Elsevier

Biomaterials

Volume 170, July 2018, Pages 1-11
Biomaterials

Cytokine induced killer cells-assisted delivery of chlorin e6 mediated self-assembled gold nanoclusters to tumors for imaging and immuno-photodynamic therapy

https://doi.org/10.1016/j.biomaterials.2018.03.048Get rights and content

Abstract

The cytotoxicity and unique tumor-tropic properties of cytokine-induced killer (CIK) cells render them promising in the field of cancer immunotherapy and delivery systems. Here, we report a novel and facile approach to assemble gold nanoclusters (GNCs) into stable and monodispersed nanoparticles (NPs) using Chlorin e6 (Ce6) molecules. Notably, the fluorescence intensity of the GNCs-Ce6 NPs was about 4.5 folds stronger than the GNCs counterparts. The as-prepared GNCs-Ce6 NPs were conjugated with CD3 antibody (Ab) and further employed to label CIK cells to create a CIK cell-based drug delivery system (Ce6-GNCs-Ab-CIK). The Ce6-GNCs-Ab-CIK exhibited high tumor-targeting efficiency and excellent therapeutic efficacy toward MGC-803 tumor-bearing mice. Benefiting from the synergistic therapeutic effect between GNCs-Ce6-Ab NPs and CIK cells, the GNCs-Ce6-Ab-CIK strategy may present an ideal cancer theranostic platform for tumor targeted imaging and combination therapy.

Introduction

Photodynamic therapy (PDT) has been approved for clinical use and holds great promise for treating various cancers [1,2]. PDT uses photosensitizer (PS) and light in combination with oxygen to generate cytotoxic singlet oxygen (1O2) that induces cancer cell death by necrosis or apoptosis [3]. In particular, activatable PDT agents have weak fluorescent and phototoxicity without laser irradiation, but become highly fluorescent and phototoxic in regions that receive the appropriate wavelength of light [1,4]. Thus, the accumulation of PS in non-specific tissues displays little systemic toxicity [5,6]. In spite of the advantages of this technique, PS has severe limitations, including poor water solubility, non-specific distribution in tumors, and low bioavailability [[7], [8], [9]]. To address these problems, it is necessary to find a new strategy for delivering PS to tumors to improve their cancer therapy efficiency.

The unique physicochemical characteristics of gold nanoclusters (GNCs), such as ultrasmall size, good photophysical properties, and biocompatibility make them ideal platforms for bio-sensing, drug delivery, and imaging [10,11]. Recently, GNCs have been introduced as building blocks for self-assembly, since self-assembled nanoparticles are regarded as an efficient strategy to deliver drugs for clinical applications [12,13]. Different agents have been used to cross-link GNCs into large assemblies, such as Gd2+ ions, Gd3+ ions, Zn2+ ions, Cu2+ ions, and cationic polymers [[14], [15], [16], [17]]. However, the self-assembled nanoparticles synthesized based on the above method can not be used for tumor treatment. Development of new approaches to assemble GNCs is of great interest to improve their biological applications. PDT agents, such as Chlorin e6 (Ce6), have not been employed in GNCs self-assembly. Thus incorporating Ce6 in nanoparticle delivery systems may expand the therapeutic applications of GNCs.

Nowadays, tumor-tropic cells (including mesenchymal stem cells, induced pluripotent stem cells (iPS cells) and immune cells)-directed drug delivery systems have aroused great interest in the field of nanomedicine [[18], [19], [20]]. Specifically, engineering of cytokines-induced killer (CIK) cells for adoptive cell therapy (ACT) has become one of the most effective approaches to trigger an antitumor immune response [[21], [22], [23], [24], [25]]. CIK cells possess MHC-unrestricted cytotoxicity and demonstrate enhanced cell proliferation rate than lymphokine activated killer (LAK) cells [26,27]. More importantly, CIK cells have low graft-versus-host disease (GVHD) risk. Meanwhile, clinical trials using CIK cells have demonstrated good therapeutic effect and proved that CIK cells may inhibit recurrence and improve the quality of patients' life [28].

Herein, we report a facile method of assembling monodisperse and stable self-assembled nanoparticles (NPs) in water using Ce6 molecules to cross-link GNCs. The overall size of GNCs-Ce6 NPs could be controlled by changing the ratio of GSH (on the surface of GNCs) to Ce6 molecules. The formed GNCs-Ce6 NPs possess enhanced fluorescence in comparison with GNCs. GNCs-Ce6 NPs were conjugated with CD3 antibodies (Ab) to prepare Ce6-GNCs-Ab NPs. And further CIK cells were applied as a drug carrier to deliver Ce6-GNCs-Ab NPs to gastric tumors with the purpose of investigating the feasibility of using the CIK cells as theranostic system for combined diagnosis and therapy (Scheme 1).

Section snippets

Materials

Ce6 was purchased from Frontier Scientific (Logan, UT, USA). DMSO was obtained from Sigma Chemical Corp. (St. Louis, MO, USA). NHS-PEG2k-NHS was supplied by Shanghai Yanyi Biosciences Co., Ltd. (Shanghai, China). GSH (98%), Gold chloride trihydrate (HAuCl4·3H2O, 99%), and tetrabutylammonium borohydride (TBAB; 95%) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Hoechst 33342 was obtained from Invitrogen Corp. (Carlsbad, CA, USA). Fetal bovine serum was purchased from

Characterization of GNCs, GNCs-Ce6 NPs and GNCs-Ce6-Ab NPs

GNCs with a uniform sphere shape (Fig. 1A) were synthesized by a modified method described elsewhere [29]. The self-assembly GNCs-Ce6 NPs were prepared by adding different concentrations of Ce6 molecules dropwise to the GNCs solution (pH 7) and stirred for 60 min at room temperature. The self-assembly of GNCs might be caused by the rehydration process that mediated by Ce6 molecules [30]. Fig. S1 and S2 indicated that the prepared GNCs-Ce6 NPs (obtained at different Ce6/GSH ratios: 0.08, 0.18

Conclusion

In summary, self-assembled GNCs-Ce6 NPs were synthesized in a simple and fast procedure. By conjugating CD3 antibody with GNCs-Ce6 NPs, the loading efficiency of GNCs-Ce6 NPs to CIK cells was improved. The in vitro assays showed that GNCs-Ce6-Ab NPs (5 μg/mL Ce6 equiv) were efficiently internalized by CIK cells with little cytotoxicity and GNCs-Ce6-Ab-CIK possessed highly anti-cancer potency upon laser irradiation. Furthermore, in vivo fluorescence imaging confirmed that GNCs-Ce6-Ab NPs laden

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

This work is supported by the National Key Research and Development Program of China (Grant No. 2017FYA0205301), the National Basic Research Program (973 Project) (Project No. 2015CB931802), the National Natural Scientific Foundation of China (Grant No. 81401458, 81225010, 81028009, and 31170961), the 863 projects of China (Project No. 2014AA020701), China Postdoctoral Science Foundation (Grant No. 2017M621486), the Shanghai Science and Technology Fund (Grant No. 14ZR1432400 and 13NM1401500).

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