Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES
Article
Inhibiting Metastasis and Preventing Tumor Relapse by Triggering Host Immunity with Tumor-Targeted Photodynamic Therapy Using Photosensitizer-Loaded Functional Nanographenes Xinhe Yu, Duo Gao, Liquan Gao, Jianhao Lai, Chenran Zhang, Yang Zhao, Lijun Zhong, Bing Jia, Fan Wang, Xiaoyuan Chen, and Zhaofei Liu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b04736 • Publication Date (Web): 13 Sep 2017 Downloaded from http://pubs.acs.org on September 14, 2017
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 free 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 accessible to all readers and 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.
ACS Nano 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 45
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 Nano
Inhibiting Metastasis and Preventing Tumor Relapse by Triggering Host Immunity with Tumor-Targeted Photodynamic Therapy Using Photosensitizer-Loaded Functional Nanographenes Xinhe Yu#1, Duo Gao#1, Liquan Gao1, Jianhao Lai1, Chenran Zhang1, Yang Zhao1, Lijun Zhong2, Bing Jia1,2, Fan Wang1,3, Xiaoyuan Chen4, Zhaofei Liu*1 1
Medical Isotopes Research Center and Department of Radiation Medicine, School of
Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China 2
Medical and Healthy Analytical Center, Peking University, Beijing 100191, China
3
Key Laboratory of Protein and Peptide Pharmaceuticals, CAS Center for Excellence in
Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 4
Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical
Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, USA.
#
These authors contributed equally to this work.
*Corresponding Author: Prof. Zhaofei Liu, Medical Isotopes Research Center and Department of Radiation Medicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China. E-mail:
[email protected] 1 ACS Paragon Plus Environment
ACS Nano
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
Page 2 of 45
ABSTRACT Effective cancer therapy depends not only on destroying the primary tumor, but also on conditioning the host immune system to recognize and eliminate residual tumor cells and prevent metastasis. In this study, a tumor integrin αvβ6-targeting peptide (the HK peptide)-functionalized graphene oxide (GO) was coated with a photosensitizer (HPPH). The resulting GO conjugate, GO(HPPH)-PEG-HK, was investigated whether it could destroy primary tumors and boost host antitumor immunity. We found that GO(HPPH)PEG-HK exhibited significantly higher tumor uptake than GO(HPPH)-PEG and HPPH. PDT using GO(HPPH)-PEG suppressed tumor growth in both subcutaneous and lung metastatic mouse models. Necrotic tumor cells caused by GO(HPPH)-PEG-HK PDT activated dendritic cells, and significantly prevented tumor growth and lung metastasis by increasing the infiltration of cytotoxic CD8+ T lymphocytes within tumors as evidenced by in vivo optical and single-photon emission computed tomography (SPECT)/CT imaging. These results demonstrate that tumor-targeted PDT using GO(HPPH)-PEG-HK could effectively ablate primary tumors and destroy residual tumor cells, thereby preventing distant metastasis by activating host antitumor immunity and suppressing tumor relapse by stimulation of immunological memory.
KEYWORDS: Graphene oxide; Photodynamic therapy; Immunotherapy; Immunological memory; Tumor vaccine
2 ACS Paragon Plus Environment
Page 3 of 45
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 Nano
Primary tumors and tumor metastasis usually occur concurrently. Although the primary tumor may be removed successfully by surgery, it is difficult to completely eliminate the metastatic lesions. Metastasis and associated complications account for over 90% of mortalities from several types of cancers.1,2 Therefore, effective cancer treatment relies not only on destroying the primary tumor, but more importantly on attacking and preventing tumor metastasis. Theoretically, immunotherapy, in which the inherent host immune system is trained or stimulated to recognize and attack tumor cells, is an ideal cancer therapeutic strategy because it prompts host immunological surveillance and defense to generate an antitumor immune response capable of eliminating any remaining lesions (either the local tumor or metastatic lesions) and preventing their recurrence.3,4 Cancer immunotherapy includes immunomodulatory antibody-based treatments, adoptive cellular therapy, and cancer vaccines.5,6 Although significant advances have been achieved in cancer immunotherapy, it still suffers from limitations, such as benefits restricted to certain subsets, dose-limiting systemic autoimmune side effects, and circumscribed antitumor efficacy.7,8 To achieve improved antitumor efficacy for both well-established tumors and tumor micrometastasis, combinatorial approaches have been extensively investigated.9 The efficacy of combination strategies depends on appropriate timing, dosage regimens and administration schedules of the therapeutic agents. However, a corresponding increase in serious adverse effects may occur for combinatorial therapies in order to reach a satisfactory antitumor effect.10 Therefore, a method that offers fewer side effects but can destroy the primary tumor more selectively, eliminate the metastatic lesions, and prevent tumor recurrence by building long-lived antitumor immunological memory, is highly desirable.
3 ACS Paragon Plus Environment
ACS Nano
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
Page 4 of 45
Photodynamic therapy (PDT) is a traditional cancer treatment strategy that combines a photosensitizer, light irradiation, and oxygen to destroy cancer cells via production of reactive oxygen species (ROS), and constitutes a promising strategy for local treatment of a variety of cancers.11 In addition to local tumor ablation by generating powerful ROS such as singlet oxygen, the role of PDT in antitumor immunity is gaining increasing attention.12-15 PDT can stimulate antitumor immunity by inducing the rapid release of tumor cell debris, which may increase immunogenicity and improve tumor antigen presentation by antigen-presenting cells. This, in turn, leads to the activation of cytotoxic T lymphocytes and their infiltration into the tumor, and could result in the destruction of residual tumor cells thereby reducing the risk of distant metastasis.16-18 Most photosensitizers for PDT have low solubility in water and suboptimal selectivity in vivo.19 To overcome these limitations, photosensitizers have been conjugated with nanocarriers such as graphene oxide (GO),20 which has a large surface area and can be used as a biosensor as well as for drug and gene delivery.21-23 Various photosensitizers have been loaded onto the surface of GO via π-π stacking and hydrophobic interactions for tumor imaging and PDT in animal models.19,24,25 However, most photosensitizer-GO nanoconjugates that have been described to date function in vivo by passive targeting to tumors via an enhanced permeability and retention (EPR) effect. Conjugating GO with tumor-selective molecules such as peptides, ligands, and antibodies could potentially improve in vivo targeting and PDT efficacy.26,27 In this study, we developed polyethylene glycol (PEG)ylated GO conjugated with an HK peptide28 that binds specifically to integrin αvβ6,12,29 a receptor that is highly expressed in a variety of tumor types.30 The functionalized GO was then coated with a
4 ACS Paragon Plus Environment
Page 5 of 45
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 Nano
photosensitizer, Photochlor (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-alpha, HPPH). We investigated whether PDT with the GO conjugate could trigger host antitumor immunity, thereby inhibiting tumor growth and metastasis, as well as building long-term host immunological memory to prevent tumor recurrence in a highly aggressive 4T1 murine breast cancer model.
RESULTS Synthesis and Characterization of GO(HPPH)-PEG-HK GO was synthesized from graphite and its biocompatibility was improved by conjugation with NH2-PEG3500-maleimide (NH2-PEG-Mal) via amide formation. The integrin αvβ6-specific HK peptide was then functionalized to GO-PEG-Mal via a maleimide-thiol coupling reaction before HPPH was loaded onto the surface of GO-PEGHK via hydrophobic interactions and π-π stacking (Figure 1a). Atomic force microscopy (AFM) imaging revealed a size range of 10–100 nm for the final nanographene product, GO(HPPH)-PEG-HK (Supplementary Figure S1a). An ultraviolet-visible light absorption peak at 665 nm (the characteristic peak for HPPH) was observed for GO(HPPH)-PEG-HK (Supplementary Figure S1b), indicating the successful loading of HPPH. HPPH fluorescence was partially diminished after loading onto GO-PEG-HK (Supplementary Figure S1c), consistent with a previous report.25 Fourier transform infrared spectroscopy (FT-IR) results confirmed the modification processes and successful conjugation of HK to GO-PEG, and successful coating of HPPH onto GOPEG-HK (Supplementary Figure S1d). GO(HPPH)-PEG-HK was highly stable in water, phosphate-buffered saline (PBS), fetal bovine serum (FBS), and cell culture medium
5 ACS Paragon Plus Environment
ACS Nano
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
Page 6 of 45
(Supplementary Figure S1e). Moreover, HPPH was firmly absorbed on the GO-PEG surface, and no significant release (< 10%) of HPPH from GO(HPPH)-PEG-HK in any of the four solutions after incubation for 96 h was observed (Supplementary Figure S2). In the in vivo situation, the amount of free HPPH released from GO(HPPH)-PEG-HK was < 20 % in the serum of mice at 24 h post-injection (Supplementary Figure S3). These results suggested the favorable in vitro and in vivo stability of GO(HPPH)-PEG-HK. The generation of singlet oxygen by GO(HPPH)-PEG-HK was assessed with the Singlet Oxygen Sensor Green (SOSG) assay. Singlet oxygen levels increased with irradiation time for HPPH, GO(HPPH)-PEG, and GO(HPPH)-PEG-HK, and were quenched by addition of NaN3 (Figure 1b). This confirmed that GO(HPPH)-PEG-HK can generate singlet oxygen upon light irradiation and suggested that GO(HPPH)-PEGHK might be used for PDT. The integrin αvβ6-binding affinity of GO(HPPH)-PEG-HK was assessed with a competitive binding assay using BxPC-3 tumor cells that highly express integrin αvβ6 and had been previously successfully used for the integrin αvβ6 receptor binding assay.28 GO(HPPH)-PEG-HK and HK peptide inhibited the binding of 125I-HYK to integrin αvβ6 expressed in these tumor cells in a concentration-dependent manner; the IC50 values were (42.02 ± 16.51) nM and (205.17 ± 124.22) nM, respectively. There was no inhibitory effect with GO(HPPH)-PEG (Figure 1c). These results suggest that GO(HPPH)-PEGHK has greater receptor binding affinity than the HK peptide. This is most likely due to the multiple HK peptides conjugated to the GO surface. To further evaluate the specificity of GO(HPPH)-PEG-HK for integrin αvβ6 in vitro, we stained integrin αvβ6positive 4T1 cells with 0.5 µM HPPH-equivalent concentrations of HPPH, GO(HPPH)-
6 ACS Paragon Plus Environment
Page 7 of 45
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 Nano
PEG, and GO(HPPH)-PEG-HK. Cell staining was carried out at 4 °C instead of 37 °C because HPPH accumulates in tumors by passive diffusion at higher temperatures.25,31 HPPH and GO(HPPH)-PEG showed low binding to 4T1 cells; in contrast, the cells were strongly stained by GO(HPPH)-PEG-HK (Figure 1d). Pre-incubating 4T1 cells with an excess of the nonfluorescent HK peptide abolished GO(HPPH)-PEG-HK staining (Figure 1d), confirming the receptor-binding specificity of the GO conjugate to 4T1 cells.
Tumor-Specific Targeting of GO(HPPH)-PEG-HK In Vivo The in vivo targeting of GO(HPPH)-PEG-HK was investigated in mouse models with subcutaneous 4T1 and pulmonary metastatic 4T1 cells stably transfected with firefly luciferase (4T1-fLuc). Representative optical images of subcutaneous 4T1 tumors after intravenous injection of HPPH, GO(HPPH)-PEG, or GO(HPPH)-PEG-HK are shown in Figure 2a. Subcutaneous 4T1 tumors were visible 4–24 h after injection of each of the three probes. The quantified tumor fluorescence intensity of GO(HPPH)-PEG-HK was significantly higher than that of the other probes at 24 h post-injection (Figure 2b). After the final scan at 24 h post-injection, mice were euthanized and the tumors and major organs were dissected and scanned ex vivo. The tumor uptake (%ID/g) of GO(HPPH)PEG-HK was significantly higher than that of free HPPH and GO(HPPH)-PEG (Supplementary Figure S4a, b; P
