreased with increasing PV-TS concentrations. Compared to PV-T, both PV-S and PV-TS showed stronger potency to suppress CcO activity (Figure 2E). These results reconfirmed that the introduced SNP could inhibit cellular respiration and thus help to save cellular O2 consumption. To prove that PV-TS can alleviate the cellular oxygen tension under hypoxic condition resulting from the inhibited cell respiration, hypoxia inducible factor-1α (HIF-1α), the hallmark of hypoxic cells,55 was examined by immunofluorescence assay. Hypoxic cells cultured in the sealed chamber presented a higher level of HIF-1α expression than normoxic cells in the open chamber (Figures 2F, G). Under hypoxic condition in the former case, the addition of PV-T did not affect HIF-1α expression significantly while PV-TS led to considerably weakened immunofluorescence, indicating the higher O2 content in the PV-TS treated hypoxic cells. The same trend was observed when western blot was applied for the analysis of cellular HIF-1α (Figure S13, Supporting

Information).

Further

analysis

using

the

ROS-ID®

Hypoxia/Oxidative stress detection kit also validated that PV-TS indeed alleviated the oxygen tension in hypoxic cells like an “O2-economizer” (Figures 2H, I, Figure S14, Supporting Information). The PV-TS caused inhibition of cell respiration is thought to spare more O2 for therapy usage (i.e., the PDT discussed here), thus providing possibility to


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combat the hypoxia obstacle in tumor treatments. To prove this assumption, we first studied the ex vitro singlet oxygen generation ability of PV-TS by 1,3diphenylisobenzofuran (DPBF) and 2,2,6,6-tetramethylpiperidine (TEMP). As shown in Figure 3A, B, irradiation to PV-TS solution led to the decreased absorption of DPBF, in contrast to the minimal changes in the absence of PVTS, indicating the irradiation-dependent generation of singlet oxygen for PVTS. The same conclusion was obtained when the electron spin resonance (ESR) probe of TEMP was used for the detection of singlet oxygen (Figure 3C). Then the assays concerning the ROS production and the corresponding toxicity were performed in cellular levels. 2’,7’-dichlorofluorescein diacetate (DCFH-DA) was used to detect the produced ROS under CLSM observation.56 Upon NIR irradiation, PV-TS and PV-T exhibited similarly strong ability to generate ROS under normoxic condition (Figure 3D). As expected, hypoxic condition largely hindered the photodynamic conversion of PV-T, as evidenced by the sharp reduction of fluorescence intensity inside cells (Figure 3D). In comparison, PVTS displayed much better performance with respect to ROS generation under hypoxic condition. The same trend was observed by the quantitative analysis using flow cytometry (Figure 3E, Figure S15, Supporting Information). Typically, PV-TS generated more than 4-fold ROS in hypoxic cells as PV-T did under the same condition. It is evident that the introduction of SNP into PV-TS could effectively combat the hypoxia-induced inhibition of ROS production. This is because the SNP-caused respiration inhibition spares more O2 for


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photodynamic transduction. The inspection using CLSM over the intracellular singlet oxygen by singlet oxygen sensor green (SOSG) under hypoxia gave the similar result. PV-TS generated more singlet oxygen inside cells than PV-T in terms of the fluorescence intensity (Figure S16). The conclusion was reconfirmed by the data of cell viability to evaluate PDT efficacy. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT)

assay

was

applied to assess the cytotoxicity in 4T1 cells. After 24h incubation, both PVTS and PV-T exhibited similarly negligible dark-toxicity (Figure S17, Supporting Information), suggesting minimal toxicity of the NO produced from PV-TS within the tested concentration range up to 80 μM. The low toxicity of SNP observed here is consistent with the results reported in references. It was reported that 100 μM of SNP caused minimal cytotoxicity to human colonic adenocarcinoma cells and human gastric cancer cell while about 90% of PC12 cells remained living when cultured with 200 μM SNP.57-59 The NO production of PV-TS involved two processes: 1) SNP is released from the nanovesicles and then react with cellular thiols to form RSNO, e.g. CSNO and GSNO; 2) Thereafter, the formed RSNO intermediates produce NO spontaneously or under the catalysis of cellular enzyme (termed as denitrosation).42-44 It has been documented that thiols can react with NO, which is named as S-nitrosation.44,60 In addition to the factor of SNP release, the balance between S-nitrosation and denitrosation during the NO production process of SNP can be taken as one of the major reasons accounting for the drastically low toxicity of SNP.61 To


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exclude the toxicity interference to the study, the dosage of SNP used in this study was limited within a safe range. Under 660 nm irradiation for 3 min, both PV-TS and PV-T killed almost all the treated 4T1 cells under normoxic condition due to the sufficient O2 supply for PDT (Figure 3F). Under hypoxic condition, the same irradiation operation led to dramatically reduced cytotoxicity of PV-TS and PV-T. However, it is noted that PV-TS exhibited apparently stronger potency to kill cells than PV-T under hypoxic condition. Prolonging the irradiation time to 10 min induced continuous cell death under hypoxic condition, and the superiority of PV-TS over PV-T in PDT efficacy was represented more profoundly. Typically, almost all the PV-TS treated cells died whereas about 40% cells remained alive after the treatment with PV-T when the TPP dosage was 20 mg L-1. In comparison, negligible celltoxic effects were detected in the PV-S treated cells regardless of the exposure to irradiation, indicating that the liberated NO caused minimal cytotoxicity under the conditions (Figure S18 and S19, Supporting Information). The visual inspection using live cell staining technique and the flow-cytometry analysis of apoptosis and necrosis agreed fairly well with the data of MTT assay (Figure 3G, H). The encouraging in vitro outcomes stimulated us to study the in vivo PDT performance toward hypoxic tumors. By monitoring the plasma concentration of TPP after the administration of PV-TS, it was found that the half-life of TPP in vivo was apparently prolonged as the result of the encapsulation in PV-TS


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(Figure S20, Supporting Information), suggesting the favorable stability of PVTS during blood circulation. The metabolic profile of PV-TS was determined by monitoring the urinary and fecal excretion of TPP, as shown in Figure S21. The result also indicated the slower elimination profile of PV-TS compared to free TPP. After a long period postinjection, the total excretion amount of TPP in the PV-TS and the TPP treated mice was 6.63 ± 0.49 μg and 6.81 ± 0.74 μg respectively, implying that the loaded cargoes can be ultimately eliminated from the body. A 4T1 tumor-bearing female BALB/c mice model was established to investigate the in vivo bio-distribution of PV-TS by small animal imaging system. At 6h post intravenous injection of PV-TS, tumors started to display much stronger fluorescence from PV-TS than normal tissues (Figure 4A, B, Figure S22, Supporting Information). The stronger fluorescence in tumors than in other organs after 96h was mainly due to the EPR effect of tumors and the faster metabolic rate of the metabolic organs like liver and kidney (Figure 4B and Figure S23, supporting information).62 To confirm that PV-TS can alleviate tumorous hypoxic tension, the 4T1 tumor bearing mice were administrated with PBS, PV-TS and PV-T, respectively. At 24h, the administrated mice were sacrificed for immunofluorescence imaging with HIF-1α staining. As shown in Figure 4C, the administration of PV-TS led to a lower level of HIF-1α expression in tumors than the PV-T treatment and blank control. In addition, at 24h after the administration with PV-TS or PV-T, the tumor bearing mice were administrated with a hypoxic pimonidazole probe for immunofluorescence


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imaging.63 The intensity of the fluorescence signal in the tumors of PV-TS treated group was much weaker than that of the controls (Figure 4D), verifying again the alleviated hypoxia in the PV-TS treated tumors owing to the inhibited respiration of tumor cells. The 4T1 tumor bearing female BALB/c mice were divided randomly into five groups: PBS (blank control), PV-TS, PV-T plus NIR irradiation, and PV-TS plus NIR irradiation. The mice were intravenously injected with PV-TS or PV-T at a fixed TPP dose of 3 mg kg-1. At 24h post-injection, NIR irradiation was operated at tumor sites for 5 min at 660 nm with a power density of 150 mW cm-2. The treatment with PV-T plus NIR displayed moderate anticancer effect in the initial 5 days, but afterward there appeared an evident tumor relapse (Figure 4E). This outcome was associated with the compromised PDT efficacy caused by the tumorous hypoxia, which is a major reason accounting for the poor prognosis.64,65 In contrast, the tumors in the group of PV-TS plus NIR were gradually shrunken till to complete elimination (Figure 4E, G), and no tumor relapse occurred during 14 days. Even under irradiation, the administration with photosensitizer-free PV-S caused nearly no antitumor effects. During the treatment, there was no significant variation in the body weight in all the treated groups (Figure 4F). At the 14th day, all the mice were sacrificed. The tumor and the major organs were harvested for the histological examination using H&E staining technique. Likewise, group of PV-TS plus NIR exhibited apparently better antitumor efficacy than other groups, based on the comparison on the


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amounts of dead cells and the corruption extent of extracellular matrix (Figure 4H). H&E analyses toward major organs showed no discernible pathological abnormalities in the five groups (Figure S24, Supporting Information), partly indicating minimal systemic toxicity.

CONCLUSIONS In conclusion, instead of the currently prevalent anti-hypoxia strategy by providing extra O2 supply into tumors, we put forward an “O2-economizer” solution to combat the hypoxia-induced tumor resistance for therapeutic interventions. To prove the concept, a vesicular PDT-specific PV-TS nanoeconomizer was constructed. PV-TS can respond to the tumorous reductive condition and release NO to inhibit cellular respiration, thus reducing O2 consumption and sparing more O2 for PDT purpose. Consequently, PV-TS offered better antitumor performance toward hypoxic tumors than the traditional PDT nano-agents, such as PV-T. The marked superiority of PV-TS over PV-T suggests the potentials of this O2-econimizer concept in overcoming the hypoxia obstacle in many tumor therapeutic means, including but not limited to PDT.

EXPERIMENTAL SECTION


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Materials. Poly(D,L-lactide-co-glycolide) (PLGA, lactide:glycolide 50:50, Mw: 38000-54000 Da), Sodium nitroprusside dihydrate (SNP),

Polyvinyl

alcohol (PVA, Mowiol® PVA-105 ) were purchased from Aladdin Reagent (Shanghai, China). 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FM DA), 2΄,7΄-dichlorodihydrofluorescein diacetate (DCFH-DA), Griess agent

assay

kit,

ATP

assay

kit

and

5,5’,6,6’-tetrachloro-1,1’,3,3’-

tetraethylbenzimidazolocarbocyanine iodide (JC-1) were purchased from Beyotime Company (China). ROS-ID® Hypoxia/Oxidative stress detection kit was purchased from Enzo Life Sciences. TPP was synthesized by our lab. And all the other agents were analytical grade and used as received. Mitoxpress® Xtra oxygen consumption assay kit (HS method) was purchased from Amsbio. Cytochrome c oxidase activity detection assay kit was purchased from Solarbio. Singlet oxygen sensor green (SOSG) was purchased from Invitrogen. 1,3diphenylisobenzofuran (DPBF) and 2,2,6,6-tetramethylpiperdine (TEMP) were purchased from Adamas Reagent. Instruments. A probe sonicator (LC-1000) was adopted in preparation of PLGA vesicles. Dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, UK) was used to determine the hydrodynamic diameter and zeta potential of the product in each preparation process. Transmission electron microscopy (TEM, JEOL-2100, Tokyo, Japan) and Scanning Electron Microscope (SEM, Zeiss Sigma FESEM) were used to observe the morphology of each product. UV-Vis spectrometer (Lambda Bio40, PerkinElmer) was used


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to record the UV-Vis absorption. X-ray photoelectron spectrometer (XPS, Thermo Fisher Scientific) was applied to ensure the successful preparation of the product. Inductively coupled plasma atomic emission spectroscopy (ICPAES, IRIS Intrepid II XSP, USA Thermo Elemental) was used to determine the iron content in the product. cytometry analysis.

BD AccuriTM C6 Flow Cytometer was used for flow-

A 660 nm NIR laser (STL808T1-7.0W, Beijing STONE

Laser) and a 660 nm LED panel (20 mW cm-2) were used for NIR irradiation. A confocal laser scanning microscope (CLSM, C1-Si, Nikon, Tokyo, Japan) was used to obtain the fluorescence image. IVIS imaging system (PerkinElmer) was used here to obtain in vivo NIRF imaging image. The bioluminescence was detected using ELIASA (Spark 10M, Tecan). Dissolved oxygen electrode (JPSJ-605F, INESA, Shanghai) was adopted to evaluate the cell respiration. HPLC analysis was performed by LC-6AD Liquid chromatograph (Shimadzu). Preparation of PV-TS and PV-T. Double emulsion (W/O/W) technique was applied to fabricate PV-TS. Briefly, 40 mg of sodium nitroprusside dihydrate (SNP) was dissolved in 1 mL aqueous PVA solution (20 mg mL-1). Then the mixture was emulsified with PLGA solution (50 mg dissolved in 4 mL DCM) containing 5 mg TPP with a probe sonicator under an ice bath for 2 min to form W/O primary emulsion in the dark. The primary emulsion was subsequently transferred into 12 mL of PVA solution (50 mg mL-1) followed by another 8 min sonication under ice bath in darkness to form W/O/W double emulsion. The resultant was poured into 100 mL ultrapure water and stirred


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overnight at room temperature to solidify the vesicles. Finally, the resultant was harvested after centrifugation (10,000 rpm, 30 min) and washing steps. PV-T and PV-S were fabricated with the same procedure except the addition of SNP or TPP. Cell Culture. Mouse breast cancer (4T1) cells were incubated in RPMI1640 medium containing 10% FBS and 1% antibiotics (penicillin-streptomycin, 10,000 U mL-1) at 37oC in a humidified atmosphere containing 5% CO2. NO Release. NO release was detected using the Griess assay. Briefly, 1 mL PV-TS (2 mg mL-1) was dialyzed against 3 mL PBS (pH=7.4, 10 mM) buffer (molecular cut off: 3000 Da) containing cysteine (0.25 mM) or GSH (5 mM) and placed in the table concentrator. 50 μL of the solution from the suspension at different time point was analyzed by Griess assay according to the protocol using a plate reader. The NO concentration was calculated by the standard curve of NO determined from the Griess assay. The control group did not contain cysteine or GSH. TPP and SNP Release. TPP release was detected by fluorescent spectrometer (RF-5301pc, Shimadzu, Japan), and SNP release was detected measuring the NO content using Griess assay. Briefly, the 1 mL of PV-TS, PV-T and PV-S (80 ug TPP and 35 ug SNP) solutions of PBS (pH=7.4, 10 mM) and 5% serum containing solution were dialyzed against 10 mL PBS solution and placed in the table concentrator at 37 ℃ . The solutions were collected and


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replaced with fresh PBS at different time points. To quantify the released TPP content, 1 mL of the solution collected at different time point was measured by fluorescence spectrometer using the excitation of 410 nm and emission of 650 nm, the fluorescence intensity at 650 nm was recorded to calculate the released TPP content. To quantify the released SNP content, 1 mL of the solution collected at different time point was added with cysteine to fully release the NO from SNP, then griess assay was applied to measure the NO content, which is identical to SNP content. Light Induced NO Release. 1 mL of PV-TS and PV-S solutions of PBS containing 100 ug of SNP were dialyzed against 5 mL of PBS and placed in the table concentrator. The dialyze solutions were collected and replaced with fresh PBS at different. For light irradiation, 20 mW cm-2 660 nm light irradiation was given for 8 min at 1 hour time point. Then the collected solution was analyzed by griess assay to determine the released NO content. Co-localization of Hydrophilic and Hydrophobic Molecule with PLGA Vesicle. To conveniently observe the co-localization by CLSM, micro-scale PLGA vesicle was prepared. Since SNP is a non-fluorescence hydrophilic molecule, the hydrophilic fluorescence molecule rhodamine B was used as the model drug. The fluorescence of rhodamine B could interference with TPP in the red region, thus we first loaded these two molecules into PLGA vesicle respectively, then co-loading of rhodamine B and TPP into PLGA vesicle was also applied.


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Photostability of PV-TS. To determine the photostability of PV-TS, PVTS solution was irradiated with 30 mW cm-2 660 nm light irradiation for 15 min, then the morphology of irradiated PV-TS was characterized by SEM and TEM. Oxygen Consumption Evaluation. To mimic the process of hypoxia formation, the oxygen supply of cells was cut off by sealing the culture medium with liquid paraffin. This means was thus used to establish hypoxia condition in this study unless otherwise specified. To evaluate the oxygen content in the cultured cells with different treatments, Mitoxpress® Xtra oxygen consumption assay kit (HS method) was applied. Briefly, cells were seeded in 96 well plate at the density of 5000 cells per well with 100 μL of culture medium and cultured for 24 hours, then 100 μL of nanoparticle solution at the relative SNP concentration of 35 μM was added to each well and cultured in 21% oxygen for 8 hours followed by the addition of oxygen consumption assay and liquid paraffin according to seal the wells according to the provider’s protocol, then the fluorescence intensity of oxygen consumption assay at different time points were measured by ELIASA (Spark 10M, Tecan). The partial pressure of oxygen (pO2) at different time points was calculated through the standard fluorescence curve. Measurement of Cytochrome c Oxidase Activity. Cytochrome c oxidase (CcO) activity was evaluated by its activity detection assay kit. Briefly, CcO was isolated by the provided method from the manufacturer. Then, the cytochrome c oxidase solution was incubated with different amount of the nanoparticles in


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the presence of cysteine in the dark at 25 oC for 6 hours, then the assay was added and the absorption at different time point was recorded. The activity was calculated according to the provider’s formula. In vitro NO Detection. 4T1 cells were cultured with PV-T or PV-TS (TPP: 20 μg mL-1) for 8 hours followed by DAF-FM DA staining for 30 min, then cells were washed and transferred for flow-cytometry analysis. Evaluation of Cell Respiration. 4T1 cells were seeded in 6-well plate at a density of 1×106 cells per well and incubated in 2 mL RPMI-1640 medium for 24 hours followed by the replacement of the culture medium with 3 mL culture medium containing PV-TS or PV-T at a relative TPP concentration of 20 mg mL-1 and incubated for 10 hours under 21% O2 condition. Then the culture medium was sealed with liquid paraffin and the O2 level of each chamber was measured over time with an oxygen electrode. The oxygen electrode was immersed in the culture medium for 10-min stabilization followed by an addition of 3 ml of liquid paraffin to cover the culture medium. Thereafter, the oxygen content was recorded every 20s. The relative oxygen content was calculated to be the ratio of the oxygen content measured at each time point versus that measured at 20s in the initial period. ATP Detection. 4T1 Cells were seeded in 24-well plate at a density of 1×105 cells per well and incubated for 24 hours followed by the replacement of the culture medium with that containing PV-TS/PV-T (TPP: 20 mg L-1) and


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cultured for a further 10 hours, then the cells were treated with ATP assay kit according to the provider’s protocol. The bioluminescence was detected and recorded using ELIASA (Spark 10M, Tecan). Mitochondria Membrane Potential (ΔΨm) Characterization. 4T1 Cells were cultured with PV-TS/PV-T (equivalent to TPP: 20 mg L-1, the nanovesicle do not contain TPP was prepared due to the fluorescence of which is overlapped with that of JC-1) for 10 hours followed by the replacement of the culture medium with serum free medium containing 10 μM JC-1 and incubated for another 30 min in the dark, and then the culture media were removed and the cells were washed thrice with PBS. The fluorescence of JC-1 (green: ex: 488 nm, em: 514 nm; red: ex: 543 nm, em: 650 nm) were observed by CLSM. The images were processed with Image-Pro Plus 6.0 software by mapping the ratio of gray intensity of green to that of red. In Vitro HIF-1α Evaluation. Cells were cultured with PV-TS/PV-T (TPP: 20 mg L-1) for 8 hours under normoxia and the wells were sealed for further 2 hours culture in the dark. Then the culture medium was replaced with paraformaldehyde to fix the cells for immunofluorescence staining of HIF-1α. Cellular Oxygen Detection. To detect the cellular oxygen degree, the nanovesicle do not contain TPP was prepared due to the fluorescence of which is overlapped with that of the oxygen probe ROS-ID. Cells were cultured with PV-TS or PV-T (both do not contain TPP) for 8 hours under normoxia, then


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ROS-ID was loaded and the culture dish was sealed for further 1 hour incubation. An unsealed dish was used as normoxic control. Afterward the culture media were removed and cells were washed for confocal and flowcytometry analysis. ROS Production. Cells were incubated with PV-TS or PV-T for 10 hours and then the culture medium was replaced with fresh serum free medium containing 5 μM of DCFH-DA for a further incubation of 30 min in darkness under normoxia, then the culture medium was replaced with fresh medium and cultured for 1 hour under normoxia. For hypoxia evaluation, cells were incubated with PV-TS or PV-T (TPP: 20 mg L-1) for 10 hours followed by the replacement of the culture medium with serum free medium containing DCFHDA for further incubation of 30 min in darkness, then the culture medium was replaced with fresh medium and the wells were sealed for further incubation of 1 hour in hypoxia. A 660 nm LED panel at a power density of 20 mW cm-2 was applied for 5 min irradiation to induce ROS generation. Then flow cytometry and CLSM were applied for ROS analysis. The red light of TPP (ex: 543 nm, em: 650 nm) and green light of DCF (ex: 488 nm, em: 515 nm) were observed using CLSM. The singlet oxygen detection by SOSG was applied in the similar manner to that of detecting ROS by DCFH DA. In Vitro Cytotoxicity. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to assess the cytotoxicity. 4T1 cells were seeded in 96-well plate at a density of 5000 cells per well and incubated in 100


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μL RPMI-1640 medium with 10% FBS and 1% antibiotics at 37 oC in a humidified atmosphere containing 5% CO2 for 24 hours. Then, another 100 μL of medium containing different concentrations of PV-TS, PV-T or PV-S were added into each well and incubated for 8 hours at normoxia followed by sealing the plates or without sealing for another 2 hours incubation to simulate a hypoxic or normoxic condition respectively in the dark. Then the wells were illuminated with 660 nm 20 mW cm-2 light for 3 min or 10 min followed by another 24 hours of incubation. The dark toxicity was evaluated without illumination. After that, 20 μL of MTT solution (5 mg mL-1 in PBS) were added into each well and incubated for another 4 hours. Finally, the medium was replaced with 150 μL DMSO. The absorbance of the solution then measured by a microplate reader at the fixed wavelength of 570 nm. Live and Dead Cell Staining. To determine the toxicity of the nanoplatform, cells were seeded in 6 well plate and cultured with PV-TS, PVT,and PV-S (TPP: 20 mg L-1) for 8 hours at normoxia followed by sealing the plate for another 2 hours incubation in the dark. Then cells were received with light irradiation (20 mW cm-2) for 10 min and cultured for a further 2 hours followed by staining with calcein-AM and PI. Then cells were washed carefully for CLSM imaging. The dark toxicity was also analyzed without light irradiation. Animals and Tumor Models. 4-5 weeks old female BALB/c mice were purchased from Wuhan University Animal Biosafty Level III Lab in this study. All research protocols were approved by the Institutional Animal Care and Use


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Committee (IACUC) of the Animal Experiment Center of Wuhan University (Wuhan, China). 4T1 murine breast tumor models were established by subcutaneously injection of 4T1 tumor cells (1 × 107 cells suspended in 100 μL of PBS) into the flank of each mouse. The tumors were allowed to grow to 90100 mm3 for further use. In the in vivo tumor inhibition experiment, mice were divided randomly into five groups, and each group contained 6 mice. Pharmacokinetic and Metabolic Evaluation. Pharmacokinetic and metabolic studies were performed according to the previous method.66 Briefly, mice were divided into two groups and were intravenously injected with 200 μL of TPP and PV-TS solution at TPP dose of 3 mg kg-1. For pharmacokinetic study, at designated time points, blood samples (30 μL) were collected from tail vein and suspended in 50 μL EDTA disodium solution (0.1 M), then 200 μL of acetonitrile was added followed by sonication and centrifugation, the supernatant was collected and filtered through 220 nm filter. The filtrate was subjected for HPLC analysis to identify the TPP concentration. For the metabolic study, mice were housed individually in the metabolic cage and were intravenously injected with 200 μL of TPP and PV-TS PBS solution at TPP dose of 3 mg kg-1, TPP was dissolved in 10% of DMSO solution. Then urine and feces samples were collected at specific time period. To determine the TPP concentration in urine and feces, urinary samples were lyophilized and then dispersed in 100 μL of acetonitrile with sonification for 10 min followed by centrifugation, the supernatants were collected for HPLC analysis to identify the


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TPP content. Feces samples were weighed and a portion was suspended in 1mL of acetonitrile with sonification for 20 min followed by centrifugation, the supernatant was collected and subjected for HPLC analysis to identify the TPP content in feces. In Vivo Distribution of PV-TS. To study the in vivo distribution of PV-TS, 4T1 tumor bearing mice were adopted for NIRF imaging when the tumor volume reached ~200 mm3. The mice were treated with intravenous injection of PV-TS. Then the mice were placed in IVIS imaging systems for the observation of the fluorescence image at different time points. 96 hours later, the mice were sacrificed and the major organs (liver, heart, lung, spleen and kidney) and tumor were harvested to analyze the ex vivo fluorescence distribution in major organs and tumor. In Vivo Hypoxia Evaluation. Tumor bearing mice were divided into three groups and intravenously injected with 200 μL of saline, PV-T or PV-TS for each group at a relative TPP dose of 3 mg kg-1. 24 hours later pimonidazole solution was intravenously injected to each mouse at a dose of 60 mg kg-1. Then 90 min after injection, mice were sacrificed and tumors were obtained for HIF-1α staining and pimonidazole immunofluorescence staining. In Vivo Tumor Inhibition. To study the anti-tumor effect, 4T1 tumor bearing mice were randomly divided into five groups (PBS (control), PV-TS without light irradiation, PV-T with light irradiation, PV-TS with light irradiation)


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and each group contained 6 mice.The mice were intravenously injected with 200 μL of PBS, PV-T, PV-S or PV-TS for each group at a relative TPP dose of 3 mg kg-1 per mouse, respectively. 24 hours after injection, mice were received with 5 min irradiation (660 nm, 150 mW cm-2). Mice weight and tumor size were measured every two days. The tumor volume was calculated as length × (width)2 × 1/2. 14 days later the mice were sacrificed and the main organs (heart, liver, spleen, lung, kidney) and the tumors were collected for H&E staining analysis. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.XXX. Supplementary figures regarding the characterizations of nanoparticles: stability test and TEM and SEM images of the nanoparticles, release of SNP and TPP, oxygen content quantification, mitochondrial membrane potential analysis, analysis of the fluorescence intensity, western blot analysis of HIF-1α, intracellular singlet oxygen detection by SOSG, dark-toxicity of PV-TS and PVT and light-toxicity of PV-S in cells, metabolic profile of PV-TS, in vivo distribution of PV-TS, H&E staining of major organs.


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AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

Author Contributions †W. Y. Yu and T. Liu contributed equally to this work. ACKNOWLEDGEMENTS This work was financially supported by the National Key Research and Development Program of China (2016YFC1100703) and the National Natural Science foundation of China (51533006, 21374085). All of the animal experiments were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Animal Experiment Center of Wuhan University (Wuhan, China).


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Scheme 1. (A) Comparison between traditional therapeutic means and the O2ecomizer way for tumor treatments. (B) Schematic illustration of PDT-specific O2-economizer for inhibiting cell respiration to combat hypoxia obstacle in tumor treatments. PV-TS accumulates in tumors and functions like an economizer to save O2 from mitochondrion respiration for the enhanced PDT performance. RSH stands for the thiol substrates such GSH and cysteine.


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Figure 1. Characterizations and NO release profile of PV-TS. (A) DLS profile of PV-TS, inset is the TEM image. (B) SEM image of PV-TS. (C) Cumulative NO release from PV-TS in the presence and absence (control) of thiols (GSH and cysteine). Data were shown as means ± SD (n=3). (D) Flow-cytometry analysis of intracellular NO after different treatments for 8 hours (TPP: 20 mg L-1). The cells were stained by DAF-FM DA.


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Figure 2. Effect of cell respiration inhibition by PV-TS. (A) Relative oxygen content in the cell-containing medium under hypoxic condition (TPP: 20 mg L1).

Blank control was detected in the cell-free medium. (B) Relative ATP content

in the cells after 10 hours co-culture with PV-T or PV-TS (TPP: 20 mg L-1, n=6). *P

O2-Economizer for Inhibiting Cell Respiration to Combat Hypoxia

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