Hollow carbon nanospheres as a versatile platform for co-delivery of siRNA and chemotherapeutics
Graphical abstract
We construct a novel amine-coated hollow carbon nanospheres (HCNs), which serve as a versatile platform for co-delivery of siRNA and chemotherapeutics (DOX or Cis) to fight drug-resistant cancers. The nanostructures exhibit enhanced nucleic acid and chemotherapeutics delivery, effective gene regulation, and synergistically inhibit drug-resistant cancers in vitro and in vivo.
Introduction
The chemotherapeutics, such as Doxorubicin (DOX) and Cisplatin (Cis) are used in clinical cancer treatment extensively [1], [2], [3]. However, the rapid emergence of drug resistance against these chemotherapeutics and subsequent treatment failures greatly hamper the therapy [4]. The major reason for the development of drug resistance is the overexpression of multidrug resistance gene (MDR1) on the cancer cells or encoding of an efflux pump protein, P-glycoprotein (Pgp) on the cell membrane, which exports the drugs and decreases intracellular concentration of the drugs to ineffective levels [5], [6], [7]. The selective modulation of the MDR1 gene is considered to be an effective way to reverse the drug resistance [8].
Small interfering RNA (siRNA)-based gene silencing emerges as a therapeutic strategy for down-regulating the expression of targeting proteins, such as MDR1. However, unmodified siRNA is unstable and immunogenic in the human body and is not easy to enter cells. Thus, adequate delivery systems are required to bring siRNA to its site of action without adverse effects. The widely studied gene-delivery vehicles, such as dendrimer, polyetherimide, and polylysine are usually limited by their high cytotoxicity [9]. Oligonucleotide-nanoparticles (NPs) conjugations are emerging as an effective structures for gene regulation, especially in the pathways of RNAi and antisense [10]. Without the aid of any other gene carriers, the oligonucleotide-NPs can be internalized in large quantity by the cells, which results in effective gene regulation [11]. Furthermore, the oligonucleotide-NPs show resistance against degradation by the nuclease, for the high oligonucleotide surface density results in more negative charges, leading to recruitment of more charge-balancing counterions and the formation of high local salt concentration at the nanoparticle surface [12]. This effect can prevent the nucleases from catalyzing hydrolysis of the oligonucleotides. Furthermore, the steric hindrance may also retard the nuclease-catalyzed degradation. Consequently, the oligonucleotide-NPs show great promise for siRNA delivery and gene regulation.
The advantage of the oligonucleotide-NPs, however, has not been fully explored. For example, nanoparticles used as cores play a minor role in drug loading but contribute to a metallic mass [13], [14], and their long term toxicity is not fully characterized [15], [16]. Hence, replacing the solid core of nanoparticle with hollow cavity is desirable for increasing capacity of drug loading. Furthermore, materials for the nanoparticle with improved safety are required.
Herein, we construct hollow carbon nanospheres (HCNs)-cored therapeutic nucleic acid nanostructure for delivering both siRNA (for gene regulation) and chemotherapeutics. In this system, novel HCNs are fabricated with chitosan and silica template-assisted strategy. The HCNs are highly dispersed, hydrophilic, spherical, primary amine-modified, and non-cytotoxic. This kind of HCN shows unique features in the structure, which is useful in the applications: i) their outer surface is activated by as-formed primary amine groups without any modification, avoiding the tedious work in activation but with low yield; ii) the large hollow cavities increase the storage space, and are particularly useful for π-π stacking in improving the loading of drug molecules [17], [18]; iii) the hydrothermal reaction forms mesoporous carbon shell, through which drug molecules can easily diffuse in or out. The HCNs act as a multifunctional platform for co-delivering siRNA targeting MDR1 mRNA (siMDR1) and chemotherapeutics (DOX or Cis) to fight drug-resistant cancers. In this study, to address the drug-resistant breast cancer and ovarian cancer [19], [20], we choose MCF-7/ADR cells or Cis-resistant human ovarian adenocarcinoma cell line (CoC1/DDP cells) as cell models (MDR1 is overexpressed in both cell lines, Fig. S1A and S1B). The chemotherapeutics and siMDR1 are loaded into the HCNs (the products are named siMDR1@HCNs/DOX or siMDR1@HCNs/Cis) (Scheme 1), and utilized to restore DOX- or Cis-sensitivity of these drug-resistant cancer cells. The siMDR1@HCNs/DOX and siMDR1@HCNs/Cis show excellent performance of treating drug-resistant cancers both in vitro and in vivo.
Section snippets
Materials
Low molecular chitosan, doxorubicin hydrochloride (DOX), silica nanoparticles were from Sigma-Aldrich Co. Ltd. Fetal bovine serum (FBS) were from by Invitrogen. D-Salt<SUP>TM</SUP> Dextran Desalting Column was from Pierce Biotechnology, Inc. Anti-P Glycoprotein antibody (CAS number: ab103477) was from Abcam Inc. (Cambridge, MA). Cisplatin was from Selleck Chemicals (Houston, USA).
Preparation of the HCNs
We prepared the primary amine functionalized HCNs based on our previous work [16]. Chitosan solution was prepared in
Characterization of HCNs
High resolution transmission electron microscopy (HR-TEM) shows a large hollow cavity of ∼40 nm (diameter) and an intact shell of ∼10 nm (thickness) in the HCNs (Fig. 1A and B). Dynamic light scattering (DLS) indicates that the HCNs are well dispersed with a diameter of ∼80 nm and polydispersity index (PDI) of 0.248 (Fig. 1C). We use X-ray photoelectron spectroscopy (XPS) spectra to test the HCNs. The N1s peak in chitosan centered at 399.100 eV is associated with nitrogen in an 
NH2 state [23].
Conclusions
In conclusion, a newly designed and amine modified HCNs were prepared. We used the HCNs to co-deliver chemotherapeutics and therapeutic nucleic acids for the treatment of drug resistant tumours. This work demonstrated, to our best knowledge, the first example that HCNs can serve as a versatile drug co-delivery platform for synergistic cancer treatment with unique advantages over traditional methods. The innate primary amines on the HCNs surface facilitate facile conjugation of functional
Acknowledgements
The financial support was provided by National Science Foundation of China (81361140345, 21535001, 31470911, and 81673039), “Strategic Priority Research Program” of the Chinese Academy of Sciences (XDA09030305, XDA09030307), and the CAS/SAFEA International Partnership Program for Creative Research Teams. We thank Dr. Zhonglin Fu and Xuefang Zhang at National Center for Protein Sciences, Peking University for flow cytometry analysis. We thank the professors in Tohoku University, including
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These authors contributed to the work equally.