Construction of Ang2-siRNA chitosan magnetic nanoparticles and the effect on Ang2 gene expression in human malignant melanoma cells

Abstract

Abstract. The aim of the present study was to construct angiopoietin-2 (Ang2)-small interfering (si)RNA chitosan magnetic nanoparticles and to observe the interference effects of the nanoparticles on the expression of the Ang2 gene in human malignant melanoma cells. Ang2-siRNA chitosan magnetic nanoparticles were constructed and transfected into human malignant melanoma cells in vitro. Red fluorescent protein expression was observed, and the transfection efficiency was analyzed. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to assess the inhibition efficiency of Ang2 gene expression. Ang2-siRNA chitosan magnetic nanoparticles were successfully constructed, and at a mass ratio of plasmid to magnetic chitosan nanoparticles of 1:100, the transfection efficiency into human malignant melanoma cells was the highest of the ratios assessed, reaching 61.17%. RT-qPCR analysis showed that the magnetic chitosan nanoparticles effectively inhibited Ang2 gene expression in cells, and the inhibition efficiency reached 59.56% (P<0.05). Ang2-siRNA chitosan magnetic nanoparticles were successfully constructed. The in vitro studies showed that the nanoparticles inhibited Ang2 gene expression in human malignant melanoma tumor cells, which laid the foundation and provided experimental evidence for additional future in vivo studies of intervention targeting malignant melanoma tumor growth in nude mice. IntroductionThe overall incidence of cancer is continually increasing, with an average annual incidence rate of 3-5% (1). Melanoma is a human malignancy characterized by invasion and metastasis, in which the cells are malignantly transformed from melanocytes. As the melanoma demonstrates rapid progression, the prognosis is poor. There is no effective treatment for melanoma at present, with no progress made over the last 13 years (2,3). The angiopoietin (Ang)-endothelial-specific receptor tyrosine kinase 2 (Tie2) system is widely expressed in human tumor vasculature remodeling, but no notable expression occurs in normal tissues, which results in the Ang-Tie2 system being an attractive potential target for anti-angiogenic cancer therapy (4). Ang is expressed at a high level in malignant melanoma cells compared with normal tissues (5). Thus, it is possible to inhibit angiogenesis by blocking Ang expression and to inhibit the growth of malignant melanoma, therefore achieving a therapy for malignant melanoma (6).Preliminary findings indicated that RNA interference (RNAi) technology may silence Ang2 gene expression, thereby inhibiting malignant melanoma xenograft angiogenesis and tumor growth (7-11). However, in the preliminary studies (8,10), a local intratumoral injection was used, which is not suitable for routine clinical use, but the intravenous injection of lentiviral vectors has safety and efficacy issues that affected the feasibility and significance of its use in additional studies. Based on the large specific surface area of nanoscale genic carriers compared with vector, target cell surface ligand o...

Targeting of angiopoietin 2-small interfering RNA plasmid/chitosan magnetic nanoparticles in a mouse model of malignant melanoma in vivo

“…The particles resist the permeability reduction of nanoparticles induced by the increased tumor interstitial pressure when particle size is small, thereby increasing the targeting of nanoparticles ( 26 ). The average particle size of Ang2-siRNA plasmid vector/chitosan magnetic nanoparticles prepared by the present study was 67 nm ( 9 ). Therefore, they were used as a suitable targeting drug delivery system.…”

“…The formed magnetic nanoparticles would be able to accumulate directly towards the target organs under external magnetic field, thus serving its roles ( 7 , 8 ). Using this technique, our previous in vitro experiments confirmed that angiopoietin 2-small interfering RNA (Ang2-siRNA) chitosan magnetic nanoparticles could inhibit the expression of Ang2 gene in human malignant melanoma (MM) cells, and the inhibition efficiency was 59.56% ( 9 ). In the present study, Ang2-siRNA plasmid/chitosan magnetic nanoparticles were injected into the nude mouse MM model to observe the targeting characteristic of these particles under external magnetic field, in order to determine certain foundations for further in vivo targeting intervention studies investigating the tumor growth in MM-transplanted nude mice.…”

<p>Effect of Chitosan Magnetic Nanoparticles Loaded with Ang2-siRNA Plasmids on the Growth of Melanoma Xenografts in Nude Mice</p>

“…The Ang2-siRNA plasmid particles were encapsulated by chitosan, which does not increase systemic toxicity and is stable in vivo. 27 Additionally, the combination of the Ang2-siRNA system and the magnetic targeted drug delivery system can improve the function of siRNAs in vivo. Moreover, when the siRNA nucleic acid transfer system is used in combination with the chitosan magnetic nanoparticles, the serum pharmacokinetics of the targeted drug delivery system and the half-life of the composite nanoparticles can prolong the siRNA interference effect, which may result in lasting RNA interference effects and enhance the efficacy of gene interference treatment.…”

Manipulation of immune‒vascular crosstalk: new strategies towards cancer treatment

“…Here we reviewed the strategies to enhance delivery efficiency and efficacy of nanomedicine by focusing on normalization of vasculature and the enhancement of extravasation of nanomedicine into tumors. By presenting insights into the basics of nanomedicine–vasculature interactions in tumors, we hope it would provide novel strategies to engineer cancer nanomedicine to accelerate their translation and fulfill the promises of this field ( Table 2 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 ).…”

“… Target/strategy Cargo/engineered nanoparticle Ref. Nanomedicine to normalize tumor vasculature Angiogenesis signalling pathway VEGFA-VEGFR2 Knockdown of VEGFA by: siRNA/PLCP 94 siRNA/PEI-SWNTs 95 shRNA/dtACPP-modified PEG-DGL 96 Inhibition of VEGFR2 by: Antibody/MSV 97 Ang2 Knockdown of Ang2 by: siRNA/chitosan magnetic nanoparticles 98 Blockage of Ang2-Tie2 interaction by: T4 peptide (K(DEAP)-AAN-NLLMAAS)/PEG 99 PDGF PDGF/PLGA–pSi-ES 100 Immune-associated cell populations and signalling: TAMs Targeting TAMs-specific ligands by: MTX/FOLR2 conjugated G5-dendrimer 101 HA/Mannose receptor conjugated MnO 2 NPs 102 siVEGF/M2pep conjugated AuNPs 103 Enhance extravasation of nanomedicine into tumor Transcytosis-based delivery Adsorptive-mediated transcytosis (AMT) CPT/PBEAGA zwitterionic polymer 104 Receptor-mediated transcytosis (RMT) Targeting iRGD/integrins α v by: siPLK1 and miR-200c/iRGD conjugated MSN 105 CA4 and DOX/iRGD conjugated MSN 106 Targeting GD16/Dll4 by: PTX/GD16 conjugated aldehyde-PEG-PLA and MPEG-PLA block copolymers 107 Immune cell-based delivery PTX/cationic liposomes (PTX-CL)-NEs to form PTX-CL/NEs …”