Chitosan layered gold nanorods as synergistic therapeutics for photothermal ablation and gene silencing in triple-negative breast cancer
Graphical abstract
Treatment options for triple-negative breast cancer (TNBC) are limited, as patients do not respond to endocrine or targeted therapies, making this a difficult disease to treat. Multifunctional Chit-Au NRs were fabricated using a layer-by-layer assembly approach for delivery of siRNA gene silencing agents and thermal therapy to TNBC cells. This platform successfully suppressed oncogene expression in vitro and in vivo, resulting in anticancer activity, which was further enhanced through NIR irradiation.
Introduction
Breast cancer is the second most common cancer worldwide after lung cancer, and the leading cause of cancer death in women [1]. With the availability of modern diagnostic tools and increased use of adjuvant systemic therapies, significant progress has been made on early stage breast cancer treatment, and consequently the overall survival rates in breast cancer patients [2]. However, triple-negative breast cancer (TNBC), characterized by tumors that do not express the estrogen receptor (ER), the progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2), represent clinical challenge as these cancers do not respond to hormonal or targeted therapies [3], [4], [5], [6]. Current treatment strategies include chemotherapy agents, such as anthracyclines, taxanes, ixabepilone, and platinum agents, as well as select biologics [3]. However, patients with TNBC are in a vulnerable position as treatment options frequently fail to completely suppress tumor growth.
Ever since RNA interference (RNAi) was discovered by Fire et al. in 1998 [7], this technology has emerged as a promising treatment strategy for viral infections, genetic diseases, and cancer [8]. However, small interfering RNAs (siRNAs) are unstable in the blood stream, unable to penetrate cell membranes, and potentially immunogenic, due to their large size and negative charge. Successful application of siRNA for cancer therapy is highly dependent on the development of delivery vehicles that are nontoxic and enable the selective and efficient transport of siRNA to a specific tissue. Although viral vectors can be highly efficient tools for the delivery of RNAi agents, there are several safety concerns regarding their use [8]. On the contrary, non-viral vectors are generally regarded safer and they usually carry a lower risk of provoking an immune response. Accordingly, nanoparticle-mediated siRNA delivery holds great potential for overcoming biological barriers upon systemic delivery. In particular, it is important that nanodelivery systems can reduce uptake by the reticuloendothelial system (RES), enhance tumor accumulation through e.g. the enhanced permeability and retention (EPR) effect, and enhance endosomal/lysosomal escape [9], [10], [11], [12].
Recent studies indicate that metallic nanoparticles are useful for nanomedicine applications owing to their optical and electronic properties [13]. Especially gold nanoparticles show great promise due to biocompatibility, photothermal responsiveness, and ease of preparation and modification. In particular, gold nanorods (Au NRs) have been extensively used for biomedical applications, including photothermal therapy, drug delivery, and biosensing [13], [14], [15], [16], [17], [18], [19].
As previous reported works, starting with gold nanorods as a core, layer-by-layer degradable polymers enable the vectors to delivery the negative gene drugs [19]. However, most of these systems have the limitation in application for in vivo delivery due to high toxicity and low efficacy. In this study, monodisperse Au NRs were used as a template for a layer-by-layer strategy to produce a nanoparticle platform modified with a safety and biodegradable polymer chitosan for gene silencing and photothermal therapy. The goal was to successfully suppress TNBC by combining the effects of siRNA and thermal ablation. The platform was designed to provide a protective environment against enzymatic and serum degradation, promote cellular internalization, and induce endosomal/lysosomal escape of siRNA to achieve the down regulation of the protein and mRNA levels.
Section snippets
Materials and reagents
All reagents were purchased from Sigma–Aldrich and used without additional treatment unless otherwise indicated. Dulbecco’s Modified Eagle Medium high glucose (DMEM) and fetal bovine serum (FBS) were acquired from Fisher Scientific. Pyruvate kinase isozymeM2 (PKM2) and β-actin antibodies were obtained from Cell Signaling. siRNA-Alexa Fluor 555 and siRNA-FAM Fluor 488 were purchased from Qiagen. Scrambled siRNA and PKM2 siRNA were ordered from Sigma. PKM2 sense and anti-sense sequences were
Preparation and characterization of the Chit-Au NRs system
CTAB-Au NRs were synthesized as previously described through a seed-mediated growth method [13]. A three step procedure to obtain the final hybrid and multifunctional Chit-Au NRs platform via a well-developed layer-by-layer assembly approach is shown in Scheme 1A. Briefly, a PSS layer assembled onto the original CTAB-Au NRs in order to obtain negatively charged NRs which could further assemble the polymeric materials. Chitosan was then coated on the PSS-Au NRs to enable electrostatic bonding
Conclusion
Here, a layer-by-layer approach has been used to prepare a multifunctional Chit-Au NRs platform for gene silencing and photothermal therapy. The experimental results show that the delivery vehicle protects siRNA from degradation and displays low cytotoxicity. The efficient cellular uptake of Chit-Au NRs/siRNA was demonstrated by flow cytometry and confocal microscopy. Furthermore, the results indicate that the delivery vehicle can successfully escape from endosomal/lysosomal structure. Gene
Conflict of interest
The authors declare no competing interests.
Acknowledgements
This study was supported by a Grant from the Major Projects Foundation of Nanjing Military Region (12Z32), the Medical Science Foundation for Young Cultivation Project of PLA (13QNP038), the Natural Science Foundation of Jinling Hospital (2013023 and 2014004), the National Natural Science Foundation of China (No. 21231007), the Ministry of Education of China (Nos. 20100171110013 and 313058), the Guangdong Provincial Natural Science Foundation (No. 9351027501000003), and the Fundamental Research
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These authors contributed equally to this work.