Hierarchical Multiplexing Nanodroplets for Imaging-Guided Cancer

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Hierarchical Multiplexing Nanodroplets for ImagingGuided Cancer Radiotherapy via DNA Damage Enhancement and Concomitant DNA Repair Prevention Wei Jiang, Quan Li, Liang Xiao, Jiaxiang Dou, Yi Liu, Wenhao Yu, Yinchu Ma, Xiaoqiu Li, Ye-Zi You, Zhuting Tong, Hang Liu, Hui Liang, Ligong Lu, Xiaoding Xu, Yandan Yao, Guoqing Zhang, Yucai Wang, and Jun Wang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01508 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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Hierarchical Multiplexing Nanodroplets for Imaging-Guided Cancer Radiotherapy via DNA Damage Enhancement and Concomitant DNA Repair Prevention Wei Jiang,†,⊥ Quan Li,‡,⊥ Liang Xiao,§,⊥ Jiaxiang Dou,ǁ Yi Liu,ǁ Wenhao Yu,# Yinchu Ma,# Xiaoqiu Li,╧ Ye-Zi You,# Zhuting Tong,§ Hang Liu,# Hui Liang,╧ Ligong Lu,╧ Xiaoding Xu,∆ Yandan Yao,*,‡ Guoqing Zhang,*,† Yucai Wang,*,ǁ and Jun Wang▼ †

Hefei National Laboratory for Physical Sciences at the Microscale, University of

Science and Technology of China, Hefei 230027, China ‡

Breast Tumor Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University,

Guangzhou 510120, China §

Department of Radiotherapy, the First Affiliated Hospital of Anhui Medical

University, Hefei 230022, China ǁ

The CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life

Sciences and Medical Center, University of Science and Technology of China, Hefei 230027, China #

Department of Polymer Science and Engineering, University of Science and

Technology of China, Hefei 230027, China ╧

Center of Intervention radiology, Zhuhai Precision Medicine Center, Zhuhai

People's Hospital of Jinan University, Zhuhai 519000, China. ∆

Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene

Regulation, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China

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▼

Institutes for Life Sciences, School of Medicine and National Engineering Research

Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006, China *Corresponding

Authors:

[email protected]

(Yandan

Yao),

[email protected] (Guoqing Zhang) and [email protected] (Yucai Wang)

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ABTRACT: Clinical success of cancer radiotherapy is usually impeded by a combination of two factors, i.e., insufficient DNA damage and rapid DNA repair during and after treatment, respectively. Existing strategies for optimizing the radiotherapeutic efficacy often focus on only one facet of the issue, which may fail to function in the long-term trials. Herein, we report a DNA-dual-targeting approach for enhanced cancer radiotherapy using a hierarchical multiplexing nanodroplet, which can simultaneously promote DNA lesion formation and prevent subsequent DNA damage repair. Specifically, the ultra-small gold nanoparticles encapsulated in the liquid nanodroplets can concentrate the radiation energy and induce dramatic DNA damage as evidenced by the enhanced formation of γ-H2AX foci as well as in vivo tumor growth inhibition. Additionally, the ultrasound-triggered burst release of oxygen may relieve tumor hypoxia and fix the DNA radical intermediates produced by ionizing radiation, prevent DNA repair and eventually result in cancer death. Finally, the nanodroplet platform is compatible with fluorescence, ultrasound, and magnetic

resonance

imaging

techniques,

allowing

for

real-time

in

vivo

imaging-guided precision radiotherapy in an EMT-6 tumor model with significantly enhanced treatment efficacy. Our DNA-dual-targeting design of simultaneously enhancing DNA damage and preventing DNA repair presents an innovative strategy to effective cancer radiotherapy. KEYWORDS: Cancer radiotherapy, DNA damage and repair, Hierarchical nanostructure, Tumor hypoxia, Multimodal imaging

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Radiotherapy (RT) constitutes a primary modality in cancer treatment where it is used in more than 60% of all cancer patients. The operating principle of RT is to employ an external high-energy X-ray or γ-ray beam to induce DNA lesions, by causing direct cellular DNA damage or by indirect effects mediated by the reactive radicals formed via ionization reactions in cells. The most lethal DNA lesions include double-strand breaks (DSBs), single-strand breaks (SSBs), and nucleotide damages.1, 2 However, the clinical success of cancer RT has been limited by a combination of two factors, i.e., insufficient DNA damage and rapid DNA repair during and after treatment, respectively.3, 4 For the former, a major attribution is that, since only a fraction of the ionizing radiation can be absorbed by cancer tissues, the beam intensity must be curbed in order to minimize the inevitable collateral damage on healthy tissues. In addition, oxygen (O2) deficiency, known as hypoxia, is a regular cancer complication and has been linked to RT failure, presumably due to the inability of DNA breaks to form stable oxidative adducts with O2.1, 5-10 As for the second limiting factor, DNA damage inflicted by ionizing radiation subsequently triggers the DNA repair, which involves a series of interlocking mechanisms that intrinsically maintain genomic integrity. There are several biological pathways such as non-homologous end-joining (NHEJ)/homologous recombination (HR) and base excision repair (BER), which mend DSBs and SSBs, respectively.11 As a result of these repair mechanisms, the RT-induced DNA lesions can be removed before they become lethal to cancer cells.12 Therefore, modulating the cellular responses to ionizing radiation through the inhibition of DNA repair has also been a longstanding focus in translational RT 4


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research. All of these above mentioned processes are attractive targets for effective therapeutic strategies. To promote the RT treatment efficacy, a number of strategies have been proposed. Among them, many efforts have been devoted to developing nanostructures from elements with high atomic numbers (e.g., Au, Ta, rare earth elements, W, Pt and Bi), which can serve as radiosensitizers to concentrate local radiation dose, enhance the photoelectric and Compton effects, and thus amplify the DNA damage effect of RT.13-20 Regarding the issue of tumor hypoxia in RT, some other approaches have been reported to increase the tumoral O2 level, or tumor oxygenation, to enhance RT-induced DNA damage. The tumor oxygenation strategies include, but are not limited to, tumor vascular normalization,21 intentional O2 transport and delivery,22 and local O2 generation through catalase (Cat),23, 24 manganese dioxide (MnO2),25 and water splitting materials.26 On the other hand, targeting DNA repair using, such as DNA alkylators, platinum chemotherapies,27 inhibitors of apurinic/apyrimidinic (AP) endonuclease,28 and histone deacetylase inhibitors (HDACIs) to inhibit specific pathways that are preferentially required for repairing, can make cancer cells more vulnerable to RT and has proven efficacious to cause subsequent tumor cell death.29, 30 Despite the respective promising results of previous strategies, most of them focused only on one side of the issue factors (i.e., either by enhancing DNA damage or inhibiting DNA repair), whereas inadequate attention has been given to the combination of the two. Furthermore, to reduce the unintended injuries of nearby healthy tissues during 5


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RT, it is critical to probe accurate characteristics of the tumors using in vivo imaging techniques prior to the treatment, such as the size and shape, number, and location of the tumor within the organ relative to blood vessels.31-34 Additionally, imaging-guided RT with improved spatial accuracy also makes low dose RT treatment feasible, with an improved therapeutic ratio for tumor sites versus side effects on normal tissues.35 More importantly, the combination of multiple imaging modalities, if possible, can potentially yield complementary and more accurate physiological and spatial information and offers synergistic advantages over individual techniques alone.36-40 Having realized the key role of both DNA damage and repair as well as precision imaging in RT-mediated antitumor efficacy, we herein propose a systematic methodology that can simultaneously address all the above issues to achieve optimal RT therapeutic outcomes. In particular, we rationally designed a DNA-dual-targeting nanodroplet (NDr) platform, which can enhance the DNA lesion formation via ultra-small Au nanoparticles (~3.6 nm, AuNPs) and prevent the subsequent DNA repair by cancer oxygenation. Our current design includes the following components (Scheme 1): I) a slightly positively charged surface that can target tumor vasculature and provide high tumor accumulation; II) ultra-small AuNPs that can concentrate passing-by radiation and enter cell nuclei with close proximity to targeted DNA; III) a liquid perfluorooctyl bromide (PFOB) core acting as an O2 reservoir, which can rapidly release O2 upon ultrasound (US) treatment, generate more reactive oxygen species (ROS), and prevent DNA repair. Furthermore, the NDrs are active under optical (fluorescence), anatomical (US imaging), and functional (magnetic resonance 6


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images (MRI)) imaging modalities, allowing for accurate and real-time tracking of the NDrs and precision RT. Consequently, a significantly enhanced RT therapeutic outcome was achieved via this NDr-based system, presumably due to its ordered hierarchical structure and multiplexing capability.

Scheme 1. (A) Schematic diagrams illustrating the design and preparation of hierarchical multiplexing nanodroplets (NDrs). (B) Schematic diagrams illustrating 7


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the use of hierarchical multiplexing NDrs for multimodal imaging-guided cancer radiotherapy via enhanced DNA damage and concomitant DNA repair prevention. With the incorporation of cationic lipid BHEM-Chol, the NDrs can efficiently accumulate at the tumor site through the enhanced vasculature targeting. The ultrasound (US) treatment triggers the burst release of O2 and ultra-small AuNPs from the NDrs. The cell nucleus accumulated AuNPs concentrate high energy X-ray and enhance DNA damage induced by RT. The O2 relieves hypoxia of the tumors and reacts with DNA radical (DNA•) and further fixes DNA damage to form more stable DNA peroxides (DNA-OO•), which further prevent DNA repair of cells and thereby improves RT therapeutic outcomes.

RESULTS AND DISCUSSION Preparation and Characterization of Hierarchical Multiplexing NDrs. NDrs containing AuNPs (NDr(Au)) composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol, aminoethyl)

N,N-bis(2-hydroxyethyl)-N-methyl-N-(2-cholesteryloxycarbonyl ammonium

bromide

(BHEM-Chol),

2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (DSPE-PEG2000), and AuNPs at a 6: 4: 0.5: 0.5: 0.5 molar ratio was prepared via a thin-film hydration method (Scheme 1A). The molar ratio of AuNPs was calculated based on the molar concentration of Au particles, instead of Au atoms. Cholesterol helps maintain the integrity of the lipid layer and fluidity as well,41 while DSPE-mPEG2000 ensures long blood circulation following administration.42 The cationic cholesteryl lipid BHEM-Chol was used to modulate the surface properties of 8


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NDrs, based on the discovery from us and others that slightly positively charged nanomedicines showed higher binding and internalization by angiogenic endothelial cells of tumors, leading to preferential tumoral accumulation.43, 44 For the next step, PFOB was added gradually to NDr(Au) under high-speed homogenization at 20,000 rpm, generating NDr(Au+PFOB). The NDr(Au+PFOB) was then oxygenated by directly bubbling O2 (5.0 L/min) into its aqueous suspension for 10 min, which produced the multiplexing NDr(Au+PFOB+O2) (Scheme 1A). Cryogenic transmission electron microscopy (cyro-TEM) observation of NDr(PFOB) confirmed that the mixed lipids could stabilize PFOB, which entered inside the lighter lipid shell and appeared darker because of its high electronic density (Figure S1). The successful encapsulation of AuNPs (~3.6 nm in size as measured by TEM, Figure S2) inside the NDr(Au) was confirmed by TEM (Figure 1A) and by the presence of the characteristic peaks of the Au element in energy dispersive X-ray spectroscopy (EDX, Figure 1B). The TEM images of NDr(Au+PFOB) revealed its hierarchical structure, in which AuNPs were dispersed predominantly at the rim of the PFOB core probably due to the chemical incompatibility between the perfluoroalkyl of PFOB and long alkyl ligands on the AuNPs (Figure 1A). The number of AuNPs per NDr(Au+PFOB) was calculated to be 58 ± 15, as measured from TEM images. All the NDrs had hydrodynamic diameters of ~100-130 nm, with a polydispersity index of ~0.15 (Figure 1C), indicating the suitability of the as-prepared NDrs for systemic drug delivery via enhanced permeation and retention (EPR) effect.45-47 Zeta potential measurements indicated the successful incorporation of BHEM-Chol, as the 9


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values changed from ~-18.7 mV (without BHEM-Chol) to ~+8.5 mV (with BHEM-Chol, Figure 1D). The loading of PFOB/O2 did not affect the surface zeta potentials of NDrs. Colloidal stability of nanoparticles is a crucial requirement for their systemic intravenous administration. We have observed no significant size changes of NDrs during the incubation with 10% fetal bovine serum for up to 120 h in buffer solutions (Figure 1E). Moreover, NDrs maintained their size and morphology upon radiation (5 Gy, Figure S3). In addition, all the NDrs without AuNPs showed good biocompatibility to EMT-6 mouse mammary cancer cells (data not shown). While we observed a slight decrease of cell viabilities for NDr(Au) at high AuNPs concentrations (100 µg/mL) (Figure S4), which might be associated with oxidative stress.48, 49

Figure 1. Physicochemical characterization of the hierarchical multiplexing nanodroplets (NDrs). (A) Typical TEM images (scale bar = 100 nm) and frequency distributions of particle size of NDr(Au), NDr(Au+PFOB) and NDr(Au+PFOB+O2) 10


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treated with ultrasound (US, 130 W, 20 kHz) for 5 min. (B) EDX spectra of NDrs. (C) Hydrodynamic sizes (diameter) of NDrs measured by dynamic light scattering. (D) Zeta potential of NDrs and NDrs without BHEM-Chol incorporation. (E) Size changes of NDrs incubated with 10% fetal bovine serum at 37 °C. NDr(Au) Enhanced RT-Induced DNA Damage and Sensitized Cancer RT. We used AuNPs since they have been reported to show significant dose-dependent radiosensitization effects, attributing to their capability of concentrating local radiation dose and increasing the production of photoelectrons, auger electrons and low energy secondary electrons.15, 50 In addition, the ultra-small size of AuNPs (~3.6 nm) was to avoid affecting the integrity of the lipid layer,51 and to take advantage of better nucleus translocation capability of smaller nanostructures (the diameter of the nuclear pore complex is 20-50 nm depending on the cell types).52 Moreover, nanostructures with smaller sizes can achieve uniform intratumoral delivery owing to their stronger diffusion ability in interstitial spaces in tumors.53 We first investigated the sensitization effect of these AuNPs loaded in NDr(Au) in vitro. NDr(Au) efficiently entered EMT-6 cells, presumably owing to the slightly positive nature of surface with BHEM-Chol incorporation (Figure S5). EMT-6 cells were then incubated with NDr(Au) at varying concentrations of AuNPs (0, 25, 50, and 100 µg/mL) in a modular incubator chamber flushed with humidified 1% O2, 5% CO2, and 94% N2, which mimicked the tumoral hypoxic condition. The cells were then subjected to a single-fraction X-ray irradiation (5 Gy) and immunostained with the antibody of γ-H2AX, a rapid and sensitive biomarker for the detection of 11


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radiation-induced DSBs, at different time points (0, 1, 4, 12, and 24 h) after treatment (Figure 2A). The extent of DNA lesions was quantified in terms of the accumulation of γ-H2AX foci per cell following confocal laser scanning microscopy (CLSM) observation (Figure 2B). The irradiated cells exhibited augmented DNA lesion formation after incubation with NDr(Au) at an AuNPs-dose-dependent manner (Figure 2C), confirming the radiosensitization effects of AuNPs encapsulated in NDr(Au). It is worth mentioning that the DNA damage was rapidly repaired 4 h after irradiation, as evidenced by the fast drop in the number of γ-H2AX foci. For cells without NDrs pre-incubation, γ-H2AX foci per cell decreased by 44.7% and 76.0% within 4 h and 24 h, respectively. Even for cells pre-incubated with NDr(Au) of 100

µg/mL AuNPs, an average of 62.5% of DNA damage was repaired after 24 h. This, again, confirms that the prevention of DNA damage repair is critical for effective RT. Colony formation evaluation on the self-renewal efficiency of EMT-6 cells revealed a similar dose-dependent radiosensitization effects of NDr(Au) (Figure 2D and Figure S6). The sensitization enhancement ratios (SER) calculated by the multitarget single-hit model,54 were 1.10, 1.23, and 1.36, corresponding to the treatments of NDr(Au) at the AuNPs concentration of 25, 50, and 100 µg/mL, respectively (Figure 2E). Encouraged by the capability of NDr(Au) to enhance DNA damage and RT efficiency in vitro, we then further investigated their radiosensitization effects in vivo. Syngeneic mouse mammary EMT-6 tumors were grown subcutaneously into the right flank of Balb/c mice. The mice were i.v. administrated with NDr(Au) (AuNPs doses at 12


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0, 5, or 10 mg/kg body weight) and treated with a single-fraction X-ray irradiation (5 Gy or 8 Gy) 12 h post administration (Figure 2F). The tumor growth curves were monitored post treatment (Figure 2G). On day 18 post treatment, the tumors grew to an average of more than 10 times their initial sizes without any treatment (Group I). NDr(Au+PFOB) (Group II) alone did not retard tumor growth and had no therapeutic effect, indicating their biocompatibility. The condition NDr(Au) (10 mg/kg)+RT 5 Gy (Group V) effectively inhibited tumor growth by 65.4% on day 18 post treatment, while NDr(Au) (5 mg/kg)+RT 5 Gy (Group IV) showed moderate inhibition effect (49.1% inhibition) compared with RT alone (Group III, 40.4% growth inhibition), illustrating the efficient dose-dependent radiosensitization of NDr(Au). The presence of PFOB in NDr(Au+PFOB) (Group VI, 71.9%) did not affect radiosensitization compared to NDr(Au) (Group V, 65.4%) at the same dose of 5 Gy. Moreover, different ionization radiation dosages (5 Gy for Group VI and 8 for Gy Group VII) exhibited the dose-dependent effects. Tumor weight measurement and direct observation of tumors morphologies further confirmed the antitumor efficacy of NDr(Au) and NDr(Au+PFOB)-mediated RT enhancement (Figure 2H and 2I). Notably, no significant body weight fluctuation was observed in any of the groups (Figure 2J).

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Figure 2. NDr(Au) enhanced RT-induced DNA damage (A-C) and sensitized cancer RT in vitro (D-E) and in vivo (F-J). (A) Treatment schedule for RT treatment of EMT-6 cells pre-incubated with NDr(Au) or PBS. Cells were incubated with NDr(Au) at varying AuNPs doses under a hypoxic condition for 4 h, and then subjected to X-ray irradiation (5 Gy). The cells were stained with DAPI (cell nuclei) and immunostained for γ-H2AX at different time points post irradiation. (B) Time dependent change of γ-H2AX foci (red) in cell nuclei (blue) of EMT-6 cells preincubated with NDr(Au) (AuNPs does of 50 µg/mL) and then exposed to 5 Gy irradiation. (C) Quantitative analysis of γ-H2AX foci density (in terms of number of γ-H2AX foci per cell, n = 100 cells) for EMT-6 cells pre-incubated with varying doses 14


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of NDr(Au) and then exposed to 5 Gy irradiation, mean ± SEM. (D) Survival fraction (SF) of NDr(Au)-pretreated EMT-6 cells received RT treatment. Data was calculated from colony formation assay and fitted to the multi-target single-hit model: SF =1-(1-eD/D0)N (SF, cell survival fraction; D, radiation dose; e, the bottom of the natural logarithm; D0, the mean death dose; N, extrapolate number), mean ± SEM. (E) The sensitization enhancement ratio (SER) of each treatment group as calculated by the multitarget single-hit model. Three independent experiments were carried out for panels D. (F) Treatment schedule for NDr(Au) sensitized cancer RT of Balb/c mice bearing EMT-6 tumors. The mice were i.v. administrated with NDr(Au) (5 or 10 mg/kg) or PBS and the tumors were exposed to 5 or 8 Gy irradiation 12 h post injection (n = 5). (G) Tumor volume changes for various treatment groups post-irradiation as indicated. (H) Images of excised tumors after sacrificing the mice on day 18 post-irradiation. (I) Tumor weights measured after sacrificing the mice. (J) Body weight monitoring of the mice during treatment. All the data are shown as mean ± SEM (*P

Hierarchical Multiplexing Nanodroplets for Imaging-Guided Cancer

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