**Lili Jin 1, Qiuyu Wang 1, Jiayu Chen 2, Zixiang Wang 1, Hongchuan Xin <sup>3</sup> and Dianbao Zhang 2,\***


Received: 22 August 2019; Accepted: 15 November 2019; Published: 17 November 2019

**Abstract:** The incidence of oral cancer is increasing due to smoking, drinking, and human papillomavirus (HPV) infection, while the current treatments are not satisfactory. Small interfering RNA (siRNA)-based therapy has brought hope, but an efficient delivery system is still needed. Here, polyethyleneimine (PEI)-modified magnetic Fe3O4 nanoparticles were prepared for the delivery of therapeutic siRNAs targeting B-cell lymphoma-2 (BCL2) and Baculoviral IAP repeat-containing 5 (BIRC5) into Ca9-22 oral cancer cells. The cationic nanoparticles were characterized by transmission electronic microscopy (TEM), scanning electronic microscopy (SEM), dynamic light scattering (DLS), and vibrating sample magnetometer (VSM). By gel retardation assay, the nanoparticles were found to block siRNA in a concentration-dependent manner. The cellular uptake of the nanoparticle/siRNA complexes under a magnetic field was visualized by Perl's Prussian blue staining and FAM labeling. High gene silencing efficiencies were determined by quantitative real-time PCR and western blotting. Furthermore, the nanoparticle-delivered siRNAs targeting BCL2 and BIRC5 were found to remarkably inhibit the viability and migration of Ca9-22 cells, by cell counting kit-8 assay and transwell assay. In this study, we have developed a novel siRNA-based therapeutic strategy targeting BCL2 and BIRC5 for oral cancer.

**Keywords:** magnetic nanoparticle; iron oxide; siRNA delivery; BCL2; BIRC5/survivin; oral cancer

#### **1. Introduction**

The incidence of oral cancer has increased due to risk factors such as tobacco, alcohol, and human papillomavirus (HPV), resulting in nearly 180 thousand deaths worldwide in 2018 [1,2]. The clinical treatment of oral cancer mainly depends on surgery, radiotherapy, chemotherapy, and several targeted drugs, but the prognosis is poor [3,4]. Therefore, it is necessary to develop novel therapeutic strategies to overcome the limitations of current therapies for oral cancer.

Recent progress in nanotechnology-based gene therapy has brought hopes for cancer treatment [5]. RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing process in eukaryotes [6]. RNAi could be triggered by microRNA (miRNA) and small interfering RNA (siRNA), which could be designed to target almost any gene [7]. It is exploited by researchers for loss-of-function studies and holds promise for the development of therapeutic gene silencing [8]. The first siRNA drug Onpattro (patisiran) targeting transthyretin (TTR) has been approved by the U.S. Food and Drug Administration (FDA) in 2018, for the treatment of peripheral nerve disease polyneuropathy in adults. This major progress, with the elucidation of more and more disease-related target genes, has greatly stimulated the research and development of siRNA drugs. However, how to effectively deliver siRNA drugs is a bottleneck to clinical practice [6,9]. The currently developed siRNA delivery strategies mainly include siRNA conjugation, lipid-based, and polymer-based delivery systems [10,11]. In our previous studies, the prepared polyethyleneimine (PEI)-coated Fe3O4 nanoparticles exhibited siRNA protection and delivery capacities for mesenchymal stem cells and glioblastoma cells [12,13]. However, the feasibility of these type of nanoparticles for delivering siRNA to oral cancer cells remains unknown, and the uniformity and efficiency of delivery needs to be improved.

An increasing number of cancer target genes has been reported in recent years. BCL2 (B-cell lymphoma-2) is a gene that is overexpressed in many cancers to escape cell death [14]. It is a promising cancer therapeutic target, but there are few targeting agents with clinical significance [14–16]. For oral cancers, BCL2 were proved to be important in cancer progression and chemoradiation resistance [17]. Inhibition of BCL2 in oral cancer cells inhibited proliferation and induced apoptosis, and also augmented the inhibitory effects of cisplatin in vitro and in vivo [18]. BIRC5 (Baculoviral IAP repeat-containing 5, also named survivin) is a conserved gene essential for cell proliferation. It is expressed in proliferating cells and upregulated in most cancers, the inhibition of BIRC5 leads to apoptosis or sensitization to chemotherapy and radiotherapy [19,20]. BIRC5 is rarely mutated in oral cancer samples and upregulated compared to non-cancerous tissue [21]. In this study, we prepared Fe3O4 nanoparticles and design siRNAs targeting BCL2 and BIRC5, aiming to explore the efficient delivery of therapeutic siRNA into oral cancer cells by Fe3O4 nanoparticles, which might provide a novel strategy for the future therapy of oral cancer.

#### **2. Materials and Methods**

#### *2.1. Synthesis and Characterization of Nanoparticles*

The magnetic nanoparticles were synthesized based on the oxidative hydrolysis method [22]. Briefly, FeSO4 and PEI dissolved in H2SO4 solution were dripped into KNO3 and NaOH solutions under nitrogen bubbling in a triple neck flask. After precipitation, the PEI–Fe3O4 nanoparticles were obtained by ultrafiltration (100 KDa, UFC910096, Millipore, Beijing, China). The particle size and morphology were analyzed by transmission electronic microscopy (TEM) and scanning electronic microscopy (SEM) [12]. The particle size was analyzed by ImageJ software. The zeta potential and hydrodynamic size of nanoparticles were analyzed by dynamic light scattering (DLS, Mastersizer 2000, Malvern, Worcestershire, UK). The amino group density was determined by the conductivity meter, and the elemental content was analyzed by energy dispersive spectroscopy (EDS). The magnetization of nanoparticles was measured by the vibrating sample magnetometer (VSM, Lakeshore7404, Westerville, OH, USA).

#### *2.2. Gel Retardation Assay*

The binding capacity of the nanoparticles to siRNA was analyzed by gel retardation assay. In general, 1 μg siRNA was mixed with 0, 0.5, 1, 1.5, and 2 μg nanoparticles in Opti-MEM Reduced Serum Medium (51985034, Gibco, Shanghai, China) and incubated at room temperature for 10 min. The mixtures were subjected to 2% agarose gel electrophoresis at 100 V for 20 min. The gels were stained with Biosafe nucleic acid dye (170-3001, Tanon, Shanghai, China) and imaged under Tanon-1600 imaging system (Tanon).

#### *2.3. Cell Culture*

Human oral cancer cell Ca9-22 and CAL 27 (Procell, Wuhan, China) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with high glucose (SH30022.01, Hyclone, Beijing, China) supplemented with 10% Certified Fetal Bovine Serum (FBS) (04-001-1A, Bioind, Kibbutz Beit-Haemek, Israel) and 1% Penicillin Streptomycin (SV30010, Hyclone) at 37 ◦C in a humidified atmosphere containing 5% CO2.
