Reactive oxygen species and enzyme dual-responsive biocompatible drug delivery system for targeted tumor therapy
Graphical abstract
Six-arginine-tailed anti-EGFR affibody has been incorporated into a dual responsive drug delivery system, MAMA, which could release MTX effectively to the cells with high hROS and trypsin activity.
Introduction
Nanotechnology-based drug delivery systems (DDSs) hold enormous potential to efficiently encapsulate chemotherapeutic drugs and achieve targeting drug release to tumor tissues with limited cytotoxicity to normal cells [1]. An ideal DDSs design needs several essential elements including biocompatibility with negligible toxicity, stable drug encapsulation, and specific targeting [2]. Gold nanostructures have been extensively studied as DDSs in cancer therapy, due to their good biocompatibility and easy in surface modification [3]. Shells and coatings have been usually used for encapsulating the loaded drug into the gold nanostructure [4]. Antibodies and affibodies of protein overexpressed on the cancer cells have been added to the surface of the DDSs shells and coatings for specifically targeting tumor biomarkers [5,6]. However, complicated functionalization of DDSs requires multiple reactions with repeated purifications, resulting in drug leaking and low yields of the final product. It remains a demanding challenge to simply fabricate DDSs without sophisticated modifications.
Meanwhile, compared to conventional nanocarriers in clinical applications or under clinical trials, DDSs are superior in controllability which reduces adverse off-target effects [7]. Since proliferation and progression of cancer are closely related to reactive oxygen species (ROS), various responsive groups of ROS have been exploited to synthesize DDSs for controlled drug release in tumors [8]. However, due to insufficient intracellular levels of endogenous ROS usually result in relatively low therapeutic efficiency, inducing the generation of high dosage ROS in tumors targeted by ROS-responsive DDSs is a promising strategy to potentiate the therapeutic outcome in cancer therapy. Besides, highly ROS (hROS) as strong oxides including hydroxyl radical (•OH), peroxynitrite (ONOO−), and hypochlorite (ClO−) can oxidize nucleic acids, proteins, and lipids, damaging the living cells and activating the programmed cell death [9]. Therefore,
developing a new DDS which can control release drug into targeted tumors, as well as increase the in situ levels of hROS in tumors are highly desired.
The other method to minimize the potential side effects of nanocarriers is providing the DDS with high affinity and selectivity to bind the aim targets. Epidermal growth factor receptor (EGFR) has been reported as an overexpressed biomarker in many cancers, especially certain gastrointestinal carcinomas [10,11]. Antibodies for EGFR have been used as targeting moieties for high-contrast tumor imaging and as potential drugs for cancer treatment because of their superior affinities with target and increased accumulation on the tumor surface [12,13]. However, the antibodies have low clearance rates and low tumor penetration capability due to their large size, resulting in the low signal-to-noise ratio for tumor imaging and unsatisfactory effectiveness for cancer treatment [14]. Affibody has been developed as a class of engineered protein that retains the desirable high affinity and specificity of the antibody, but with small size and chemical robustness for medical application [15,16]. The specific selectivities, short circulation, high renal clearance, biocompatibility, and ease of synthesis make affibody suitable for use in DDS.
Herein, we report a novel strategy to easily synthesize a highly biocompatible tumor-targeting DDS that exhibits the desired properties of hROS and trypsin dual responsive drug-controlled release, as well as inducing hROS generation to accelerate drug release and cell damage in tumors for enhanced cancer therapy. Methotrexate (MTX) is selected as the loaded chemotherapeutic drug, which has been used clinically in high doses as a folate antagonism to block the folate pathway and stimulate ROS production for decreasing metastasis and inducing apoptosis of cancer [17]. As shown in Scheme 1, hROS-responsive 11-mercaptoundecanoic acid-modified gold nanoclusters (MUA-Au NCs) are one-pot synthesized, then cancer-targeting ligand, six-arginine-tailed anti- EGFR affibodies are prepared. MTX is loaded into MUA-Au NCs to form MUA-Au NCs-MTX (MAM). Finally, positively charged polyarginine tails of affibodies easily self-assemble onto the negatively charged surface of MAM, forming a shell to seal in the loaded drug, whereas EGFR targeting fragments of affibodies remains on the surface of MUA-Au NCs-MTX-Affibody (MAMA) for tumor targeting. Upon active targeting to EGFR overexpressed tumor, endogenous upregulated trypsin in cancer cells can digest polyarginine chains of affibodies to release MTX. In situ released MTX further induces hROS production, achieving subsequent amplification of high-dosage MTX release by destroying Au NCs structure, which is favorable for both chemotherapy and high oxidative stress therapy of cancer.
Section snippets
Materials
Human cancer cell lines HepG2, BxPC3, and normal cell line hTERT-HPNE were obtained from Institute of Biochemistry and Cell Biology (SIBS, CAS, Shanghai, China) and grown in Dulbecco's Modified Eagle medium (DMEM) and RPMI 1640 media with 10% FBS subsequently maintained in a 37 °C with 5% CO2 incubator, respectively. Female Nude mice (18–20 g, 6 weeks old, purchased from Charles River Laboratories) were selected for establishing the xenograft mice models. 5 × 107 HepG2 or BxPC3 cells in 100 μL
Results and discussion
MAMA was prepared in three steps and the characterization of the product coming from each step as shown in Fig. 1. The average diameter of MUA-Au NCs, MAM, and MAMA was 1.1 nm, 3.4 nm, and 9 nm, respectively (Fig. 1A). The molecular weight of synthesized affibody was 7519.1 g/mol with 99.9% purity after high-performance liquid chromatography (HPLC) (Fig. S1A, B). The pronounced IR absorption bands of affibody occurred at 1650 cm−1 (ν-CO-NH-), 1590 cm−1 (νasCOO-), 1210 cm−1 (νCN) and 1100 cm−1 (ν
Conclusions
In summary, a polyarginine-tailed anti-EGFR affibody has been successfully synthesized and has been incorporated into the MTX loaded MUA-Au NCs to form theranostic system MAMA by self-assembly based on charge effect between negatively charged -COOH terminal of MUA and positively charged polyarginine tails. MAMA has excellent biocompatibility, the adequate loading capacity of MTX, precise targeting to EGFR-overexpressed cells, and hROS and trypsin dual-responsive drug release in cells. The red
Declaration of Competing Interest
The authors declare no competing financial interest.
Acknowledgment
This work was supported, in part, by the National Natural Science Foundation of China (81201141) and the Clinical Capability Construction Project for Liaoning Provincial Hospitals (LNCCC-D50-2015 and LNCCC-C09-2015). All experiments involving animals were approved by the Administrative Panel on Laboratory Animal Care of Stanford University. We are very grateful to Michael Moseley (Stanford University, USA) for helping with the revised manuscript. We are also pleased to acknowledge the Stanford
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