Tumor Oxygenation and Hypoxia-Inducible-Factor-1 Functional

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Tumor Oxygenation and Hypoxia-Inducible-Factor-1 Functional Inhibition via a Reactive-Oxygen-Species Responsive Nanoplatform for Enhancing Radiation Therapy and Abscopal Effects Lingtong Meng, Yali Cheng, Xiaoning Tong, Shaoju Gan, Yawen Ding, Yu Zhang, Chao Wang, Lei Xu, Yishen Zhu, Jinhui Wu, Yiqiao Hu, and Ahu Yuan ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b03590 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

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Tumor Oxygenation and Hypoxia-Inducible-Factor-1 Functional Inhibition via a Reactive-Oxygen-Species Responsive Nanoplatform for Enhancing Radiation Therapy and Abscopal Effects Lingtong Meng1,#, Yali Cheng1,#, Xiaoning Tong1, Shaoju Gan1, Yawen Ding1, Yu Zhang1, Chao 1

1,4

4

1,2,3

Wang , Lei Xu , Yishen Zhu , Jinhui Wu

1,2,3,

*, Yiqiao Hu

1,2,3,

*, Ahu Yuan

*

Affiliations: 1

State Key Laboratory of Pharmaceutical Biotechnology, Medical School and School of life science, Nanjing University, Nanjing 210093, China;

2

Jiangsu Key Laboratory for Nano Technology, Nanjing University, Nanjing 210093, China;

3

Institute of Drug R&D, Medical School of Nanjing University, Nanjing 210093, China;

4

College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211800, China.

# These authors contributed equally. *Author for correspondence: Jinhui Wu, Ph.D. Associate Professor Address: 22 Hankou Road, Nanjing 210093, China Phone: +86-25-83596143; E-mail: [email protected] Yiqiao Hu, Ph.D. Professor Address: 22 Hankou Road, Nanjing 210093, China Phone: +86-25-83596143; E-mail: [email protected] Ahu Yuan, Ph.D. Assistant Professor Address: 22 Hankou Road, Nanjing 210093, China Phone: +86-25-83596143; E-mail: [email protected]

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Abstract Hypoxia, and hypoxia-inducible factor-1 (HIF-1), can induce tumor resistance to radiation therapy. To overcome hypoxia-induced radiation resistance, recent studies have described nano-systems to improve tumor oxygenation for immobilizing DNA damage and simultaneously initiate oxygen-dependent HIF-1α degradation. However, HIF-1α degradation is incomplete during tumor oxygenation treatment alone. Therefore, tumor oxygenation combined with residual HIF-1 functional inhibition is crucial to optimizing therapeutic outcomes of radiotherapy. Here, a reactive oxygen species (ROS) responsive nanoplatform is reported to successfully add up tumor oxygenation and HIF-1 functional inhibition. This ROS-responsive nanoplatform, based on manganese dioxide (MnO2) nanoparticles, delivers the HIF-1 inhibitor acriflavine and other hydrophilic cationic drugs to tumor tissues. After reacting with overexpressed hydrogen peroxide (H2O2) within tumor tissues, Mn2+ and oxygen molecules are released for magnetic resonance imaging and tumor oxygenation, respectively. Cooperating with the HIF-1 functional inhibition, the expression of tumor invasion-related signaling molecules (VEGF, MMP-9) are obviously decreased to reduce the risk of metastasis. Furthermore, the nanoplatform could relieve T-cell exhaustion via downregulation of PD-L1, whose effects are similar to the checkpoint inhibitor PD-L1 antibody, and subsequently activates tumor-specific immune responses against abscopal tumors. These therapeutic benefits including increased X-ray


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induced damage, downregulated resistance and T-cell exhaustion related proteins expression, achieved synergistically the optimal inhibition of tumor growth. Overall, this designed ROS-responsive nanoplatform is of great potential in the sensitization of radiation for combating primary and metastatic tumors.

Keywords ROS-responsive nanoplatform, tumor oxygenation, HIF-1 functional inhibition, abscopal effects, metastasis


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Radiotherapy is a universal and essential approach to cancer treatment, and about 50% of all cancer patients have received radiotherapy.1 However, not all patients benefit from radiotherapy because hypoxia, especially hypoxia-inducible factor-1 (HIF-1), can induce tumor resistance to radiation.2 To overcome hypoxia-induced radiation resistance, researchers have developed some intelligent strategies to improve tumor oxygenation and enhance radiation therapy. These strategies provide oxygen to immobilize DNA damage to sensitize radiotherapy, but the degradation of HIF-1α is incomplete because the oxygen supply is transient due to the rapid oxygen consumption of proliferative cancer cells.3-5 Current tumor oxygenation agents including catalase, manganese dioxide, and perfluorocarbon nanoparticles, can only induce 25%–70% of HIF-1α degradation during radiation or photodynamic therapy.6-10 The residual HIF-1α will further dimerize with HIF-1β to form HIF-1 and initiate downstream gene transcription, including that of GLUT-1, PD-L1, and VEGF, which are mainly involved in cellular proliferation, immune exhaustion, and metastasis, respectively.11,12 Therefore, tumor oxygenation cooperation with residual HIF-1 functional inhibition is crucial to obtaining optimal radiation therapeutic outcomes. Recently, manganese dioxide nanoparticles (MnO2-NPs) have been widely

studied

as

a

catalase-like

nanoenzyme

to

decompose

the

overexpressed H2O2 within the tumor microenvironment into oxygen molecules,


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which can relieve tumor hypoxia and enhance the therapeutic efficacy of radiation or photodynamic therapies.13-15 Moreover, these biocompatible MnO2-NPs take advantage of T1-weighted magnetic resonance imaging for tumor detection and image-guided therapy.16,17 Therefore, MnO2-NPs are promising tumor oxygenation agents for radiation sensitization. In addition, acriflavine (ACF) is a potent HIF-1 functional inhibitor that prevents HIF-1α/β dimerization. It is also a safe and FDA-approved antibacterial.18 Acriflavine is hydrophilic and cationic, and exhibits a short pharmacokinetic half-life, since the concentration of acriflavine in the blood decreases by 90% over 5 min after intravenous injection.19 The rapid clearance and poor tumor accumulation of acriflavine determine that repeated injections at a large dose offer mild tumor suppression.18 Therefore, integrating hydrophilic and cationic molecules like acriflavine into catalytic MnO2 nanoparticles in a simple way to improve their pharmacokinetic characteristics is an urgent challenge in the fields of both radiation therapy and drug delivery. Inspired by the application of MnO2 to eliminate metal cation pollution (Cu2+, Pb2+) via electrostatic adsorption, we surprisingly found high affinity between anionic MnO2 nanoparticles and cationic small molecules (i.e., the HIF-1 functional inhibitor acriflavine, photosensitizer methylene blue, CXCR4 inhibitor AMD3100, and others) which could be potentially applied in multiple tumor therapeutics.20-25 Subsequently, we established a reactive oxygen species

(ROS)-responsive

nanoplatform

to

successfully


encapsulate

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hydrophilic and cationic acriflavine into MnO2 nanoparticles (ACF@MnO2) (Figure 1a). After accumulation within the tumor tissue, these nanoparticles could be efficiently endocytosed by tumor cells and mainly located within the lysosomes. Once reacted with H2O2 in the acidic conditions, Mn2+ was released within the tumor tissues for magnetic resonance imaging (MRI) to guide radiation therapy. The nanoparticles would then produce oxygen to relieve the hypoxic microenvironment and implement direct radiation sensitization. Meanwhile, the released acriflavine would gradually transfer into the cellular nucleus to inhibit HIF-1’s transcription function to synergistically inhibit HIF-1 downstream signaling molecules (e.g., PD-L1) against abscopal tumors. Our work thus presented an ROS-responsive nanoplatform with great efficiency for tumor oxygenation and HIF-1 functional inhibition to combat primary and metastatic tumors (Figure 1b).


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Figure 1. (a) Schematic illustration of two-step method to prepare ROS-responsive nanoplatform, e.g., ACF@MnO2. (b) The mechanism of ACF@MnO2 for enhancing MRI guided radiation therapy and abscopal effects via tumor oxygenation and HIF-1 function inhibition simultaneously to combat primary and metastatic tumors. Results Preparation and Characterization of MnO2-ACF Intermediate. As MnO2 was widely applied in the field of sewage treatment, we speculated that there would be strong affinity between MnO2 nanoparticles and the heterocyclic aromatic compound acriflavine that was positive charge in aqueous solution (Figure 2a and Figure S1). The interaction between MnO2 and acriflavine was observed by isothermal titration calorimetry (ITC). Acriflavine binding to MnO2 instead of HSA was an exothermal and spontaneous process (∆H= -2.55


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kcal/mol, ∆G= -5.91 kcal/mol) with a negative entropy change. The process was driven by both enthalpy and entropy due to electrostatic attraction and hydrogen bond formation (Figure 2b).26 The MnO2-ACF intermediate was formed via the processes of attraction and deposition where acriflavine and KMnO4 solution were added dropwise alternately into MnO2 nanoparticles. The unadsorbed acriflavine was then removed by an ultrafiltration device to obtain the MnO2-ACF intermediate. To confirm the force between MnO2 and acriflavine, the resulting MnO2-ACF intermediate was diluted with various concentrations of NaCl solution. At physiological concentration of NaCl (0.9%), 15.3% acriflavine was substituted from MnO2-ACF intermediate. In addition, the release of acriflavine from MnO2-ACF intermediate was dependent on the concentration of NaCl, but there was no observable acriflavine release diluted with distilled deionized water (Figure 2c). These results indicated that electrostatic attraction played an important role in forming the MnO2-ACF intermediate. Urea was a potent hydrogen breaker.27 As the concentration of urea increased, the acriflavine released from the MnO2-ACF intermediate gradually increased. The results in Figure 2d suggested that the hydrogen bond also mediated the affinity between acriflavine and MnO2. Overall, the mechanism of forming the MnO2-ACF intermediate was mainly attributed to the interaction force between MnO2 and the cationic acriflavine including electrovalent and hydrogen bonds.


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Figure 2. Preparation and characterization of MnO2-ACF intermediate. (a) Schematic illustration of formation of MnO2-ACF intermediate attributed to electrostatic attraction and hydrogen bond. (b) Isothermal titration calorimetry (ITC) of MnO2 with acriflavine. The upper panel of graph indicated heat flow of each injection (µcal/s) as a function of time (min); the lower panel indicates the integration of each peak (kcal/mol) as a function of molar ratio of acriflavine to MnO2 after subtracting the heat of acriflavine dropped into HSA solution. (c) UV-absorbance spectrum of released acriflavine from MnO2-ACF intermediate ([acriflavine]=0.15 mM) in the presence of different Na+ concentrations (0, 0.15, 0.75, 1.5, 3, 15, 60, and 150 mM). (d) UV-absorbance spectrum of released acriflavine from MnO2-ACF intermediate ([acriflavine]=0.15 mM) in the presence of different urea concentrations (0, 0.15, 0.75, 1.5, 3, 75, and 150 mM). (e-f) Dynamic light scattering (DLS) and zeta potential of MnO2, MnO2-ACF intermediate, and ACF@MnO2. Data was shown as mean ±SD. Preparation and Characterization of ACF@MnO2. To prevent premature release during circulation, an external shell was formed after additional deposition of MnO2 on the MnO2-ACF intermediate to obtain ACF@MnO2. As figure 2e shown, the basal template MnO2 nanoparticles were approximately 5


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nm. With MnO2 deposition and acriflavine adsorption, the diameter of the MnO2-ACF intermediate gradually grew during the process. The size of the final ACF@MnO2 nanoparticles was about 30 nm. The MnO2 nanoparticles had a zeta potential of -21.70 mV while cationic acriflavine absorption decreased the charge to -1.60 mV. After MnO2 re-deposition, the zeta potential of ACF@MnO2 turned to -13.60 mV (Figure 2f and 3a). Moreover, this facile two-step method, was also suitable for other hydrophilic and cationic drugs including the toll-like receptor agonist imiquimod (R837), the photosensitizer methylene blue (MB), and the CXCR4 inhibitor AMD3100 (Figure 3a and 3b). These were successfully encapsulated into MnO2 nanoparticles to form R837@MnO2, MB@MnO2, and AMD3100@MnO2, respectively (Figure S2, Figure S3 and Table S1). TEM images showed that the ACF@MnO2 was spherical with a rough surface and a diameter of 20-30 nm consistent with DLS data (Figure 3c). Lattice fringes observed in the high resolution TEM images indicated the structure of MnO2 in the ACF@MnO2 with a lattice distance of 0.24 nm (Figure 3d). No characteristic lattice fringes were found after reaction with H2O2 (Figure S4). Encapsulation of acriflavine within the nanoparticles was confirmed through Fourier transform infrared spectrum (FTIR). There was a strong split peak of acriflavine at 1598 cm-1 attributed to conjugation of the aromatic ring and heterocyclic aromatic ring; no split peak was observed in the spectrum of MnO2 nanoparticles. Although FTIR of ACF@MnO2 did not show a


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related peak, a characteristic absorption peak at 1598 cm-1 was found in the subtractive spectrum of ACF@MnO2 deducted by MnO2 nanoparticles (Figure 3e). Furthermore, the status of the encapsulated acriflavine was also studied with X-ray diffraction. The diffraction pattern for pure acriflavine exhibited several sharp peaks at 10.32 º, 18.18 º, 26.70 º, 31.68 º, and 45.46 º as well as a mixture of MnO2 nanoparticles and acriflavine. However, there was no characteristic peak in the XRD spectrum for ACF@MnO2 which indicated that acriflavine was randomly dispersed within the ACF@MnO2 in an amorphous state (Figure 3f). To inspect the selective responsiveness of ACF@MnO2 to the tumor microenvironment, different concentrations of H2O2 were added to react with MnO2. As expected, the UV-absorption of MnO2 at 400 nm decreased with H2O2 addition while the characteristic peak of acriflavine at 450 nm remained constant (Figure 3g). There was no fluorescence observed in the ACF@MnO2 solution due to self-quenching. After reaction with H2O2, the fluorescence of acriflavine gradually recovered, and the intensity of fluorescence showed a positive correlation with increased H2O2 concentration (Figure 3h). There was negligible acriflavine release in the PBS, Na+ or urea solutions, which indicated the stabilization of ACF@MnO2 and little leakage during circulation (Figure S5). Once nanoparticles incubated with H2O2 (100 µM, pH=6.5), acriflavine could be gradually released from ACF@MnO2 (Figure 3i). Furthermore, the stability of the ACF@MnO2 was also investigated by DLS


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data under various conditions including PBS at 25 ºC, 37 ºC and serum at 25 ºC, 37 ºC, respectively. The size data in Figure 3j indicated that ACF@MnO2 was stable and remained ~30 nm within 24 hours. In addition, the ACF@MnO2 could maintain stability at 4 ºC for at least three months which was a crucial factor in performing a wide range of clinical applications (Figure S6).

Figure 3. The main characteristics of ACF@MnO2. (a) Schematic illustration of preparation of ACF@MnO2 nanoparticles with shelter shell. (b) Photos of the nanoparticles with the developed fabrication approach. (c-d) TEM image (scale bar=100 nm) and enlarged HRTEM image (scale bar=5 nm) of ACF@MnO2. (e) Fourier transform infrared spectrum of acriflavine, MnO2, ACF@MnO2, and subtractive spectrum of ACF@MnO2 deducted by MnO2. The black circle indicated the characteristic absorption peak of acriflavine attributed to the conjugation of aromatic ring and heterocyclic aromatic ring. (f) XRD patterns of acriflavine, MnO2, ACF@MnO2, and MnO2+ACF physical mixture. (g) UV-absorbance spectrum of ACF@MnO2 ([MnO2]=400 µM, [acriflavine]=32 µM) reacted with various concentrations of H2O2 (0, 50, 100, 150, 200, 300, and 400 µM). (h) Fluorescence spectrum of ACF@MnO2 ([MnO2]=400 µM, [acriflavine]=32 µM) reacted with different concentrations of H2O2 (0, 25, 50, 100, 150, 400, and 800 µM) (i) In vitro release profile of ACF@MnO2 in the


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presence or absence of H2O2 at pH=7.4 or pH=6.5 PBS, respectively. (j) Dynamic light scattering (DLS) data of ACF@MnO2 incubated with PBS or serum at 25 ºC or 37 ºC, respectively. Data was shown as mean ±SD. Cellular Uptake and Penetration of ACF@MnO2 in Vitro. To study the interaction between ACF@MnO2 and CT26 colorectal tumor cells, Lysotracker and DAPI were used to mark the lysosome and nucleus, respectively. After incubation of ACF@MnO2 for 0.5 h, acriflavine was mainly located in the lysosomes where many orange regions emerged. One hour later, acriflavine was partially transported into the cytoplasm. Then, more acriflavine was found in the area of the nucleus at 12 h post incubation (Figure 4a). Similarly, Mander’s colocalization coefficients of acriflavine with lysosome was 0.56 at 0.5 h and decreased to 0.07 at 12 h. Meanwhile, the colocalization of acriflavine with the nucleus was the opposite of acriflavine with the lysosome. Mander’s colocalization coefficients of acriflavine with the nucleus increased from 0.24 at 0.5 h to 0.70 at 12 h (Figure 4b). These results indicated that ACF@MnO2 nanoparticles could be endocytosed by tumor cells and mainly located in the lysosomes. Then, these nanoparticles could react with abundant H+ and H2O2 to release Mn2+ and acriflavine molecules. After that, free acriflavine might passively diffuse across the lysosomal membrane and transfer into cytoplasm/nucleus for performing HIF-1 functional inhibition.28 As the concentration of ACF@MnO2 increased, more acriflavine fluorescence was detected within CT26 cells as shown in flow cytometry (Figure 4c). To simulate tumor tissue in vitro, CT26 tumor cell 3D spheroids were


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established in ultra-low attachment round bottom 96-well plates.29 Most ACF@MnO2 were located in the marginal areas (the 50-µm section) at 6 h. After incubation with ACF@MnO2 for 12 h, both marginal area and core regions were lighted by the fluorescence of acriflavine. The core region of the cell spheroid was brighter when the incubation time was prolonged to 24 h (Figure 4d). Furthermore, the dynamic intensity of fluorescence at the 50-µm section showed that the distance of penetration of ACF@MnO2 through 3D cell spheroids was dependent on the incubation time (Figure 4e and 4f). Compared with free acriflavine, the penetration of ACF@MnO2 was remarkably increased, which could deliver more acriflavine into the depth of 3D cell spheroids. (Figure S7). These results indicated the ability of ACF@MnO2 to penetrate through tumor lesions and be effectively endocytosed by tumor cells.


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Figure 4. Cellular uptake and penetration of ACF@MnO2 in vitro. (a) Laser scanning confocal microscope (LSCM) images of CT26 cells after treatment with ACF@MnO2, DAPI and Lysotracker, respectively. Yellow regions indicated localization of acriflavine in the lysosomes, scale bar=20 µm. (b) Mander’s co-localization coefficients of acriflavine with nucleus or lysosomes at different time points. Acriflavine could be released from ACF@MnO2 in the lysosomes, then transfer into cytoplasm and nucleus. (c) Flow cytometry of CT26 cells incubated with diverse concentrations of ACF@MnO2 ([acriflavine]=0, 0.08, 0.4, 0.8, and 2 µM). (d) Penetration of ACF@MnO2 in the CT26 cell 3D spheroids. The images were obtained by LSCM after CT26 cells incubated with ACF@MnO2 ([acriflavine]=4 µM) for various incubation times; scale bar=100 µm. (e-f) Dynamic acriflavine fluorescence intensity profile of the sections labeled with white arrow and three-dimensional quantification of fluorescence intensity of cell spheroids at 50 µm. Data was shown as mean ± SD. *p

Tumor Oxygenation and Hypoxia-Inducible-Factor-1 Functional

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