Development and Application of an Atomic Absorption Spectrometry-Based Method to Quantify Magnesium in Leaves of Dioscorea polystachya.

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.

Correction: Panjideh et al. Improved Therapy of B-Cell Non-Hodgkin Lymphoma by Obinutuzumab-Dianthin Conjugates in Combination with the Endosomal Escape Enhancer SO1861. Toxins 2022, 14, 478.

The authors wish to make corrections to their paper [...].

Sapovaccarin-S1 and -S2, Two Type I RIP Isoforms from the Seeds of Saponaria vaccaria L.

Type I ribosome-inactivating proteins (RIPs) are plant toxins that inhibit protein synthesis by exerting rRNA N-glycosylase activity (EC 3.2.2.22). Due to the lack of a cell-binding domain, type I RIPs are not target cell-specific. However once linked to antibodies, so called immunotoxins, they are promising candidates for targeted anti-cancer therapy. In this study, sapovaccarin-S1 and -S2, two newly identified type I RIP isoforms differing in only one amino acid, were isolated from the seeds of Saponaria vaccaria L. Sapovaccarin-S1 and -S2 were purified using ammonium sulfate precipitation and subsequent cation exchange chromatography. The determined molecular masses of 28,763 Da and 28,793 Da are in the mass range typical for type I RIPs and the identified amino acid sequences are homologous to known type I RIPs such as dianthin 30 and saporin-S6 (79% sequence identity each). Sapovaccarin-S1 and -S2 showed adenine-releasing activity and induced cell death in Huh-7 cells. In comparison to other type I RIPs, sapovaccarin-S1 and -S2 exhibited a higher thermostability as shown by nano-differential scanning calorimetry. These results suggest that sapovaccarin-S1 and -S2 would be optimal candidates for targeted anti-cancer therapy.

Improved Therapy of B-Cell Non-Hodgkin Lymphoma by Obinutuzumab-Dianthin Conjugates in Combination with the Endosomal Escape Enhancer SO1861.

Immunotoxins do not only bind to cancer-specific receptors to mediate the elimination of tumor cells through the innate immune system, but also increase target cytotoxicity by the intrinsic toxin activity. The plant glycoside SO1861 was previously reported to enhance the endolysosomal escape of antibody-toxin conjugates in non-hematopoietic cells, thus increasing their cytotoxicity manifold. Here we tested this technology for the first time in a lymphoma in vivo model. First, the therapeutic CD20 antibody obinutuzumab was chemically conjugated to the ribosome-inactivating protein dianthin. The cytotoxicity of obinutuzumab-dianthin (ObiDi) was evaluated on human B-lymphocyte Burkitt's lymphoma Raji cells and compared to human T-cell leukemia off-target Jurkat cells. When tested in combination with SO1861, the cytotoxicity for target cells was 131-fold greater than for off-target cells. In vivo imaging in a xenograft model of B-cell lymphoma in mice revealed that ObiDi/SO1861 efficiently prevents tumor growth (51.4% response rate) compared to the monotherapy with ObiDi (25.9%) and non-conjugated obinutuzumab (20.7%). The reduction of tumor volume and overall survival was also improved. Taken together, our results substantially contribute to the development of a combination therapy with SO1861 as a platform technology to enhance the efficacy of therapeutic antibody-toxin conjugates in lymphoma and leukemia.

Construction of Minicircle Suicide Genes Coding for Ribosome-Inactivating Proteins.

Due to the lower risks of adverse effects, nonviral gene therapy is a suitable alternative to transfect cancer cells with a suicide gene to let them kill themselves by expressing toxic ribosome-inactivating proteins. Plasmids are stable and easy-to-produce vectors, but they have some disadvantages due to the bacterial backbone. Applying the minicircle technology, this problem can be solved with manageable effort in a well-equipped laboratory. With the described methodology, minicircle-DNA can be produced at low costs. The cell killing properties are monitored following transfection using the CytoSMART® Omni system-a camera based live cell imaging device.

Suicide nanoplasmids coding for ribosome-inactivating proteins.

Conventional eukaryotic expression plasmids contain a DNA backbone that is dispensable for the cellular expression of the transgene. In order to reduce the vector size, minicircle DNA technology was introduced. A drawback of the minicircle technology are considerable production costs. Nanoplasmids are a relatively new class of mini-DNA constructs that are of tremendous potential for pharmaceutical applications. In this study we have designed novel suicide nanoplasmid constructs coding for plant derived ribosome-inactivating proteins. The suicide-nanoplasmids were formulated with a targeted K16-lysine domain, analyzed for size, and characterized by electron microscopy. The anti-proliferative activity of the suicide-nanoplasmids was investigated in vitro by real time microscopy and the expression kinetic was determined using an enhanced green fluorescent protein nanoplasmid variant. In an aggressive in vivo neuroblastoma tumor model, treated mice showed a reduced tumor growth whereby the therapy was well tolerated.

An advanced method for the small-scale production of high-quality minicircle DNA.

Minicircle DNA is a promising tool in the field of gene therapy, whose products are increasingly gaining market access. Greater transfection efficiency and longer expression time as well as lower immunogenicity contrast with cost-intensive production, which also stands in the way of a broader use of the advantages of this technology in research. Starting from a commercial minicircle production kit a simple protocol for the cost-effective small-scale production of high-quality minicircle DNA to be used at a research scale has been developed by combining and improving procedures of various publications. An optimized size-exclusion chromatography method led to almost pure minicircle DNA with a superior proportion of the desired supercoiled plasmid conformation. The pharmaceutical potential of the produced minicircle DNA was investigated in vitro by real-time impedance assays in a tumor cell model in case of coded suicide genes as well as by ELISA of the translation product in case of coded human coagulation factor IX.

Plant Extracellular Vesicles and Nanovesicles: Focus on Secondary Metabolites, Proteins and Lipids with Perspectives on Their Potential and Sources.

While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-ß-glucosidases, pectinesterases, polygalacturonases, ß-galactosidases and ß-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles.

An unusual type I ribosome-inactivating protein from Agrostemma githago L.

Agrostemma githago L. (corn cockle) is an herbaceous plant mainly growing in Europe. The seeds of the corn cockle are toxic and poisonings were widespread in the past by consuming contaminated flour. The toxic principle of Agrostemma seeds was attributed to triterpenoid secondary metabolites. Indeed, this is in part true. However Agrostemma githago L. is also a producer of ribosome-inactivating proteins (RIPs). RIPs are N-glycosylases that inactivate the ribosomal RNA, a process leading to an irreversible inhibition of protein synthesis and subsequent cell death. A widely known RIP is ricin from Ricinus communis L., which was used as a bioweapon in the past. In this study we isolated agrostin, a 27 kDa RIP from the seeds of Agrostemma githago L., and determined its full sequence. The toxicity of native agrostin was investigated by impedance-based live cell imaging. By RNAseq we identified 7 additional RIPs (agrostins) in the transcriptome of the corn cockle. Agrostin was recombinantly expressed in E. coli and characterized by MALDI-TOF-MS and adenine releasing assay. This study provides for the first time a comprehensive analysis of ribosome-inactivating proteins in the corn cockle and complements the current knowledge about the toxic principles of the plant.

A new acetylated triterpene saponin from Agrostemma githago L. modulates gene delivery efficiently and shows a high cellular tolerance.

Transfection is the process to deliver nucleic acid into eukaryotic cells. Different transfection techniques already exist. However, they can be expensive and toxic toward subjected cells. Previous research shed light on natural occurring molecules called triterpene saponins that have great potential for the non-viral gene delivery. Using a combination of different chromatographic techniques and in vitro transfection bioassays, a new triterpenoid saponin (agrostemmoside E) from the plant Agrostemma githago L. was isolated. Agrostemmoside E was characterized by mass spectrometry, intense NMR spectroscopy and was identified as 3-{O-ß-D-Galactopyranosyl-(1→2)]-[ß-D-xylopyranosyl-(1→3)]-ß-D-glucuronopyranosyl} quillaic acid 28-O-{[ß-D-4,6-di-(O-acetyl)-glucopyranosyl-(1→3)]-[ß-D-xylopyranosyl-(1→4)]-α-L-rhamnopyranosyl-(1→2)}-[3,4-di-(O-acetyl)-ß-D-quinovopyranosyl-(1→4)]-ß-D-fucopyranoside ester. Agrostemmoside E has a great potential for delivery of gene loaded nanoplexes and increased the transfection efficiency by 70% compared to 2% without agrostemmoside E. By comparative toxicity studies, we show that agrostemmoside E can be applied at high concentrations without toxicity, justifying its use as a new tool for gene transfections.

Structure-Activity Relationship of Transfection-Modulating Saponins - A Pursuit for the Optimal Gene Trafficker.

The ability of certain triterpenoid saponins to modulate the endosomal release during the process of endocytosis and to ensure a nontoxic and efficient transfection recently led to an exceptional interest in the field of nonviral gene delivery. In vitro and in vivo studies demonstrated promising results in terms of tumor growth inhibition after the delivery of a suicide gene such as saporin and dianthin. With that, the question arises which structural features are necessary or advantageous to achieve an effective endosomal escape. Former studies described certain important characteristics a potent saponin should have. Particularly SA1641 (Gypsophila paniculata) and SO1861 (Saponaria officinalis) played an utmost important role to get a first insight into the structure-activity relationship. However, a number of issues such as the purpose of functional groups on the aglycon and the substitution of sugars and their modification remain unsolved and their value needs to be specified. By conducting a screening of several diverse saponins in terms of their transfection improving ability, we aimed to examine these questions in more detail and get a better understanding of the relevant features. The transfection of Neuro-2A-cells with GFP-DNA containing peptide-based nanoplexes provided a reliable method in order to compare the activity of the saponins. With that, we were able to provide new and essential insights regarding the structure-activity relationship of transfection-modulating saponins and give an idea of how a highly potent saponin for future gene therapies may look like.

A new type 1 ribosome-inactivating protein from the seeds of Gypsophila elegans M.Bieb.

Ribosome-inactivating proteins (RIPs) are enzymes with N-glycosylase activity that remove adenine bases from the ribosomal RNA. In theory, one single RIP molecule internalized into a cell is sufficient to induce cell death. For this reason, RIPs are of high potential as toxic payload for anti-tumor therapy. A considerable number of RIPs are synthesized by plants that belong to the carnation family (Caryophyllaceae). Prominent examples are the RIPs saporin from Saponaria officinalis L. or dianthin from Dianthus caryophyllus L. In this study, we have isolated and characterized a novel RIP (termed gypsophilin-S) from the tiny seeds of Gypsophila elegans M. Bieb. (Caryophyllaceae). It is noteworthy that this is the first study presenting the complete amino acid sequence of a RIP from a Gypsophila species. Gypsophilin-S was isolated from the defatted seed material following ammonium sulphate precipitation and HPLC-based ion exchange chromatography. Gypsophilin-S-containing fractions were analysed by SDS-PAGE and mass spectrometry. The full amino acid sequence of gypsophilin-S was assembled by MALDI-TOF-MS-MS and PCR. Gypsophilin-S exhibited strong adenine releasing activity and its cytotoxicity in human glioblastoma cells was investigated using an impedance-based real-time assay in comparison with recombinant saporin and dianthin.

Plant derived triterpenes from Gypsophila elegans M.Bieb. enable non-toxic delivery of gene loaded nanoplexes.

To this date, a number of different Gypsophila species from the family of Caryophyllaceae were phytochemically characterized and tested for diverse pharmacological effects. With Gypsophila elegans M. Bieb., we investigated a scarcely explored Gypsophila species, providing a number of potential transfection enhancing triterpene saponins, and so-called sapofection agents. So far triterpene saponins have not been isolated in Gypsophila elegans M.Bieb. Crude extracts from roots and seeds, as well as each purification step were tested for delivery modulation of gene-loaded nanoplexes into neuroblastoma cells. The application of the bioassay guided isolation strategy enabled the assessment of the most active Gypsophila compound, the bisdesmosidic triterpene saponin gypsophilosid A. Gypsophilosid A was isolated by chromatographic techniques, and characterized by electrospray mass spectrometry and intense NMR-spectroscopy, using a variety of 1D and 2D-NMR experiments such as HSQC, HMBC, HQQC, TOCSY and NOESY. In neuroblastoma cells, gypsophilosid A increased the transfection efficiency of gene-nanoplexes up to 80% compared to 2% in the control group without saponin. Our results proved the successful applicability of the implemented methods to detect, isolate and identify saponins, which are biochemically active in terms of transfection.

Targeted suicide gene transfections reveal promising results in nu/nu mice with aggressive neuroblastoma.

Neuroblastoma represents the third most common malign neoplasm occurring in children and the most common in newborn. Although mortality in childhood cancer declined in the last decade, high-risk patients have poor prospects, due to the aggressiveness of the cancer. In the recent past, we underlined the potential of sapofectosid as novel and efficient transfection enhancer, demonstrating non-toxic gene delivery, but its value in tumor therapies has yet to be elucidated. A suicide gene, coding for saporin, a ribosome-inactivating protein type I, was incorporated into targeted, peptide-based nanoplexes. The nanoplexes were characterized for their size, zeta potential and appearance by electron microscopy. Gene delivery was observed via confocal imaging. In vitro transfections were conducted to monitor the real-time cell viability. After initial tolerability studies, NMRI nu/nu-mice bearing tumors from Neuro-2A-Luc-cells (murine neuroblastoma cells, transduced with a luciferase gene), were treated with targeted nanoplexes (30 µg saporin-DNA i.v./treatment) and sapofectosid (30 µg s.c. treatment). The treatment was compared to a vehicle (PBS) control and treatment without sapofectosid in terms of body weight, tumor growth and integrated density of tumor luminescence. The study revealed an anti-tumoral effect of the sapofectosid mediated gene therapy in the Neuro-2A-tumor model. The treatments were well tolerated by the animals indicating the applicability of this approach. With these results, we were able to proof the efficacy of a therapy, consisting of targeted suicide gene nanoplexes and sapofectosid, a novel and potent transfection enhancer. This study points out the enormous value for future targeted cancer and gene therapies.

A novel adenine-releasing assay for ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) are toxic enzymes that are mostly biosynthesized by plants. RIPs are N-glycosidases that cleave an essential adenine molecule from the 28S rRNA. This is followed by the irreversible inhibition of protein synthesis leading to cell death. By fusing RIPs to cancer cell specific targeting ligands RIPs have been utilized for targeted anti-tumor therapy. The anti-tumoral efficiency of such conjugates depends significantly on the N-glycosidase activity of the RIP domain. Different methods have been developed in order to determine the N-glycosidase activity of RIPs and RIP domain containing anti-tumor toxins. However the existing methods are elaborate and include radioassays, HPLC and enzymatic conversion assays. Here, a simple and cost effective N-glycosidase assay is presented, which is based on the direct determination of the released adenine by thin-layer chromatography (TLC) and TLC-densitometry. An adenine based single stranded oligonucleotide is used as substrate. Following TLC development the released adenine is quantified on silica glass plates by UV absorbance at 260nm.

Sapofectosid - Ensuring non-toxic and effective DNA and RNA delivery.

Different methods are being deployed for non-viral DNA/RNA delivery. However non-viral formulations for DNA/RNA-delivery are often accompanied by severe toxicity and thus low efficiency. Particular costly cell culture media are required as well. Here we introduce sapofection as a valuable enhancing method for non-viral DNA/RNA delivery. Sapofection is based on the application of DNA/RNA nanoplexes and sapofectosid, a plant derived natural transfection reagent. Sapofectosid was produced from plant raw material by chromatographic methods and characterized by tandem mass spectrometry and intensive one and two dimensional NMR-spectroscopy. Sapofectosid did enhance the transfection efficiency of different DNA- and RNA-nanoplexes formulated with liposomes, polyethylenimine (PEI) or targeted and non-targeted oligo-lysine peptides. All nanoplexes were characterized physicochemically and the influence of sapofectosid on the nanoplex integrity was determined by DNA complexation assays. The nanoplexes and sapofectosid were administered to a variety of cancer cell lines and the transfection efficiency was investigated by flow cytometry and confocal microscopy. Dependent on the cell line the transfection efficiencies varied from 6 to 76%. The saponin- and receptor-mediated endocytosis of nanoplexes was investigated by flow cytometry. As demonstrated by impedance based live cell imaging sapofection was non-toxic. The findings show the great potential of sapofection to be used as an effective and non-toxic transfection enhancing method.

Targeted dianthin is a powerful toxin to treat pancreatic carcinoma when applied in combination with the glycosylated triterpene SO1861.

Targeted cancer therapy provides the basis for the arrest of tumor growth in aggressive pancreatic carcinoma; however, a number of protein-based targeted toxins lack efficacy due to insufficient endosomal escape after being endocytosed. Therefore, we tested a fusion protein of the ribosome-inactivating protein dianthin and human epidermal growth factor in combination with a glycosylated triterpene (SO1861) that serves as an endosomal escape enhancer. In vitro investigations with the pancreatic carcinoma cell lines BxPC-3 and MIA PaCa-2 revealed no significant differences to off-target cells in the half maximal inhibitory concentration (IC50 ) for the fusion protein. In contrast, combination with SO1861 decreased the IC50 for BxPC-3 cells from 100 to 0.17 nm, whereas control cells remained unaffected. Monotherapy of BxPC-3 xenografts in CD-1 nude mice led to a 51.7% average reduction in tumor size (40.8 mm3 ) when compared to placebo; however, combined treatment with SO1861 resulted in a more than 13-fold better efficacy (3.0 mm3 average tumor size) with complete regression in 80% of cases. Immunohistochemical analyses showed that tumor cells with lower target receptor expression are, in contrast to the combination therapy, able to escape from the monotherapy, which finally results in tumor growth. At the effective concentration, we did not observe liver toxicity and saw no other side effects with the exception of a reversible skin hardening at the SO1861 injection site, alongside an increase in platelet counts, plateletcrit, and platelet distribution width. In conclusion, combining a targeted toxin with SO1861 is proven to be a very promising approach for pancreatic cancer treatment.

Glycosylated Triterpenoids as Endosomal Escape Enhancers in Targeted Tumor Therapies.

Protein-based targeted toxins play an increasingly important role in targeted tumor therapies. In spite of their high intrinsic toxicity, their efficacy in animal models is low. A major reason for this is the limited entry of the toxin into the cytosol of the target cell, which is required to mediate the fatal effect. Target receptor bound and internalized toxins are mostly either recycled back to the cell surface or lysosomally degraded. This might explain why no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date although more than 500 targeted toxins have been developed within the last decades. To overcome the problem of insufficient endosomal escape, a number of strategies that make use of diverse chemicals, cell-penetrating or fusogenic peptides, and light-induced techniques were designed to weaken the membrane integrity of endosomes. This review focuses on glycosylated triterpenoids as endosomal escape enhancers and throws light on their structure, the mechanism of action, and on their efficacy in cell culture and animal models. Obstacles, challenges, opportunities, and future prospects are discussed.

Dianthin-30 or gelonin versus monomethyl auristatin E, each configured with an anti-calcitonin receptor antibody, are differentially potent in vitro in high-grade glioma cell lines derived from glioblastoma.

We have reported that calcitonin receptor (CTR) is widely expressed in biopsies from the lethal brain tumour glioblastoma by malignant glioma and brain tumour-initiating cells (glioma stem cells) using anti-human CTR antibodies. A monoclonal antibody against an epitope within the extracellular domain of CTR was raised (mAb2C4) and chemically conjugated to either plant ribosome-inactivating proteins (RIPs) dianthin-30 or gelonin, or the drug monomethyl auristatin E (MMAE), and purified. In the high-grade glioma cell line (HGG, representing glioma stem cells) SB2b, in the presence of the triterpene glycoside SO1861, the EC50 for mAb2C4:dianthin was 10.0 pM and for mAb2C4:MMAE [antibody drug conjugate (ADC)] 2.5 nM, 250-fold less potent. With the cell line U87MG, in the presence of SO1861, the EC50 for mAb2C4:dianthin was 20 pM, mAb2C4:gelonin, 20 pM, compared to the ADC (6.3 nM), which is >300 less potent. Several other HGG cell lines that express CTR were tested and the efficacies of mAb2C4:RIP (dianthin or gelonin) were similar. Co-administration of the enhancer SO1861 purified from plants enhances lysosomal escape. Enhancement with SO1861 increased potency of the immunotoxin (>3 log values) compared to the ADC (1 log). The uptake of antibody was demonstrated with the fluorescent conjugate mAb2C4:Alexa Fluor 568, and the release of dianthin-30:Alexa Fluor488 into the cytosol following addition of SO1861 supports our model. These data demonstrate that the immunotoxins are highly potent and that CTR is an effective target expressed by a large proportion of HGG cell lines representative of glioma stem cells and isolated from individual patients.

Delivery of ENaC siRNA to epithelial cells mediated by a targeted nanocomplex: a therapeutic strategy for cystic fibrosis.

The inhibition of ENaC may have therapeutic potential in CF airways by reducing sodium hyperabsorption, restoring lung epithelial surface fluid levels, airway hydration and mucociliary function. The challenge has been to deliver siRNA to the lung with sufficient efficacy for a sustained therapeutic effect. We have developed a self-assembling nanocomplex formulation for siRNA delivery to the airways that consists of a liposome (DOTMA/DOPE; L), an epithelial targeting peptide (P) and siRNA (R). LPR formulations were assessed for their ability to silence expression of the transcript of the gene encoding the α-subunit of the sodium channel ENaC in cell lines and primary epithelial cells, in submerged cultures or grown in air-liquid interface conditions. LPRs, containing 50 nM or 100 nM siRNA, showed high levels of silencing, particularly in primary airway epithelial cells. When nebulised these nanocomplexes still retained their biophysical properties and transfection efficiencies. The silencing ability was determined at protein level by confocal microscopy and western blotting. In vivo data demonstrated that these nanoparticles had the ability to silence expression of the α-ENaC subunit gene. In conclusion, these findings show that LPRs can modulate the activity of ENaC and this approach might be promising as co-adjuvant therapy for cystic fibrosis.