Cancer Chemoradiotherapy Duo: Nano-Enabled Targeting of DNA

Subscriber access provided by University of Sunderland

Biological and Medical Applications of Materials and Interfaces

Cancer Chemo-Radiotherapy Duo: Nano-Enabled Targeting of DNA Lesion Formation and DNA Damage Response Wei Jiang, Quan Li, Zhengchun Zhu, Qin Wang, Jiaxiang Dou, Yingming Zhao, Weifu Lv, Fei Zhong, Yandan Yao, Guoqing Zhang, Hang Liu, Yucai Wang, and Jun Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10901 • Publication Date (Web): 26 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cancer Chemo-Radiotherapy Duo: Nano-Enabled Targeting of DNA Lesion Formation and DNA Damage Response Wei Jiang,†,⊥ Quan Li,‡,⊥, Zhengchun Zhu,ǁ,⊥ Qin Wang,† Jiaxiang Dou,† Yingming Zhao,# Weifu Lv,# Fei Zhong,ǁ Yandan Yao,‡ Guoqing Zhang,† Hang Liu,*,† Yucai Wang,*,† and Jun Wang╧ †

Division of Molecular Medicine, Hefei National Laboratory for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences, University of Science and Technology of China ‡ Breast Tumor Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China ǁ Department of Radiotherapy, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China # Department of Oncology, Anhui Provincial Hospital, the First Affiliated Hospital of University of Science and Technology of China, Hefei 230001, China ╧ 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

1


Page 2 of 34

Page 3 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABTRACT: Both production of DNA damage and subsequent prevention of its repair are crucial in concluding the therapeutic outcome of radiotherapy. However, nearly all current strategies for improving radiotherapy focus only on one of the two aspects and overlook the necessity of their combinations. In this work, we introduce a concept of DNA-dual-targeting nanomedicine to simultaneously enhance DNA lesion formation and prevent the succeeding repair. Briefly, the cisplatin prodrug loaded in nanomedicine can form platinated DNA in cell nuclei, making DNA more vulnerable to

the

ionizing

radiation

generated

by

radiotherapy.

Concomitantly,

the

spatial-temporally co-delivered vorinostat, a histone deacetylase inhibitor, prolongs the build-up of double-strand breaks and causes cell apoptosis en masse, probably due to the suppressed expression of DNA repair proteins. Furthermore, this nanoplatform is suitable for fluorescence and magnetic resonance imaging techniques, enabling accurate trafficking of the nanomedicine as well as reliable real-time imaging-guided precision radiotherapy. Finally, results from in vitro and in vivo jointly reveal that this dual-action system attains a remarkably enhanced radiotherapeutic outcome. In conclusion, our imaging-guided DNA-dual-targeting design represents a novel strategy for efficient cancer precision radiotherapy. KEYWORDS: Chemo-radiotherapy, DNA damage, DNA damage response, DNA-dual-targeting, Polymeric nanomedicines

2



INTRODUCTION Radiotherapy (RT) is used as a commonly and effective clinical treatment to prolong patient survival. RT exerts its clinical effects through the induction of DNA damage in cancer cells, which leads to the generation of DNA lesions and ultimately cell deaths.1,2 Various forms of DNA lesions are generated during RT, and among them double-strand breaks (DSBs) are the most deleterious type. However, inadequate DNA damage and rapid DNA damage response during and post RT, respectively, have limited the success rate and therapeutic outcome.3 For the factor of inadequate DNA damage, the major cause is the intrinsic or microenvironment-induced radio-resistance of tumor cells, which makes them less sensitive to radiation. In these cases, the maximal RT dose received by tumors is to be determined by normal tissue tolerance since elevated radiation dose is often unfeasible as increased adverse effects in healthy tissues emerge.4-7 Therefore, a number of strategies, such as using high atomic number elements (Z) radio-sensitizer to concentrate local radiation dose,8-14 relieving hypoxic tumoral microenvironment to improve the production of lethal radicals,15,16 and perturbing cell cycles to arrest cells in the more sensitive G2/M phase,17,18 have been reported to augment the DNA damage effect and thus to bolster the treatment potency of RT. Out of these strategies, combining RT with chemotherapeutics (such as DNA alkylator, antimetabolites, topoisomerase inhibitors, replication inhibitors),19-22 known as chemo-radiotherapy, can enhance the formation of DNA lesions through a variety of mechanisms and make cancer cells more vulnerable to RT.23,24 As such, a number of chemo-radiotherapy sensitizers have been shown to have favorable clinical 3


Page 4 of 34

Page 5 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


results in combination with RT.20 With regard to the second limitation of rapid and unwanted repair of DNA lesions, the DNA damage imposed by ionizing radiation can activate the subsequent DNA damage response (DDR) and initiate DNA repair.3 DDR involves a network of evolutionarily conserved signaling cascades, which can sense, respond, and repair damaged DNA with the purpose of maintaining genomic integrity.25,26 As major DNA DDR cellular pathways, base excision repair (BER) are major pathways repair single strand DNA breaks (SSB), while non-homologous end-joining (NHEJ) and homologous recombination (HR) are major pathways to DSB repair, which enable tumor cells to survive from RT.27 Knowledge of the significance of DDR has resulted in the development of methods to inhibit or attenuate DNA damage repair in order to augment RT-induced inhibitory effect on tumors. RNA interference or small molecule chemo-inhibitors targeting DNA-dependent protein kinase, ataxia telangiectasia and Rad3-related protein (ATR) have been demonstrated promising in inhibiting specific DDR pathways preferentially required for DNA repair.23,28 As stated above, both production (i.e., the process related to DNA lesion formation) and maintenance (i.e., the process related to DDR) of DNA damage is instrumental in determining the therapeutic outcome of RT. However, most of the research on improving the outcomes was concentrated only on one aspect of the issue and ignoring the necessity of their combinations.26 DNA-dual-targeting strategies that target both DNA damage and DNA repair have been demonstrated to improve the therapeutic efficacy in chemotherapy.29-31 Herein, 4



Page 6 of 34

we propose to use a DNA-dual-targeting nanomedicine (NM) to improve RT therapeutic outcomes, in light of the importance of DNA damage and repair in RT-mediated antitumor treatment. Specifically, the encapsulated cisplatin prodrug (PtIV) in the NMs can bind with cellular DNA to form platinum-DNA adducts, concentrate the radiation in the vicinity of targeted DNA, and make cancer cells DNA more vulnerable to the ionizing radiation (as compared to unmodified DNA), which thereby induces more DNA damage (Scheme 1).32,33 Concomitantly, the release of vorinostat, a histone deacetylases inhibitor (HDACi), from the NMs can generate reactive oxygen species (ROS), which subsequently fixes intermediates of DNA lesions and accumulate the lesions. Additionally, the vorinostat has been reported to suppress expression of DNA damage repair-related proteins including Ku70/Ku80, DNA-PK, and RAD50, thereby prevent rapid DNA repair and cause cell death en masse.34 Furthermore, the nano-mediated strategy allows the spatial and temporal co-delivery of the PtIV prodrug and vorinostat to the same cancer cell, thus amplifying radio-sensitizing synergism.35,36 To further improve the therapeutic efficacy, the NMs were

surface-functionalized

with

a

cell-penetrating

peptide

derived

from

transactivator of transcription (TAT) protein of HIV-1, which can be activated in the more acidic tumoral microenvironment and thus increase tumoral accumulation and cellular uptake of NMs.37-40 It was consequently shown that, a remarkably enhanced therapeutic outcome could be achieved via this dual-action system, presumably attributing to its well-designed DNA-dual-targeting capability.

5


Page 7 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. Schematic of a tumoral acidic microenvironment-activatable nanomedicine (DANP/Pt+Vor) for targeted cancer chemo-radiotherapy via enhanced DNA lesions formation and suppressed DNA damage response. DANP/Pt+Vor can increase Pt-DNA adducts formation in nuclei and thus amplify radiotherapy-induced DNA damage by the high-Z atom effect. The DNA damage further fixes upon the release of vorinostat from DANP/Pt+Vor, which prevents rapid DNA repair and thereby causes more cell apoptosis. The NMs are active under dual-mode detection (near infrared imaging and magnetic resonance imaging), showing high sensitivity and resolution of the DANP/Pt+Vor in vivo to enhance the radiotherapy.

MATERIALS AND METHODS Preparation of NMs Encapsulating PtIV Prodrug and/or Vorinostat. The cis, cis, trans-[Pt(NH3)2Cl2(OOC(CH2)8CH3)2] (PtIV) and vorinostat co-loaded DANP/Pt+vor was prepared by dialysis method. Briefly, PtIV prodrug (2.0 mg), vorinostat (0.2 mg), and 2, 3-dimethylmaleic anhydride (DA) modified TAT peptide conjugated poly(ethylene glycol)-block-poly(lactic-co-glycolic acid) (DATAT-PEG-b-PLGA, 7.0 mg) were dissolved in 1.0 mL of DMSO and dialyzed against H2O. Free PtIV prodrug and vorinostat were removed by centrifugation. The platinum and vorinostat content in the NMs was detected via inductively coupled plasma mass spectrometry (ICP-MS) and 6



Page 8 of 34

UV-Vis spectrometry, respectively. The preparation of NP/Pt+Vor was similar except for using monomethoxy poly (ethylene glycol)-block-poly(lactic-co-glycolic acid) (mPEG-b-PLGA) instead of DATAT-PEG-b-PLGA. For magnetic resonance imaging (MRI), poly(lactic-co-glycolic acid) (PLGA) terminated with diethylene triamine pentaacetic dianhydride (DTPA) was loaded into NP/Pt+Vor and

DA

NP/Pt+Vor during preparation. The NMs were further chelated with

gadolinium (III) (Gd3+) ions by incubating with a NaOH solution (pH 8.0) containing 0.2 M Gd3+ and 0.6 M citrate for 48 h. Thereafter, free Gd3+ ions were removed by dialysis.

For

fluorescence

1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine NP/Pt+Vor and

DA

imaging, iodide

(DiR)-loaded

NP/Pt+Vor were prepared by incorporating the fluorescent dye DiR at

0.30% weight ratio during preparation. γ-H2AX Immunofluorescent Staining for DNA Damage Assay. EMT-6 cells were seeded on coverslips in a 24-well plate (5 × 104 per well) and incubated overnight. The cells were then incubated with different formulations at 37 °C at pH 6.8 for 4 h. After staining with anti-γ-H2AX antibody following a standard procedure, the γ-H2AX foci density in cell nuclei was imaged by CLSM and counted using the ImageJ software.41 Cell Cycle Analysis. EMT-6 cells were seeded in a 6-well plate (1 × 105 cells per well) and incubated overnight. The cells were treated with PBS, vorinostat, NP/Vor and DA

NP/Vor (at a vorinostat dose of 1 µM) for 4 h, respectively. The cell cycle profiles 7


Page 9 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


were detected by staining the DNA using propidium iodide and analyzed by the Modfit LT 3.1 software, according to manufacturer's instructions. Fluorescence Imaging. DiR-labeled DANP/Pt+Vor and NP/Pt+Vor were i.v. injected into EMT-6 tumor-bearing mice at a DiR dose of 0.5 mg/kg. Whole body fluorescence images were recorded using a Xenogen IVIS Lumina system (Caliper Life Sciences, Alameda, CA) at different time points. The tumors and major organs were collected and further imaged at 72 h post-injection. MRI In Vitro and In Vivo. For T1 MRI in vitro: T1 relaxation time testing was on a Siemens 3.0 T MR scanner. For T1-weighted imaging, turbo spin echo sequence was employed to collect T1 weighted images with field of view (FOV) = 144 × 220 mm2, repetition time (TR) = 500 ms, and echo time (TE) = 8.5 ms. Aqueous suspensions of DA

NP/Pt+Vor or NP/Pt+Vor chelated with different amounts of Gd3+ in plastic tubes were

placed in a head coil. For the measurement of r1, inversion recovery sequence was used, and in the obtained T1 map the relaxation time of each sample was measured. In vivo mouse MRI was performed using a customized specialized coil (Suzhou Medcoil Healthcare, China) with FOV = 320 × 320 mm2, TR = 500 ms, and TE = 8.5 ms. Anesthetized EMT-6 tumor bearing mice were fixed onto a customized holder by a medical elastic bandage. NP/Pt+Vor or

DA

NP/Pt+Vor were i.v. injected into mice at a

Gd3+ dose of 10 mg/kg. Whole body MRI was performed at the different time points, and the data were processed using the Osirix MD software. Tumor Suppression Study. The tumor-bearing mice (tumor size ~85 mm3) were 8



randomly divided into different treatment groups (n = 5 per group), and i.v. injected with different formulations with equivalent doses of platinum and vorinostat were 1.0 mg and 0.5 mg/kg, respectively. The mice in groups were received X-rays irradiation (5 Gy) or not after 24 h post injection. The tumor volume was measured using a vernier caliper and calculated with the following formulas: tumor volume = 0.5 ×l × w2, where l is the length of tumor and w is the width of tumor. At the end of the therapy, the mice were sacriﬁced and the tumor tissues were excised.

9


Page 10 of 34

Page 11 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RESULTS AND DISCUSSION

Figure 1. Characterization of DANP/Pt+Vor and its non-responsive counterpart NP/Pt+Vor. (A-C) (A) Zeta potential changes, (B) hydrodynamic diameter, and (C) Cryo-TEM images of DANP/Pt+Vor and NP/Pt+Vor in phosphate buffers at different pHs (pH 7.4 and 6.8). (D) Size changes of DANP/Pt+Vor and NP/Pt+Vor in PBS containing 10% fetal bovine serum at 37 °C at different pHs. (E, F) Release profiles of (E) PtIV prodrug and (F) vorinostat from DANP/Pt+Vor and NP/Pt+Vor at different pHs, respectively. Mean ± SD (n = 3). Preparation of tumoral acidic microenvironment-activatable NMs for co-delivery

of

cisplatin

prodrug

and

vorinostat.

The

tumoral

acidic

microenvironment activatable NMs were made up of i) a hydrophobic PLGA core that allows for the co-encapsulation of both PtIV prodrug and vorinostat; ii) a hydrophilic poly(ethylene glycol) corona that protects the NMs from clearance by the reticuloendothelial system (RES); and iii) a protected TAT peptide that can be exposed at the tumoral acidic microenvironment to enhance tumoral retention and cellular internalization of NMs. Specifically, the NMs were consisted primarily of the FDA approved block copolymer of poly (ethylene glycol) (PEG, Mw,PEG = 5,000) and PLGA (Mw,PLGA = 11,000, LA/GA = 75/25 (mol/mol)). NMs consisting of this 10



Page 12 of 34

polymer exhibited excellent blood circulation and tumoral accumulation based on our prior

in

vivo

combinatorial

screening

studies.42

The

TAT

peptide

“YGRKKRRQRRRC” was conjugated to the terminal of PEG via the reaction of maleimide with thiol groups of TAT peptide to obtain TAT-PEG-b-PLGA (Scheme S1, Figure S1 and S2). The TAT peptide was further reacted with DA to produce DA

TAT-PEG-b-PLGA (Scheme S1 and Figure S3). PtIV prodrug and vorinostat were

subsequently encapsulated into the two NMs composed of

DA

TAT-PEG-b-PLGA

(DANP/Pt+Vor) and the non-responsive mPEG-b-PLGA (NP/Pt+Vor), with drug loading content of ca. 3.0 ± 0.3% and 3.2 ± 0.5%, respectively. It suggested that surface modification of DATAT did not affect the encapsulation of PtIV prodrug and vorinostat. NMs with only PtIV or vorinostat encapsulation were also prepared and denoted as DA

NP/Pt (or NP/Pt for the non-responsive one) and NP/Vor (or NP/Vor for the

non-responsive one), respectively. The amide bond between the amine of lysine residue of the TAT peptide and DA is relatively stable under neutral physiological conditions (pH 7.4) and can be fast degraded, followed by the original form of TAT peptide at the tumor acidity (pH< 6.8). 43,44

The zeta potential of

DA

NP/Pt+Vor incubated at pH 6.8 led to a significant change

from −18.1 to 1.6 mV, which suggested the exposure of the positively charged TAT peptide upon the cleavage of amide bonds (Figure 1A). However, the zeta potential of the

DA

NP/Pt+Vor only exhibited a slightly increase to −11.3 mV after incubation at pH

7.4, confirming its stability under neutral physiological (pH 7.4) conditions. Negligible changes of zeta potential were observed for NP/Pt+Vor at different pHs. The 11


Page 13 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DA

NP/Pt+Vor and NP/Pt+Vor exhibited uniform spherical morphology with average sizes

of ~85 nm from cryogenic transmission electron microscopy observation (Figure 1B), consistent with the results of dynamic light scattering (DLS) (Figure 1C). Neither DA

NP/Pt+Vor nor NP/Pt+Vor aggregated at pH 6.8 and 7.4 for up to 120 h, indicating the

cleavage of acid-labile DA had a negligible effect on the stability of NMs (Figure 1D). Similar patterns of slow and diffusion-controlled release of PtIV prodrug from the two NMs were observed regardless of pHs. Approximately 55% of drug was released by 96 h at 37 ℃ (Figure 1E). Consistently, the release of the vorinostat (~58% of the drug being released by 96 h) was similar to PtIV prodrug (Figure 1F) and the UV-Vis spectrometry data confirmed the release of intact vorinostat (Figure S4). These results demonstrated the DA protection on the TAT peptide underwent rapid degradation and charge reversal under mild acidic conditions, without affecting other physicochemical properties or the stability of the NMs.

12



Figure 2. DANP/Pt enhanced the formation of platinated-DNA and thereby RT-induced DNA lesions. (A) Representative CLSM images of EMT-6 cells treated with rhodamine B-labeled DANP/Pt and NP/Pt at two pHs for 4 h. The cytoskeleton and cell nuclei and were stained with Alexa Fluor 488 phalloidin (green) and DAPI (blue), respectively (scale bar = 50 µm). (B) ICP-MS determination of the intracellular platinum of EMT-6 cells incubated with PtIV, NP/Pt, and DANP/Pt for 4 h at an equivalent platinum amount of 10 µM. (C) Quantitative analysis of platinated DNA of EMT-6 cells after treatment with PtIV, NP/Pt, and DANP/Pt at a platinum dose of 10 µM for different time points. (D) Treatment schedule for EMT-6 cells treated with different NMs immunostained for γ-H2AX after RT (5 Gy) treatment. (E) Images of immunofluorescent staining for γ-H2AX foci (red) in cell nuclei (blue) of EMT-6 cells pre-incubated with NMs (equivalent platinum amount of 10 µM) and followed with RT treatment or not. (F) Quantitative analysis of γ-H2AX foci density (γ-H2AX foci per cell) for n = 50 cells in each treatment group. (G) Apoptosis induced by PBS, RT, NP/Pt+RT, and DANP/Pt+RT in EMT-6 cells. (H) Percentage of viable, apoptotic, and necrotic cell assessed by Annexin V/FITC-PI staining at pH 6.8. Mean ± SD. *p

Cancer Chemoradiotherapy Duo: Nano-Enabled Targeting of DNA

Recommend Documents