Elsevier

Journal of Controlled Release

Volume 350, October 2022, Pages 448-459
Journal of Controlled Release

DNA-cloaked nanoparticles for tumor microenvironment-responsive activation

https://doi.org/10.1016/j.jconrel.2022.08.044Get rights and content

Abstract

Although progress has been made in developing tumor microenvironment-responsive delivery systems, the list of cargo-releasing stimuli remains limited. In this study, we report DNA nanothread-cloaked nanoparticles for reactive oxygen species (ROS)-rich tumor microenvironment-responsive delivery systems. ROS is well known to strongly induce DNA fragmentation via oxidative stress. As a model anticancer drug, hydrophobic omacetaxine was entrapped in branched cyclam ligand-modified nanoparticles (BNP). DNA nanothreads were prepared by rolling-circle amplification and complexed to BNP, yielding DNA nanothread-cloaked BNP (DBNP). DBNP was unmasked by DNA nanothread-degrading ROS and culture supernatants of LNCaP cells. The size and zeta potential of DBNP were changed by ROS. In ROShigh LNCaP cells, but not in ROSlow fibroblast cells, the uptake of DBNP was higher than that of other nanoparticles. Molecular imaging revealed that DBNP exhibited greater distribution to tumor tissues, compared to other nanoparticles. Ex vivo mass spectrometry-based imaging showed that omacetaxine metabolites were distributed in tumor tissues of mice treated with DBNP. Intravenous administration of DBNP reduced the tumor volume by 80% compared to untreated tumors. Profiling showed that omacetaxine treatment altered the transcriptional profile. These results collectively support the feasibility of using polymerized DNA-masked nanoparticles for selective activation in the ROS-rich tumor microenvironment.

Introduction

As our understanding of tumor pathophysiology has grown, tumor microenvironment-responsive anticancer drug delivery systems have increasingly been investigated. Compared to tumor cell-specific ligand-modified nanoparticles, tumor microenvironment-responsive delivery systems offer the advantage of overcoming the heterogeneity of individual tumor cells in tumor tissues [1,2]. Anticancer drug delivery systems have been designed to respond to various stimuli in the tumor microenvironment, including acidic pH [[3], [4], [5]], peptide-specific enzymes [1], and hypoxia [6,7]. One of most actively studied stimuli is the acidic pH of the tumor microenvironment, and several studies have reported delivery systems that have pH-responsive features for activation in an acidic tumor microenvironment [[3], [4], [5]].

Although progress has been made in these approaches, there are still unmet needs. In the case of acidic pH-based activation systems, activation in the tumor microenvironment can overlap with that in lysosomes [8]. Moreover, local inflammation can form a tumor-like environment that includes low pH and hypoxic conditions [9]. Regarding other strategies, there is only a limited repertoire of enzymes that are exclusively overexpressed in the tumor microenvironment [10]. Among them, matrix metalloproteinase-responsive peptides have been used to modify nanomaterials carrying anticancer drugs [1]. To camouflage delivery systems in the circulation, researchers have surface-tethered their delivery systems with polyethylene glycol (PEG) via pH-labile or enzyme-responsive peptide linkers [1,4].

Reactive oxygen species (ROS) are known to play important roles in oncogenic signaling, cancer progression, and metastasis [11], and upregulation of extra/intracellular ROS has been observed in the tumor microenvironment [12]. Although ROS-mediated DNA damage has been studied for several decades [13,14], relatively little effort has been made to develop a DNA-based tumor microenvironment-responsive anticancer delivery system.

In this study, we hypothesized that polymerized DNA nanothread masking could provide serum stability and selective activation for an anticancer drug delivery system targeting the ROS-rich tumor microenvironment. To test the hypothesis, we formulated lipid nanoparticles loaded with omacetaxine (OMT; Synribo®) as a model anticancer drug, which can be inserted into a lipid bilayer due to its hydrophobic moiety. The nanoparticles were decorated with branched cyclam ligand to increase cellular uptake of OMT. Recently, cyclam-derived ligands have been reported to improve a delivery efficiency of nanoparticles [15]. Polymeric cyclam was formulated with siRNA [16], and miRNA [17] to simultaneously inhibit CXCR4 and silence target mRNA.

Next, we masked the surface of branched cyclam ligand-modified nanoparticles carrying OMT (BNP) with DNA nanothreads (DNT). DNT produced by rolling circle amplification (RCA) can interact with branched cyclam via electrostatic interaction. DNT has been coated onto a surface of graphene oxide nanosheet to enhance a biocompatibility of nanoparticles [18]. Human prostate adenocarcinoma LNCaP cells were selected as a target cancer cells due to the ROS-rich tumor microenvironment. DNT-cloaked BNP (DBNP) were designed to undergo unmasking at the ROS-rich tumor microenvironment, thereby exposing BNP (Fig. 1). Here, we report the ROS-selective unmasking of DBNP and enhanced anticancer effects against tumors.

Section snippets

Preparation of DNA nanothreads

Single-stranded DNT was prepared by rolling-circle amplification (RCA), as previously described [19]. Briefly, to generate the RCA template, 0.5 μM of phosphorylated linear single-stranded DNA (5’-ATC TGA CTA GTA TAT ACA AAA CTA ATG AGG CGT TGG AAG TGT AGT GGG GCG GTG CGC TCG GTC ATA GTA AT-3′, 45% GC content) was circularized using a primer and annealed with T4 DNA ligase (125 units/mL) (Thermo Scientific, Waltham, MA, USA). The resulting circular template was incubated for 2 h with 1 μg/mL of

ROS-mediated unmasking of DBNP

The structures of the various nanoparticles are illustrated in Fig. 2A. UBNP indicates DNA cloaking-unmasked BNP. The ROS-mediated selective unmasking of active cyclam moieties on BNP was demonstrated by morphological and physicochemical property changes. The surface coating of BNP with DNT increased the particle size (Fig. 2B). The average size of BNP was 75.3 ± 7.0 nm; this increased to 185.3 ± 19.3 nm for DBNP but decreased to 106.1 ± 44.5 nm after exposure to ROS-rich LNCaP cell culture

Discussion

We herein demonstrate that DBNP can be selectively unmasked at an ROS-rich tumor microenvironment, and that this enhances the anticancer efficacy of a model anticancer drug. The surface coating of BNP with DNT was unmasked by ROS and the ROS-rich conditioned medium of LNCaP prostate cells. The enhanced anticancer efficacy and transcriptome profiling results support the idea that DBNP may circulate with the active moieties hidden in the DNA cloak, and activate only in an ROS-rich tumor

Conclusion

This study demonstrated the ROS-rich tumor microenvironment-sensitive activation of DBNP. Surface cloaking of nanoparticles with DNT can reduce their interactions during blood circulation, while activation at the tumor microenvironment can increase the interaction of exposed active ligands on nanoparticles with tumor cells, providing enhanced anticancer efficacy. Although we used OMT in this study, ROS-activated nanoparticles could be used to deliver other anticancer drugs. Moreover, the

Acknowledgements

This research was supported by grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT, Republic of Korea (NRF-2018R1A5A2024425) and the Ministry of Education (NRF-2021R1A6A3A01086428; 2021R1A6A1A10044154), the Korea Medical Device Development Fund funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of

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