Reactive oxygen species and enzyme dual-responsive biocompatible drug delivery system for targeted tumor therapy

Highlights

• Arginine-tailed anti-EGFR affibody serves as the shell of the drug delivery system.

• High trypsin activity triggers drug release by digesting the cargo arginine shell.

• The hROS- and trypsin-responsive drug delivery system benefits cancer treatment.

Abstract

Spurred by newly developed drug delivery systems (DDSs), side effects of cancer chemotherapy could be reduced by using multifunctional nanoplatforms. However, the facile synthesis of effective DDSs remains a challenge. Here, a six-arginine-tailed anti-epidermal growth factor receptor (EGFR) affibody was employed to easily synthesize the highly reactive oxygen species (hROS)- and trypsin-responsive 11-mercaptoundecanoic acid-modified gold nanoclusters (MUA-Au NCs) for tumor-targeted drug delivery. The polyarginine moiety of affibody sealed methotrexate (MTX)-loaded MUA-Au NCs through charge effect, as well as leaving the rest targeting fragment of the affibody to specifically bind tumor overexpressed EGFR. As the shell of MUA-Au NCs-MTX-Affibody (MAMA), polyarginine chains of affibody could be digested by trypsin, helping to release MTX from MAMA. The released MTX accelerated destroying MUA-Au NCs through inducing the generation of hROS. Specifically targeting EGFR-overexpressed tumors, quickly delivering a sufficient amount of drug to the tumor, subsequently increasing the local MTX and hROS levels, and safely eliminating the biocompatible structure from kidney, endowed MAMA greater treatment effectiveness and lower side effect than chemotherapy, especially in pancreatic cancer due to its high trypsin level. This simply fabricated DDS may find applications in high effective cancer therapy, especially for tumors with high 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.