Target delivery of MYCN siRNA by folate-nanoliposomes delivery system in a metastatic neuroblastoma model

Cancer Cell International Target delivery of MYCN siRNA by folate-nanoliposomes delivery system in a metastatic neuroblastoma model Qiqi Zhu 0 Chen Feng 0 Weiwei Liao 0 Yan Zhang 0 Suoqin Tang 0 0 Department of Pediatrics, Hainan Brach of Chinese PLA General Hospital , Haitang Bay, Sanya, Hainan Province 572013 , China Background: Folate-nanoliposomes delivery system has emerged recently as a specific and safety delivery method and gradually used as the carrier of a variety kinds of drugs including compounds, plasmids and siRNAs. Methods: In this study, we established a bone marrow and bone metastasis xenograft mouse model by injecting the LA-N-5 cell into the bone marrow cavity. Fluorescence microscopy, TUNEL Assay, Quantitative RT-PCR and western blot were conducted to analysis the distribution of folate-nanoliposomes entrapped MYCN (V-myc myelocytomatosis viral related oncogene) siRNA in mice and the relevant suppression effect. Results: The folate-nanoliposomes entrapped MYCN siRNA can be specifically distributed in tumor tissues. Further study shows that folate-nanoliposomes entrapped MYCN siRNA lead to MYCN mRNA expression significantly down-regulated (>50%, and p < 0.05) compared with negative control siRNA treatment. MYCN protein expression was inhibited about 60% in vivo, thus induced tumor cell apoptosis markedly. Conclusion: This study point to a new way for treatment of metastatic neuroblastoma and could widen the application of folate-nanoliposomes delivery system in tumor therapy. Folate-nanoliposomes; Metastatic neuroblastoma; MYCN; siRNA - Introduction Neuroblastoma is the most common extracranial solid tumor occurring in children, which accounts for about 15% of all childhood cancer deaths [1,2]. It is generally accepted that the amplification of the MYCN protooncogene is highly relevant to the genesis and prognosis of neuroblastoma [3,4]. MYCN is an important member of the myc family that includes 3 strong oncogenes, MYC, MYCN, and MYCL [5]. Targeting MYCN gene has developed into a promising therapeutic strategy [6-8]. Small interference RNA (siRNA) is synthetic short double-stranded RNA, which can induce the destruction of homologous mRNA when introduced into a cell [9]. siRNA has been developed into an effective tool for suppressing target proteins expression by specifically digesting their mRNAs [10]. siRNAs have been widely investigated as therapeutic agents to treat a wide range of human diseases including neuroblastoma [7,11]. Folate, a nontoxic low-weigh compound, is vital for tumor cell proliferation and survival. Studies show that tumor cells can express 200-folds more folate receptor on the membrane than normal cells, which is accordance to the high intake of folate in tumor cells. Folate receptor is overexpressed in a wide range of human cancer cells, including ovarian cancer, endometrial cancer, head and neck cancers, myeloid leukaemia, and some pediatric cancers [12]. Folate conjugating on liposomes with entrapped drugs [13-16] or plasmids [17-20] has been used successfully to deliver drugs to folate-receptor -expressing cancer cells. Our previous study had revealed that the folate receptor was highly expressed on the membrane of LA-N-5 neuroblastoma cells. In this study, we want to further investigate whether folate receptor-targeted liposome can act as a good delivery tool of MYCN siRNA and exert a satisfying therapeutic effect on neuroblastoma. In this study, we established a bone marrow and bone metastasis xenograft mouse model by injecting the LAN-5 cell into the bone marrow cavity. Then we examined whether folate-nanoliposomes entrapped MYCN siRNA can specifically distribute to tumor tissues and suppress MYCN gene expression as well as induce apoptosis in neuroblastoma cells. Materials and methods Animals BALB/c nude mice (4 wk) were used for all experiments. All animals were housed in the Experimental Animal Centre of PLA General Hospital, and all procedures were approved by the Institutional Animal Care and Use Committee of PLA General Hospital. Cell culture LA-N-5 neuroblastoma cell was the generous gift of Dr. Robert Seeger from Childrens Hospital Los Angeles, and the cells were grown in RPMI 1640 medium (GIBCO BRL, USA) containing 15% fetal bovine serum (Hyclone, USA), 2 mmol/L l-glutamine, 100 IU/ml penicillin G and 100 g/mL streptomycin (all were from Huamei Company, China) in a humidified 5% CO2 and 95% air atmosphere at 37C. Establishment of the bone marrow and bone metastasis xenograft mouse model Previously described procedure was introduced in the current study [21,22]. Briefly, LA-N-5 neuroblastoma cells were harvested and washed by centrifugation and resuspended in cold serum-free medium prior to injection. Mice were immobilized in a restraining device, and cells (105 cells in 5 l) were injected into the bone marrow cavity of the femoral proximal metaphysis. About five weeks later, the diameters of tumors can be up to 5 mm and the bone metastasis model is successfully established. Preparation of the folate-nanoliposome entrapped MYCN siRNA The folate-nanoliposome entrapped MYCN siRNA with Cy-3 fluorescence tag was the generous gift from Dr. Robert J. Lee (Ohio State University, College of Pharmacy). siRNA sequences were as follows: MYCN siRNA sense: 5-CGGAGAUGCUGCUUGAGAA dtdt-3, antisense: 5-UUCUCAAGCAGCAUCUCCG dtdt-3; the negative control siRNA: sense: 5-UUCUCCGAACGU GUACGU dtdt-3, anti-sense: 5-ACGUGACAC GUU CGGAGAA dtdt-3. siRNA distribution analysis and gene therapy For siRNA distribution analysis, LA-N-5 neuroblastoma cells were injected into mice bone marrow cavity to establish the bone marrow and bone metastasis xenograft models. Then the folate-nanoliposome entrapped MYCN siRNA with Cy3 tag (3 mg/kg) was injected into mice via lateral tail vein, and mice were sacrificed 8 hours after injection. Tumors of femur and other indicated organs (heart, lung, liver, kidney) were harvested, embedded in optimum cutting temperature compound (OCT) and sectioned into 4 m frozen sections. Tissues sections were observed under fluorescence microscopy (stimulating wavelength, 554 nm). Three views of each organ were chosen randomly and subjected to calculate the average integrated optical density (IOD) with Image pro plus 6.0 Software. In the gene therapy experiment, mice were divided into two groups (6 mice for each group). LA-N-5 neuroblastoma cells were injected into mice bone marrow cavity to establish the bone marrow and bone metastasis xenograft model. Then the folatenanoliposome entrapped MYCN siRNA with Cy3 tag (3 mg/kg/d) were injected into one group of mice by lateral tail vein. The other group of mice were injected with folate-nanoliposome entrapped the control siRNA (3 mg/kg/d). Mice were sacrificed at the sixth day after 5 injections of folate-nanoliposome entrapped siRNAs. Tumors frozen sections were analyzed by TUNEL Assay. Quantitative RT-PCR analysis The cells were harvested and RNA was extracte (...truncated)