| Melanoma is the deadliest form of skin cancer with a very poor prognosis and a high mortality rate.Small interfering RNA(siRNA)has become a promising clinical therapy as it can silence the expression of pathogenic genes without affecting the expression of genes in healthy cells.Due to the its large molecular weight,strong water solubility and negative charge characteristics,there are obstacles on siRNA delivery in system circulation,tissue penetration,cell absorption,and endosome escape.How to deliver siRNA safely and effectively into target cells to exert the effect of RNA interference is currently the biggest challenge facing the clinical application of siRNA drugs.In this study,a multifunctional Wulff-type phenylboronic acid(PBA)derivative was designed as a siRNA delivery carrier by introducing phenylboronic acid functional groups into low molecular weight chitooligosaccharide(COS)with good biocompatibility.Using the property that phenylboronic acid can form a reversible phenylborate bond with cis-diol-containing compounds spontaneously,the synthetized PBA20%-COS3000 could effectively condense siRNA through a synergetic assembly of electrostatic complexation and chemical cross-linking.PBA and siRNA chemically bonded to form a phenylborate bond with a pH-sensitive property.Consequently,the phenylborate bonds in nanoparticles could be broken in the weakly acidic microenvironments of tumor tissues and cells,which can promote the release of siRNA in tumor cells.In this study,a chitooligosaccharide polymer modified with phenylboronic acid was successfully synthesized and the structure of the polymer was confirmed and verified by 1H-NMR and FTIR spectra.The degree of substitution of phenylboronic acid was determined.Three chitooligosaccharide derivatives with different PBA substitution degree were synthesized and chitooligosaccharide polymers with substitution degree of 20%with high transfection efficiency were optimized by endogenous Luciferase reporter gene model.The CCK-8 method examined the cytotoxicity of the polymers.The results showed that the biocompatibility was good at the concentration range of 5-500 μg/mL.Subsequently,siRNA-loaded nanoparticles were prepared by self-assembly method and the nanoparticle size and zeta potential were investigated.Eventually,the PBA20%-COS3000/siRNA at a w/w ratio of 90 was selected.The average size was 98.45±1.15 nm(polydispersity,PDI=0.148±0.01),and the zeta potential was+26.2±1.76 mV.The stability of nanoparticles was good.The pH sensitivity of the phenylborate bond between siRNA and PBA was investigated.As a result,as the pH decreased,part of the phenylborate bond dissociated,making the particle size and PDI gradually increased and zeta potential rise.The endogenous Luciferase reporter model was used to examine the effects of transfection medium,time and siRNA dose on the efficiency of gene silencing.The results showed that serum-free medium,4 h transfection,and 100 nM dose were suitable transfection conditions.Subsequently,using Cy5-labeled siRNA,the cellular uptake of nanoparticles in B 16F10 cells was studied.The Survivin-targeted siRNA was used to prepare nanoparticles to investigate their anti-cells.proliferation ability,ability to induce apoptosis.It was found that the nanoparticles prepared by PBA20%-COS3000/siRNA exhibited significantly improved cell uptake efficiency,anti-cell proliferation inhibitory ability,and ability to induce apoptosis compared with the use of COS3000 alone to deliver siRNA.There is no obvious difference between PBA20%-COS3000/siRNA and Lip2000/siRNA.Next,a mouse B16F10 subcutaneous melanoma model was constructed in vivo,the anti-melanoma growth and metastasis ability of PBA20%-COS3000/siRNA nanoparticles were investigated,and the apoptosis of tumor tissue and tumor metastasis of the liver and lung tissues was investigated by TUNEL staining and H&E staining method,respectively.At the same time,the safety of nano-preparations was investigated in vivo.The results showed that PBA20%-COS3000/siRNA nanoparticles had good biocompatibility in vivo.The novel nanoparticles remarkably inhibited the growth and metastasis of melanoma in vivo.TUNEL staining showed that the mechanism of nanoparticles inhibiting tumor growth was achieved by inducing apoptosis of tumor cells.This study is of great significance to the research of low molecular weight COS as siRNA delivery vectors,which can solve the low efficiency of low molecular weight chitooligosaccharide,and also provides a theoretical basis for the development of multifunctional chitooligosaccharide delivery systems in the field of pharmacy and method reference. |
