YC-1

Promoting antitumor efficacy by suppressing hypoxia via nano self-assembly of two irinotecan-based dual drug conjugates owning a HIF-1α inhibitor

Bin Zhang,‡ab Xiaochao Huang,‡ab Hengshan Wang*c and Shaohua Gou*ab

Abstract

Hypoxia inducible factor (HIF-1α) as a major transcription factor in response to hypoxia revealed that it could be a promising tumor-specific target for anticancer therapy. In view of clinical application, the formation of hypoxic microenvironment in tumors can decrease curative effect of cytotoxic chemotherapeutic drugs. To promote antitumor efficacy of chemical drugs by suppressing hypoxia, we designed and conjugated a hydrophobic HIF-1α inhibitor (YC-1) with a hydrophilic anticancer drug, irinotecan (Ir), into one molecular entity through dicarboxylate and PEG3 linkages. Benefiting from their amphiphilicity, the resulting conjugate could act as molecular building blocks to self-assemble into nanoparticles (Ir-YC-1 and Ir-PEG3-YC-1 NPs) in water, which improved the solubility of drugs. As expected, the Ir-YC-1 NPs significantly down-regulated the expression of HIF-1α and VEGF proteins, and exhibited 5.7-fold increased antitumor activity than Ir when administered to A549 cells in hypoxic condition. This novel, simple, and effective strategy for overcoming tumor hypoxia and enhancing antitumor effect of chemical drugs has great potential in cancer therapy.

Introduction

Hypoxia, caused by an inadequate oxygen supply, is increasingly recognized as a characteristic feature in most solid tumors.1,2 Tumor hypoxia severely reduces the treatment efficiency and constitutes a huge obstacle to the clinical application of chemotherapeutic agents.3,4 Recently, a few strategies have been applied to overcome tumor hypoxia for better therapeutic efficiency, e.g. hyperbaric oxygen (HBO) therapy,5 combination of oxygen-rich gas and photosensitizers6 or vasodilating agents,7 and reducing the rate of oxygen consumption.8 However, the utility of these methods in clinical application is largely confined by safety concerns, inefficient O2 loading capacity, and limited drug delivery to the hypoxic region of solid tumors. In addition, some materials, such as perfluorocarbon, carbon nitride, CaO2 and MnO2, have been used to overcome tumor hypoxia.5,6,9-13 These materials directly transport O2 molecules as a delivery vehicle or catalytically generate O2 through hydrogen peroxide or light activation. However, poor biocompatibility, transitory O2 generation effects, difficulties in systemic delivery as well as the necessity for continuous external activation have largely limited their utility. Thus, there is an urgent need for new strategies to circumvent tumor hypoxia to improve chemotherapy response in solid tumors. It was found that the cellular response to hypoxia is primarily mediated by hypoxia inducible factor 1 (HIF-1).14,15 Under hypoxic conditions, the subunit HIF-1α escapes proteolysis and rapidly accumulates in cells, and then translocates into the nucleus, in which it dimerizes with HIF-1β to form the active transcription factor HIF-1.16 Over-expression of HIF-1α has been observed in many human solid tumors including colon, lung and prostate cancers, and is significantly associated with poor response to aggressive tumor growth and therapeutic resistance, making HIF-1α an attractive target for the treatment of tumor hypoxia.16,17
In recent years, nanotechnology has emerged as a promising strategy to achieve better therapeutic outcomes and overcome the drawbacks of traditional chemotherapy. Anticancer agents can be delivered to the action sites of hypoxic regions in solid tumors through physical entrapment or chemical conjugation with the help of nanocarriers including water-soluble polymers, liposomes, polymeric nanoparticles, and inorganic materials, displaying remarkable therapeutic efficacy against tumors and fewer side effects to normal tissues compared to free drugs.18-20 However, most of nanocarriers did not possess therapeutic efficacy by themselves and had low drug loading capacity (typically less than 10%).21-23 Even worse, some nanocarriers might cause adverse effects including high toxicity and serious inflammation toward kidney, liver and other organs in the process of degradation, metabolism and excretion. Thus, instead of using any carrier materials, small amphiphilic molecules as prodrugs have attracted attention due to their simple structures for convenient assembly. Recently, some pioneering studies demonstrated that amphiphilic drug-drug conjugates (ADDC) could self-assemble into nanoparticles (NPs) in water by themselves without the help of nanovehicles, which not only avoid the concern of toxicity of drug carriers but also exhibit nanoscale advantages together with fixed ratio drug payload.23,24
In the light of above considerations, we herein report two novel ADDC strategies to overcome tumor hypoxia. As shown in Figure 1, the conjugate consists of a water-soluble anticancer drug (Ir) and a water-insoluble drug (YC-1) linked by succinate. Ir is a potent DNA topoisomerase I inhibitor in clinic use, while YC-1 is a potent HIF-1α inhibitor that has been considered as one of the most potential drug candidates due to its various potent biological activities like anti-platelet, anti-vascular and anti-cancer activities.17,25,26 Thanks to its amphiphilic structure, the Ir-YC-1 conjugate can self-assemble into nanoparticles in water to deliver themselves into tumor tissues possibly by passive accumulation via enhanced permeability and retention (EPR) effect. After the cellular internalization of Ir-YC-1 NPs, both Ir and YC-1 can be easily released to kill the tumor cells and meantime inhibit HIF-1α to overcome tumor hypoxia via the hydrolysis of the ester bond in the cancer cells.

Results and discussion

Design and synthesis of Ir-YC-1 and Ir-PEG3-YC-1 conjugates

The amphiphilic Ir-YC-1 conjugate, synthesized through esterification using DCC/DMAP as shown in Fig. 1View Article Onlin and Schemee 1, was confirmed by 1H NMR, 13C NMR DOI: 10.1039/C9TB00541Bspectra and high resolution mass spectroscopy (HR-MS) (Fig. S1-9). Considering the introduction of a hydrophilic PEG chain could increase the water solubility of the conjugates, the PEG3 linker was also designed to obtain another conjugate. The synthetic route of IrPEG3-YC-1 was shown in Scheme 2, and the structures data of all the synthesized compounds were shown in (Fig. S10-18). IrYC-1 and Ir-PEG3-YC-1 conjugates were also characterized by ultraviolet−visible spectrophotometer (UV-vis) (Fig. S19 and 20). The target compound Ir-YC-1 with the purity greater than 95%, analyzed by HPLC (Fig. S21).

Preparation and characterization of Ir-YC-1 and Ir-PEG3-YC-1 NPs

The inherent amphiphilicity of Ir-YC-1 and Ir-PEG3-YC-1 ADDCs provide an opportunity for themselves to self-assemble into IrYC-1 and Ir-PEG3-YC-1 NPs in aqueous solution. Thus, a dimethyl sulfoxide (DMSO) solution of the Ir-YC-1 ADDC was added dropwise into water, followed by dialysis against water to remove DMSO to obtain the final Ir-YC-1 conjugate concentration of 0.5 mg/mL. The method for preparing Ir-PEG3YC-1 NPs was similar with that of Ir-YC-1 NPs. To confirm the formation of Ir-YC-1 and Ir-PEG3-YC-1 NPs, the dynamic light scattering (DLS) and transmission electron microscopy (TEM) were performed to determine the size and morphology of Ir-YC1 and Ir-PEG3-YC-1 NPs. The DLS measurement revealed the average diameter of Ir-YC-1 NPs is ~75.6 nm with a polydispersity index (PDI) of 0.219 (Fig. 2a). Notably, the introduction of a hydrophilic PEG chain increased the average diameter of Ir-PEG3-YC-1 NPs (~106.4 nm), with a more potent PDI (0.081) (Fig. S22a). The TEM image of Ir-YC-1 NPs in Fig. 2b exhibited the spherical nanoparticles with an average size of approximate 50.5 nm, while the average size of Ir-PEG3-YC-1 NPs was about 74.0 nm (Fig. S22b), which were somewhat smaller than that measured by DLS due to the shrinkage of nanoparticles in a drying state during TEM sample preparation. In addition, the surface charge of the Ir-YC-1 NPs solution, investigated by DLS, indicated that the value of zeta potential was positive (+5.9) in phosphate buffer solution (PBS, pH 7.4). The DLS measurements at different time intervals demonstrated that the stability of Ir-YC-1 NPs was high enough for long storage (Fig. S23).

In vitro studies of Ir-YC-1 and Ir-PEG3-YC-1 NPs

Hypoxia was associated with human non-small cell lung cancers (NSCLC) such as A549 cells, which was strongly resistant to chemotherapy.27,28 In order to investigate the inhibition effect of cellular proliferation of Ir-YC-1 and Ir-PEG3-YC-1 NPs against HIF-1α over-expressing cancer cells and human normal liver LO2 cells, the cell viability was evaluated by MTT assay. A549 cancer cells (high expression of HIF-1α) and LO2 cells were treated with Ir, YC-1, Ir/YC-1 mixture, Ir-YC-1 and Ir-PEG3-YC-1 NPs, respectively, at the indicated concentrations for 72 h of incubation. Hypoxic condition was achieved by incubating the cells under a hypoxic atmosphere (1% O2, 5% CO2, and N2 94%). Under normoxic condition, the cytotoxicity of Ir against A549 cancer cells is nearly equal to the Ir/YC-1 mixture but much higher than that of YC-1, probably due to the difficult uptake of hydrophobic YC-1 by the cancer cells (Fig. 2c and Table 1). However, the Ir-YC-1 NPs displayed the highest cellular proliferation inhibition toward A549 cells among the tested samples (Fig. 2c). Under hypoxic condition, Ir caused 2.2-fold decrease in anticancer activity compared withView Article Onlin that undeer normoxic condition (Table 1), and the anticancer activity of Ir DOI: 10.1039/C9TB00541B was much lower than that of the Ir/YC-1 mixture under the same condition (Fig. 2d and Table 1). But significantly, the cytotoxity of Ir-YC-1 NPs did not obviously change under either normoxic or hypoxic condition. In addition, the Ir-PEG3-YC-1 NPs exhibited better cellular proliferation inhibition toward A549 cells compared with Ir under normoxic or hypoxic condition, but in contrast, the anticancer activity of Ir-PEG3-YC-1 NPs is lower than that of Ir-YC-1 NPs under the same tested condition (Table 1). More importantly, Ir-YC-1 and Ir-PEG3-YC-1 NPs exhibited lower cytotoxicity toward to human normal liver LO2 cells than that of Ir or Ir/YC-1 mixture in normoxic or hypoxic condition (Table S1). These results demonstrated that the Ir-YC-1 Ps or IrPEG3-YC-1 NPs could effectively overcome the hypoxia of tumor cells, consequently enhancing the therapeutic efficiency of irinotecan.

In vitro cellular uptake

Since Ir-YC-1 NPs exhibited the best antitumor activity against human lung cancer A549 cells, it was therefore selected to carry out the cellular uptake test in A549 cells by confocal laser scanning microscopy (CLSM). In order to investigative the suitable concentration of conjugate, cells were cultured with Ir- YC-1 NPs for 2 h at 10, 20 and 40 μM under normoxic condition before observation. The nuclei were stained for 30 min with propidium iodide (PI), and the prepared cells were observed in CLSM. As shown in Fig. S24, the blue fluorescence of Ir-YC-1 NPs in A549 cells was significantly strong at 20 μM, which can be attributed to the cellular uptake of Ir-YC-1 NPs by A549 cells. Then, the cells were cultured with Ir-YC-1 NPs at 20 μM for 2 and 4 h under normoxic and hypoxic condition. As shown in Fig. 3, the cellular uptake of Ir-YC-1 NPs by A549 cells was significantly strong at both 2 and 4 h. In short, these results proved that Ir-YC-1 NPs could be internalized in the A549 cells under either normoxic or hypoxic condition. Meanwhile, we investigative the cellular uptake ability of Ir-PEG3-YC-1 NPs by A549 cells under normoxic or hypoxic condition. As shown in Fig. S25, the blue fluorescence of Ir-YC-1 NPs in A549 cells was significantly stronger than Ir-PEG3-YC-1 NPs under either normoxic or hypoxic condition, probably due to the smaller average diameter of Ir-YC-1 NPs. The higher cellular uptake level of Ir-YC-1 NPs results in the higher anticancer activity than that of Ir-PEG3-YC-1 NPs. determine whether the death of cancer cells incubated with IrYC-1 NPs was induced by apoptosis. A549 cells were incubated with Ir, YC-1, Ir/YC-1 mixture, and Ir-YC-1 NPs, respectively, at the same concentration (20 μM) for 24 h and then subjected to FITC-Annexin V/PI staining. The untreated A549 cells were served as negative control. The flow cytometry analysis exhibited that the ratio of early and late apoptosis cells was37.43%, 7.42%, 41.76%, and 78.77% induced by Ir, YC-1, Ir/YC-1 mixture and Ir-YC-1 NPs, respectively, when incubation under normoxic condition (Fig. 5a). It was noted under hypoxic condition that the ratio of early and late apoptosis cells was strikingly decreased to 21.47% when induced by Ir, wheras the cells was significantly increased to 70.7% compared with Ir after24 h incubation with Ir-YC-1 NPs under the same condition,

In vitro drug release

The in vitro hydrolysis behavior of Ir-YC-1 conjugate under pHs 5.0 and 7.4 was evaluated by HPLC at different time. As shown in Fig. 4, Ir-YC-1 conjugate was hydrolysed at pH 5.0 and released Ir and YC-1 successfully at 37 °C. This result was also confirmed by HR-MS (Fig. S26). In addition, the in vitro release behavior of Ir-YC-1 NPs was also evaluated by dialysis method in PBS (pH 7.4) with or without 10% FBS and PBS (pH 5.0) at 37 °C. The cumulative release curves (Fig. S27) and the HPLC results at 48 h (Fig. S28) exhibited an obvious pH-responsive release behavior of Ir. At pH 7.4, only ~18 % of Ir was released over 24 h, indicating the high stability of Ir-YC-1 NPs in the physiological condition. In contrast, the cumulative release of Ir was more than ~45 % at pH 5.0 over 24 h, proving that the free form of Ir was released more rapidly from NPs under the acidic condition. Meanwhile, the time-dependent changes in DLS and Fig. 5 (a) Flow cytometry analysis for apoptosis of A549 cells induced by Ir, YC-1, Ir/YC-1 mixture, and Ir-YC-1 NPs, TEM of Ir-YC-1 NPs were measured at pH 5.0. As shown in Fig. respectively, at the same concentration of 20 μM for 24 h under S29, the average diameter of Ir-YC-1 NPs increases from 76 nm either normoxic or hypoxic condition. Q1, Q2, Q3, and Q4 to several hundreds of nanometers with a broad distribution. represent four different cell states: necrotic cells, late apoptotic
The expression levels of caspase-3, and (c) HIF-1α and VEGF in detachment of hydrophilic Ir shells from the micelles and the A549 cells induced by Ir, YC-1, Ir/YC-1 mixture, and Ir-YC-1 NPs enhancement of hydrophobic interaction of YC-1 core. at the indicated concentration (20 μM) for 24 h, determined by In vitro apoptosisWestern blot analysis. Data represent three individual exhibiting a similar trend to that under normoxic condition. To further verify this result, we analyzed activation and expression of caspase-3 that has been considered as a major effector of cell apoptosis and identified to be activated in response to cytotoxic agents.29-31 Western blot analysis revealed that the expression level of caspase-3 protein was slightly up-regulated by Ir, YC-1 and Ir/YC-1 mixture in comparison with untreated control, whereas the expression of caspase-3 protein was markedly upregulated in contrast to that of Ir or Ir-YC-1 mixture when treating the cells with Ir-YC-1 NPs at the same concentration under either normoxic or hypoxic condition (Fig. 5b). These results clearly suggested that despite caspase-3 can be activated by various formulations, the Ir−YC-1 NPs is the most effective one to promote the activation of caspase-3 under the same condition.

Inhibition of HIF-1α and VEGF accumulation by Ir-YC-1 NPs

As HIF-1α caused by cancer cells plays an important role in adaptive response to hypoxia by modulating various cellular functions including proliferation, apoptosis, angiogenesis andView Article Online anaerobic glycolysis,16,25,32 upon activation, DOI: 10.1039/C9TB00541Bit binds to the hypoxia responsive element, thereby promoting transcription of various genes such as VEGF. Therefore, we investigated the effect of Ir-YC-1 NPs on HIF-1α and its downstream target protein VEGF using western blotting and immunofluorescence assay under hypoxic condition in A549 cells. The cells were incubated with Ir, YC-1, Ir/YC-1 mixture, and Ir-YC-1 NPs, respectively, at the same concentration (20 μM) for 24 h. As expected, Ir-YC-1 NPs could significantly down-regulate the level of HIF-1α and VEGF protein expression under hypoxic condition, similar to the effect of YC-1 (Fig. 5c). Importantly, the similar trend was also observed in immunofluorescence assay (Fig. 6 and S30). These results indicated that down-regulation of the expression of HIF-1α and VEGF proteins could effectively alleviate tumor hypoxia and inhibit tumor progression.

Ir-YC-1 NPs induced mitochondrial depolarization and reactive oxygen species production

Many researchers demonstrated that most of small-molecule anticancer agents induce apoptosis through the mitochondrial pathway,33-35 therefore, we further investigated whether Ir-YC1 NPs induced an alteration of the mitochondrial membrane potential (MMP). MMP was monitored by flow cytometry using the dye JC-1. The untreated A549 cells were used as negative control. The cells were incubated with Ir, YC-1, Ir/YC-1 mixture and Ir-YC-1 NPs, respectively, at the same concentration (20 μM) for 24 h under both normoxic and hypoxic conditions. The results indicated that the MMP was decreased after incubation with each sample, whose ability to decrease MMP was consistent with its in vitro cytotoxicity (Fig. 7a). It was observed under normoxic condition that a conspicuous decrease in MMP happened after incubation with Ir-YC-1 NPs (77.87%) in contrast to those with Ir, YC-1 and Ir/YC-1 mixture (44.95%, 10.62% and 63.57%, respectively). Notably, decreased MMP induced by YC1 under hypoxic condition was a little higher than that under normoxic condition (17.24% vs 10.62%), but Ir caused about 2.1-fold decrease in MMP compared with its value under normoxic condition (20.93% vs 44.95%). Significantly, Ir-YC-1 NPs induced a remarkably decrease in MMP (73.15%) under hypoxia in comparison to that of Ir (20.93%). Because mitochondrial membrane depolarization was associated with mitochondrial production of reactive oxygen species (ROS),33-35 and increased ROS could damage macromolecules and alter cell function or induce apoptosis, so we used the fluorescent probe DCF-DA to evaluate intracellular ROS levels in the presence or absence of Ir-YC-1 NPs. As shown in Fig. 7b, Ir-YC-1 NPs caused the production of significant amounts of ROS in comparison with the control cells under either normoxia or hypoxia on the indicated condition. More importantly, Ir-YC-1 NPs (79.92% vs 70.05%) under two circumstances produced much more ROS than Ir did (36.7% vs 21.99%), in agreement with the decrease
NPs at the dose of 10 and 15.6 mg/kg. (b) Tumor volume of the mice in each group during the observation period. (c) Tumor inhibitory rate (IR) after treatment with Ir and Ir-YC-1 NPs in mice bearing A549 xenograft. (d) Representative plasma concentration-time profiles of free YC-1, Ir, and Ir-YC-1 NPs after i.v. injection into rats (a dose of 8 mg/kg).

Pharmacokinetics studies

In general, nanoparticles can enhance the antitumor efficacy by elongating the blood circulation of therapeutic agents, which promotes their accumulation at the tumor site. To determine whether Ir-YC-1 NPs have this property, the pharmacokinetic study was undertaken by i.v. injection of the free YC-1, Ir, and Ir-YC-1 NPs to Sprague-Dawley (SD) rats (~200 g). As shown in Fig. 8d, Ir-YC-1 NPs are retained at a higher concentration in the bloodstream up to 12 h, whereas free YC-1 and Ir could be rapidly cleared from blood after intravenous administration. As compared to that of free YC-1 and Ir, the longer blood retention time of Ir-YC-1 NPs provides the possibility of enhanced drug accumulation in the tumor tissues.

Ir-YC-1 NPs alleviate tumor hypoxia

Finally, the capability of Ir-YC-1 NPs to ameliorate tumor hypoxia in vivo was further performed using immunofluorescence staining assay, in which the tumor cell nuclei and hypoxia areas were stained with 4, 6-diamidino-2phenylindole (DAPI) (blue) and antipimonidazole antibody (green), respectively. As shown in Fig. 9, mice tumor sections of the intravenous administration of Ir-YC-1 NPs (especially at the dose of 15.6 mg/kg) showed a signifcantly weakened green fluorescence (the more pimonidazolestained green fluorescent regions indicate the more severe hypoxia of tumor tissue) compared to the control and Ir-treated groups, respectively.

Conclusions

In summary, we have developed two new “all-in-one” nanodrug deliver system, derived from the self-assembly of irinotican and YC-1, to promote the antitumor efficiency of chemotherapeutic agents against hypoxic tumors. Ir and YC-1 are linked through dicarboxylate or PEG3 to obtain two conjugates. Benefiting from the amphiphilicity of Ir-YC-1 NPs, the resulting Ir-YC-1 nanoparticles can effectively enter cancer cells via EPR-effectmediated passive targeting. As expected, the Ir-YC-1 and IrPEG3-YC-1 NPs showed stronger anticancer activities against A549 cells than that of Ir. However, the anticancer activity of IrPEG3-YC-1 NPs is lower than that of Ir-YC-1 NPs under the same conditions. Interestingly, the study clearly indicated that the IrYC-1 NPs exhibited better anticancer activity and higher apoptotic ratio than either free individual drug or a mixture of two drugs under both normoxic and hypoxic conditions. More importantly, the Ir-YC-1 NPs significantly down-regulated the expression of HIF-1α and VEGF proteins in A549 cancer cells under hypoxic condition, serving to alleviate hypoxia-induced Ir resistance in solid malignancies. These advantages of the Ir-YC1 NPs result in a higher antitumor efficacy and a significant inhibition of tumor hypoxia than irinotican in vivo. In short, this work provides a new insight into the design of smart nanodrug delivery systems to achieve more effective chemotherapeutic therapy in hypoxic tumor tissue.

Materials

All solvents and chemicals were used as purchased without further purification, unless noted specifically. N, N’Dicyclohexylcarbodiimide (DCC, 99%, J&K), 4(dimethylamino)pyridine (DMAP, 99%, J&K) and 3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) were used as received without further purification. GAPDH, caspase-3, HIF-1α and VEGF antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). All secondary antibodies were obtained from Earthox LLC. (Earthox, USA). All chemicals and solvents were of analytical reagent grade and used without further purification, unless noted specifically. Irinotecan (Ir) was purchased from Energy Chemical, NH2-PEG3-COOH was purchased from Peng Sheng Biological, Shanghai of China, and used without further purification. YC-1 was prepared according to literature report.25,36 TLC was performed on 0.20 mm silica gel 60 F254 plates and column chromatography was performed using silica gel (200-300 mesh). High performance liquid chromatography (HPLC) was performed on an Agilent 1260 system equipped with a phoenix C18 column (250 × 4.6 mm, 5 μm). The purity of the compound Ir-YC-1 was analyzed by HPLC using a mixture of solvent acetonitrile/water at the flow rate of 1.0 mL/min with peak detection at 210 and 254 nm under UV. Mass spectra were measured on an Agilent 6224 TOF LC/MS instrument. 1H NMR and 13C NMR spectra were recorded in CDCl3 or DMSO-d6 with a Bruker 400 MHz spectrometer.

Preparation and characterization of Ir-YC-1 and Ir-PEG3-YC-1 NPs

The method of preparing Ir-PEG3-YC-1 NPs was the same as that of Ir-YC-1 NPs. In brief, 5 mg Ir-YC-1 was dissolved in 2 mL of dimethylsulfoxide (DMSO) and stirred at room temperature for 10 min. Then, the solution was slowly added to 3 mL of deionized water and stirred slightly for 1 h. Subsequently, the solution was dialyzed against deionized water for 48 h (molecular weight cutoff = 1,000 g/mol), during which the water was renewed every 4 h. The volume of the solution was increased to 10 mL with the addition of deionized water to produce a solution with a concentration of 0.5 mg/mL for further experiments.

In vitro cytotoxicity measurements of Ir-YC-1 and Ir-PEG3-YC-1 NPs

A549 cells were used to evaluate the anticancer activity of IrYC-1 NPs under the normoxic or hypoxic condition. Untreated cells were used as negative control. Cells were treated with IrYC-1 and Ir-PEG3-YC-1 NPs, Ir, YC-1 and Ir/YC-1 mixture were used as positive controls. All tested compounds were dissolved in DMSO to a final concentration of 2 mmol/L and then subsequently diluted in culture medium at final concentration of 1.25, 2.5, 5, 10, 20, 40, 80 μmol/L, respectively. About 5×104 cells/mL cells, which were in the logarithmic phase, were seeded in each well of 96-well plates and incubated for 12 h at the indicated condition. Compounds at seven different concentrations were then added to the test well and the cells were incubated at 37 °C under the indicated condition (normoxia or hypoxia) for 72 h. An enzyme labeling instrument was used to read absorbance with 570/630 nm double wavelength measurement. Cytotoxicity was examined on the percentage of cell survival compared with the negative control. The final IC50 values were calculated by the Bliss method (n = 5). All of the tests were repeated in three times.

In vitro drug release

The released ability of Ir-YC-1 conjugate in PBS (pH 7.4 or pH 5.0) for different time was studied by HPLC at 37 °C. Briefly, the Ir-YC-1 conjugate was incubated in PBS (pH 7.4 or pH 5.0) for 6, 12 and 24 h. HPLC profiles were recorded on UV detection at 254 nm. Mobile phase consisted of acetonitrile/water (10:90 to 100:0, v/v), and flow rate was 1.0 mL/min. The samples were taken for HPLC analysis after filtration by 0.45 μm filter. In addition, the in vitro release behavior of Ir-YC-1 NPs was evaluated by dialysis method. Typically, 2 mL of Ir-YC-1 NPs (0.5 mg/mL) was transferred into a membrance tubing (MWCO = 3500). It was incubated at 37 °C in 60 mL PBS (pH 7.4) containing (or not) 10% fetal bovine serum (FBS) in 60 mL PBS (pH 5.0) atView Article Online predetermined time intervals. Then 4 mL DOI: 10.1039/C9TB00541Bof external buffer solution was withdrawn and replaced with 4 mL of fresh PBS (pH 7.4) containing (or not) 10% FBS or PBS (pH 5.0). The amount of released Ir was determined by using fluorescence measurement (QC-4-CW spectrometer, excitation at 360 nm) and HPLC (detection at 254 nm under UV).

Cell culture

A549 cells (human lung cancer cells) in this study were purchased from China Life Science Collage (Shanghai, PRC). Culture medium Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS, pH=7.2), and Antibiotice-Antimycotic came from KeyGen Biotech Company (China). Cells were grown in the supplemented with 10% FBS, 100 units/ml of penicillin and 100 g/ml of streptomycin in a humidified atmosphere of normoxia (20% O2, 75% N2 and 5% CO2) or hypoxia (1% O2, 94% N2 and 5% CO2) at 37 oC.

Cellular uptake of Ir-YC-1 NPs in A549 Cells

The cellular uptake behaviors were studied in A549 cells using confocal laser scanning microcopy (CLSM). A549 cells were seeded into 6-well plates at a density of 0.5 × 104 cells per well, incubated at 37°C for 24 h, and then treated with Ir-YC-1 NPs at the indicated concentration for different incubation time periods at 37°C under the normoxic or hypoxic condition. After incubation, the cells were washed thrice and then fixed by 70% ice-cold ethanol at -20°C for 30 min. Subsequently the cells were washed with PBS three times, and the nuclei were then stained by PI for 30 min. Finally, the cells were mounted and observed with a Leica TCS SP5 confocal laser scanning microscopy.

Cell apoptosis

Apoptosis was detected by flow cytometry with annexin V/PI staining. A549 cells were grown in each well of six-well plates at the density of 5×104 cells/mL of the DMEM medium with 10% FBS to the final volume of 2 mL. The plates were incubated for overnight and treated with test compounds at the indicated concentration (20 μM) for 24 h under the normoxic or hypoxic condition. Briefly, cells were harvested and washed with twice ice-cold PBS, and then suspended cells in the annexin-binding buffer at a concentration of 5× 105 cells /ml. Cells were then incubated with 5 μL of annexin V-FITC and 5 μL of PI for 30 minutes at room temperature in the dark. The cells were detected by system software (Cell Quest; BD Biosciences).

Western blot analysis

Western blot analysis was performed as described previously.37 A549 cells were treated with test compounds at the indicated concentration (20 μM) for 24 h under the normoxic or hypoxic condition. Untreated cells were used as negative control. Cells were treated with Ir, YC-1 and Ir/YC-1 mixture were used as positive control. After for 24 h, cells were collected, centrifuged, and washed twice with ice-cold PBS. The pellet was then resuspended in lysis buffer. After the cells were lysed on ice for 30 min, lysates were centrifuged at 20000 g at 4 °C for 10 min.
The protein concentration in the supernatant was detected by the BCA protein assay reagents. Equal amounts of protein per line were was separated on 12% SDS polyacrylamide gel electrophoresis and transferred to PVDF Hybond-P membrane (GE Healthcare). Membranes were incubated with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) buffer for 1 h and then the membranes being gently rotated overnight at 4 °C. Membranes were then incubated with primary antibodies against caspase-3, HIF-1α and VEGF or GAPDH for overnight at 4 °C. Membranes were next incubated with peroxidase labeled secondary antibodies for 2 h. Then all membranes were washed with TBST four times for 20 minutes and the protein blots were detected by chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). The X-ray films were developed with developer and fixed with fixer solution. Immunofluorescence assay
A549 cells were grown in each well of six-well plates at the density of 5×104 cells/mL of the DMEM medium with 10% FBS to the final volume of 2 mL. The plates were incubated for overnight and treated with test compounds at the indicated concentration (20 μM) for 24 h under the hypoxic condition. Briefly, cells were harvested and washed with twice ice-cold PBS, and fixed in 4% paraformaldehyde at 37 °C for 15 min, and then the cells were blocked for 30 min in 10% goat serum. The cells were incubated overnight with primary antibody (HIF-1α and VEGF) at 4 °C. The next day the cells were washed with icePBS three times, and cells were incubated with corresponding fluorescence-conjugated secondary antibody for 2 h. After the nuclei of cells were stained with DAPI in the dark at room temperature for 30 min. Cells were visualized using a fluorescence microscopy.

Mitochondrial membrane potential (MMP) assay

MMP was measured with the lipophilic cation probe JC-1 as literature reported.37 A549 cells were treated with test compounds at the indicated concentration (20 μM) for 24 h under the normoxic or hypoxic condition. After for 24 h, the JC-1 fluorescent probe was added 20 minutes after replacing with fresh medium. Cells were collected at 2500 rpm and washed twice with ice-cold PBS and then the MMP were detected by flow cytometer.

ROS assay

The production of ROS was examined by flow cytometry using DCFH-DA (Molecular Probe, Beyotime, Haimen, China), as literature reported.37 A549 cells were grown into six-well plates and treated with test compounds at the indicated concentration (20 μM) under the normoxic or hypoxic condition for 24 h. On the following treatment, cells were harvested at 2000 rpm and washed twice with ice-cold PBS, and then resuspend cells in 10 mM DCFH-DA dissolved in cell free medium at 37 °C for 20 min in dark, and then washed twice with PBS. Cellular fluorescence was detected by flow cytometry at an excitation of 485 nm and an emission of 538 nm, respectively.

In vivo antitumor activity

The mice conducted in vivo experiment were purchased from Shanghai Ling Chang biotechnology company (China), and of Simcere according to the guidelines for laboratory animals DOI: 10.1039/C9TB00541B approved. All surgical interventions and postoperative animal care procedures were approved by the Experimental Animal Ethics Committee of Southeast University (Nanjing, China). The in vivo antitumor activity of Ir-YC-1 NPs was evaluated using human lung cancer cells in BALB/c nude mice. Tumors of fiveweek-old male BALB/c nude mice were induced by a subcutaneous injection in their dorsal region of 5.0 × 106 cells in 100 μL of sterile PBS. Animals were randomly divided into four groups, and started on the second day. When the tumors reached a volume of 100 mm3 in all mice on day 20, the first group was injected with an equivalent volume of physiological saline via a tail vein as the vehicle control mice. No. 2 group was treated with Ir.HCl at dose of 10 mg/kg body weight once every three days for four weeks. No. 3 and No. 4 groups were treated with Ir-YC-1 NPs at the doses of 10 mg/kg (equal mass dose to Ir.HCl) and 15.6 mg/kg (equal molar dose to Ir.HCl) body weight once every three days for four weeks, respectively. All tested samples were dissolved in vehicle. Tumor volume and body weight were recorded every other day after drug treatment. All mice were sacrificed after four weeks of treatment and the tumor volumes were measured with electronic digital calipers and determined by measuring length (A) and width (B) to calculate volume (V = AB2/2). For histological study, the major organs (liver, heart, lung and kidney) were fixed with 10% formalin and were then paraffin-embedded, sectioned, and stained with H&E.

Pharmacokinetics studies

For pharmacokinetic studies, SD rats (~200 g) were randomly divided into Ir-YC-1 NPs and free YC-1, Ir groups (n = 3). The aqueous solutions of Ir-YC-1 NPs, free YC-1 and Ir were intravenously injected via tail vein at a dose of 8 mg/kg. At predetermined time intervals (5 min, 30 min, 1 h, 4 h, 8 h and 12 h), blood samples were collected from the retro-orbital plexus of eyes, then placed in heparinized tubes and centrifuged to obtain plasma. The amounts of Ir-YC-1 NPs, free YC-1 and Ir were obtained from standard curves previously obtained by analysis of known amounts of Ir-YC-1 NPs, YC-1 and Ir using high performance liquid chromatography (Waters e2695 HPLC, USA).

Hypoxia immunofluorescence assay

For immunofluorescence staining, tumor sections were incubated with antipimonidazole antibody (FITC-MBb1) (dilution 1:100, Hypoxyprobe Inc.) and horseradish peroxidase linked to rabbit anti-FITC secondary antibody (dilution 1:100) following the kit’s instructions. Nuclei were stained with DAPI (blue) and hypoxia areas were stained with antipimonidazole antibody (green). Images were obtained by confocal microscopy.

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