RESUMEN
Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.
Asunto(s)
Saponinas , Animales , Humanos , Ratones , Saponinas/química , Saponinas/farmacología , Línea Celular Tumoral , Técnicas de Transferencia de Gen , Péptidos/química , Transfección/métodos , Saponaria/química , Saporinas/química , Saporinas/farmacología , Terapia Genética , Supervivencia Celular/efectos de los fármacos , Cationes/químicaRESUMEN
Saponin-mediated endosomal escape is a mechanism that increases the cytotoxicity of type I ribosome-inactivating proteins (type I RIPs). In order to actualize their cytotoxicity, type I RIPs must be released into the cytosol after endocytosis. Without release from the endosomes, type I RIPs are largely degraded and cannot exert their cytotoxic effects. Certain triterpene saponins are able to induce the endosomal escape of these type I RIPs, thus increasing their cytotoxicity. However, the molecular mechanism underlying the endosomal escape enhancement of type I RIPs by triterpene saponins has not been fully elucidated. In this report, we investigate the involvement of the basic amino acid residues of dianthin-30, a type I RIP isolated from the plant Dianthus caryophyllus L., in endosomal escape enhancement using alanine scanning. Therefore, we designed 19 alanine mutants of dianthin-30. Each mutant was combined with SO1861, a triterpene saponin isolated from the roots of Saponaria officinalis L., and subjected to a cytotoxicity screening in Neuro-2A cells. Cytotoxic screening revealed that dianthin-30 mutants with lysine substitutions did not impair the endosomal escape enhancement. There was one particular mutant dianthin, Arg24Ala, that exhibited significantly reduced synergistic cytotoxicity in three mammalian cell lines. However, this reduction was not based on an altered interaction with SO1861. It was, rather, due to the impaired endocytosis of dianthin Arg24Ala into the cells.
Asunto(s)
Endocitosis , Saponinas , Animales , Ratones , Saponinas/metabolismo , Arginina , Endosomas/metabolismo , Línea Celular Tumoral , Mutación , Análisis Mutacional de ADN , Supervivencia Celular/efectos de los fármacosRESUMEN
The Chinese yam (Dioscorea polystachya, DP) is known for the nutritional value of its tuber. Nevertheless, DP also has promising pharmacological properties. Compared with the tuber, the leaves of DP are still very little studied. However, it may be possible to draw conclusions about the plant quality based on the coloration of the leaves. Magnesium, as a component of chlorophyll, seems to play a role. Therefore, the aim of this research work was to develop an atomic absorption spectrometry-based method for the analysis of magnesium (285.2125 nm) in leaf extracts of DP following the graphite furnace sub-technique. The optimization of the pyrolysis and atomization temperatures resulted in 1500 °C and 1800 °C, respectively. The general presence of flavonoids in the extracts was detected and could explain the high pyrolysis temperature due to the potential complexation of magnesium. The elaborated method had linearity in a range of 1-10 µg L-1 (R2 = 0.9975). The limits of detection and quantification amounted to 0.23 µg L-1 and 2.00 µg L-1, respectively. The characteristic mass was 0.027 pg, and the recovery was 96.7-102.0%. Finally, the method was applied to extracts prepared from differently colored leaves of DP. Similar magnesium contents were obtained for extracts made of dried and fresh leaves. It is often assumed that the yellowing of the leaves is associated with reduced magnesium content. However, the results indicated that yellow leaves are not due to lower magnesium levels. This stimulates the future analysis of DP leaves considering other essential minerals such as molybdenum or manganese.