Entropy-Driven Quick Loading of Functional Proteins in Nanohydrogels for Highly Efficient Tumor Targeting Therapy.

With the gradual deep understanding of the tumorigenesis and development process, nanodrug are thought to have great prospects for individualized treatment of tumors. To deliver adequate concentration of active ingredients to targeted tissues, proteins are usually used as carriers to avoid clearance by the immune system. Herein, a new strategy is developed for preparation of the protein-functionalized targeting nanodrugs; different kinds of proteins (albumin, horseradish, transferrin, and ricin) can be quickly loaded in polyacrylic acid nanohydrogels (PAA-NGs) without discrimination within 1 min under the strong driving force of entropy; and the loading efficiency can reach 99% with about 50% loading content. Meanwhile, the activity of the released protein can be well retained. After oriented binding of the targeting agent on the surface of the nanocarriers by a unique and facile technique, the protein-loaded nanodrug exhibits excellent tumor cell uptake and targeting effect. The excellent targeting ability from the oriented binding is further proved by comparing with the non-oriented targeting system. With quick loading of the anti-tumor protein of ricin and oriented binding of transferrin protein (Tf), the targeting nanodrug (PAA-BB@Ricin/Tf) shows a remarkable anti-tumor effect. This study proves a new universal delivery and targeting strategy for improving the nanodelivery system, which has great potentials for clinical application.

The low density receptor-related protein 1 plays a significant role in ricin-mediated intoxication of lung cells.

Ricin, a highly lethal plant-derived toxin, is a potential biological threat agent due to its high availability, ease of production and the lack of approved medical countermeasures for post-exposure treatment. To date, no specific ricin receptors were identified. Here we show for the first time, that the low density lipoprotein receptor-related protein-1 (LRP1) is a major target molecule for binding of ricin. Pretreating HEK293 acetylcholinesterase-producer cells with either anti-LRP1 antibodies or with Receptor-Associated Protein (a natural LRP1 antagonist), or using siRNA to knock-down LRP1 expression resulted in a marked reduction in their sensitivity towards ricin. Binding assays further demonstrated that ricin bound exclusively to the cluster II binding domain of LRP1, via the ricin B subunit. Ricin binding to the cluster II binding domain of LRP1 was significantly reduced by an anti-ricin monoclonal antibody, which confers high-level protection to ricin pulmonary-exposed mice. Finally, we tested the contribution of LRP1 receptor to ricin intoxication of lung cells derived from mice. Treating these cells with anti-LRP1 antibody prior to ricin exposure, prevented their intoxication. Taken together, our findings clearly demonstrate that the LRP1 receptor plays an important role in ricin-induced pulmonary intoxications.

Production, Characterisation and Testing of an Ovine Antitoxin against Ricin; Efficacy, Potency and Mechanisms of Action.

Ricin is a type II ribosome-inactivating toxin that catalytically inactivates ribosomes ultimately leading to cell death. The toxicity of ricin along with the prevalence of castor beans (its natural source) has led to its increased notoriety and incidences of nefarious use. Despite these concerns, there are no licensed therapies available for treating ricin intoxication. Here, we describe the development of a F(ab')2 polyclonal ovine antitoxin against ricin and demonstrate the efficacy of a single, post-exposure, administration in an in vivo murine model of intoxication against aerosolised ricin. We found that a single dose of antitoxin afforded a wide window of opportunity for effective treatment with 100% protection observed in mice challenged with aerosolised ricin when given 24 h after exposure to the toxin and 75% protection when given at 30 h. Treated mice had reduced weight loss and clinical signs of intoxication compared to the untreated control group. Finally, using imaging flow cytometry, it was found that both cellular uptake and intracellular trafficking of ricin toxin to the Golgi apparatus was reduced in the presence of the antitoxin suggesting both actions can contribute to the therapeutic mechanism of a polyclonal antitoxin. Collectively, the research highlights the significant potential of the ovine F(ab')2 antitoxin as a treatment for ricin intoxication.

Fate and action of ricin in rat liver in vivo: translocation of endocytosed ricin into cytosol and induction of intrinsic apoptosis by ricin B-chain.

Cytotoxicity of many plant and bacterial toxins requires their endocytosis and retrograde transport from endosomes to the endoplasmic reticulum. Using cell fractionation and immunoblotting procedures, we have assessed the fate and action of the plant toxin ricin in rat liver in vivo, focusing on endosome-associated events and induction of apoptosis. Injected ricin rapidly accumulated in endosomes as an intact A/B heterodimer (5-90 min) and was later (15-90 min) partially translocated to cytosol as A- and B-chains. Unlike cholera and diphtheria toxins, which also undergo endocytosis in liver, neither in cell-free endosomes loaded by ricin in vivo nor upon incubation with endosomal lysates did ricin undergo degradation in vitro. A time-dependent translocation of ricin across the endosomal membrane occurred in cell-free endosomes. Endosome-located thioredoxin reductase-1 was required for translocation as shown by its physical association with ricin chains and effects of its removal and inhibition. Ricin induced in vivo intrinsic apoptosis as judged by increased cytochrome c content, activation of caspase-9 and caspase-3, and enrichment of DNA fragments in cytosol. Furthermore, reduced ricin and ricin B-chain caused cytochrome c release from mitochondria in vivo and in vitro, suggesting that the interaction of ricin B-chain with mitochondria is involved in ricin-induced apoptosis.

Toxin effect on protein biosynthesis in eukaryotic cells: a simple kinetic model.

A model for toxin inhibition of protein synthesis inside eukaryotic cells is presented. Mitigation of this effect by introduction of an antibody is also studied. Antibody and toxin (ricin) initially are delivered outside the cell. The model describes toxin internalization from the extracellular into the intracellular domain, its transport to the endoplasmic reticulum (ER) and the cleavage inside the ER into the RTA and RTB chains, the release of RTA into the cytosol, inactivation (depurination) of ribosomes, and the effect on translation. The model consists of a set of ODEs which are solved numerically. Numerical results are illustrated by figures and discussed.

Serial ricinine levels in serum and urine after ricin intoxication.

Ricinine is an alkaloid present in the castor bean plant (Ricinus communis) that can be used as a biomarker for ricin poisoning. Serial ricinine levels are reported in the serum and urine of a patient suffering from intentional ricin intoxication. The patient was brought to the hospital 4 h after injection and oral intake of a castor bean extract, but died 38 h later, despite intensive medical care. Ricinine was isolated from the samples by solid-phase extraction and quantitatively determined by isotopic dilution liquid chromatography-mass spectrometry. The ricinine level in serum declined from 33 to 23 ng/mL between 10 and 29 h post-exposure. Three urine samples collected from 12 to 41 h after ricin intoxication showed ricinine concentrations in the range of 20-58 ng/mL. The creatinine corrected values (21-30 µg/g) indicated a concentration-time profile with a maximum ricinine level in urine between 12 and 29 h after exposure.

Inhibitors of the cellular trafficking of ricin.

Throughout the last decade, efforts to identify and develop effective inhibitors of the ricin toxin have focused on targeting its N-glycosidase activity. Alternatively, molecules disrupting intracellular trafficking have been shown to block ricin toxicity. Several research teams have recently developed high-throughput phenotypic screens for small molecules acting on the intracellular targets required for entry of ricin into cells. These screens have identified inhibitory compounds that can protect cells, and sometimes even animals against ricin. We review these newly discovered cellular inhibitors of ricin intoxication, discuss the advantages and drawbacks of chemical-genetics approaches, and address the issues to be resolved so that the therapeutic development of these small-molecule compounds can progress.

Reductive activation of type 2 ribosome-inactivating proteins is promoted by transmembrane thioredoxin-related protein.

Members of the type 2 ribosome-inactivating proteins (RIPs) family (e.g. ricin, abrin) are potent cytotoxins showing a strong lethal activity toward eukaryotic cells. Type 2 RIPs contain two polypeptide chains (usually named A, for "activity", and B, for "binding") linked by a disulfide bond. The intoxication of the cell is a consequence of a reductive process in which the toxic domain is cleaved from the binding domain by oxidoreductases located in the lumen of the endoplasmic reticulum (ER). The best known example of type 2 RIPs is ricin. Protein disulfide isomerase (PDI) was demonstrated to be involved in the process of ricin reduction; however, when PDI is depleted from cell fraction preparations ricin reduction can still take place, indicating that also other oxidoreductases might be implicated in this process. We have investigated the role of TMX, a transmembrane thioredoxin-related protein member of the PDI family, in the cell intoxication operated by type 2 RIPs ricin and abrin. Overexpressing TMX in A549 cells resulted in a dramatic increase of ricin or abrin cytotoxicity compared with control mock-treated cells. Conversely, no difference in cytotoxicity was observed after treatment of A549 cells or control cells with saporin or Pseudomonas exotoxin A whose intracellular mechanism of activation is not dependent upon reduction (saporin) or only partially dependent upon it (Pseudomonas exotoxin A). Moreover, the silencing of TMX in the prostatic cell line DU145 reduced the sensitivity of the cells to ricin intoxication further confirming a role for this enzyme in intracellular ricin activation.

Adapting yeast as model to study ricin toxin a uptake and trafficking.

The plant A/B toxin ricin represents a heterodimeric glycoprotein belonging to the family of ribosome inactivating proteins, RIPs. Its toxicity towards eukaryotic cells results from the depurination of 28S rRNA due to the N-glycosidic activity of ricin toxin A chain, RTA. Since the extention of RTA by a mammalian-specific endoplasmic reticulum (ER) retention signal (KDEL) significantly increases RTA in vivo toxicity against mammalian cells, we here analyzed the phenotypic effect of RTA carrying the yeast-specific ER retention motif HDEL. Interestingly, such a toxin (RTA(HDEL)) showed a similar cytotoxic effect on yeast as a corresponding RTA(KDEL) variant on HeLa cells. Furthermore, we established a powerful yeast bioassay for RTA in vivo uptake and trafficking which is based on the measurement of dissolved oxygen in toxin-treated spheroplast cultures of S. cerevisiae. We show that yeast spheroplasts are highly sensitive against external applied RTA and further demonstrate that its toxicity is greatly enhanced by replacing the C-terminal KDEL motif by HDEL. Based on the RTA resistant phenotype seen in yeast knock-out mutants defective in early steps of endocytosis (∆end3) and/or in RTA depurination activity on 28S rRNA (∆rpl12B) we feel that the yeast-based bioassay described in this study is a powerful tool to dissect intracellular A/B toxin transport from the plasma membrane through the endosomal compartment to the ER.

Role of the mannose receptor (CD206) in innate immunity to ricin toxin.

The entry of ricin toxin into macrophages and certain other cell types in the spleen and liver results in toxin-induced inflammation, tissue damage and organ failure. It has been proposed that uptake of ricin into macrophages is facilitated by the mannose receptor (MR; CD206), a C-type lectin known to recognize the oligosaccharide side chains on ricin's A (RTA) and B (RTB) subunits. In this study, we confirmed that the MR does indeed promote ricin binding, uptake and killing of monocytes in vitro. To assess the role of MR in the pathogenesis of ricin in vivo, MR knockout (MR(-/-)) mice were challenged with the equivalent of 2.5× or 5× LD(50) of ricin by intraperitoneal injection. We found that MR(-/-) mice were significantly more susceptible to toxin-induced death than their age-matched, wild-type control counterparts. These data are consistent with a role for the MR in scavenging and degradation of ricin, not facilitating its uptake and toxicity in vivo.

The acute toxicity, tissue distribution, and histopathology of inhaled ricin in Sprague Dawley rats and BALB/c mice.

Ricin is a highly toxic ribosome-inactivating protein derived from the castor bean (Ricinus communis). Due to the relative ease of producing ricin, it is characterized as a category B priority pathogen by the Center for Disease Control and Prevention. The purpose of this study was to compare the acute toxicity, associated histopathology, as well as the regional respiratory tract deposition and clearance kinetics of inhaled ricin in rats and mice using a single pure preparation. Acute toxicity was evaluated in five groups of six animals per species exposed nose-only to ricin aerosols and followed up to 7 days post-exposure. Tissues were collected for histopathology. The calculated median lethal doses (LD50s) were 0.24 µg/kg (rats) and 0.58 µg/kg (mice). Histological changes were noted in nose, larynges, trachea, lung, thymus, and spleen of both species. Pulmonary deposition in rats inhaling 94-99 ng/L ricin for 20 min (low dose) or 40 min (high dose) were 45.9 and 96 ng/g lung, respectively. Clearance was best described by a single-component negative exponential function. Estimated lung doses were 0.38 and 1.43 µg/g·h among the low and high dose rats, respectively. In mice inhaling 94 ng/L ricin for 20 min, pulmonary deposition was 91.1 ng/g lung and the estimated tissue dose was 1.72 µg/g·h. No ricin was detected in extra-respiratory tract tissue or in excreta. Results of this study demonstrate differences exist in pulmonary deposition, clearance rates, and tissue dose and histopathological changes between rats and mice inhaling ricin.

A phase III study of anti-B4-blocked ricin as adjuvant therapy post-autologous bone marrow transplant: CALGB 9254.

Anti-B4-blocked ricin (anti-B4-bR) is a potent immunotoxin directed against the CD19 antigen. Previous phase I and II studies suggested a possible role for anti-B4-bR as consolidation after high-dose chemotherapy and autologous stem cell transplant. Cancer and Leukemia Group B (CALGB) 9254 is a phase III study which randomized 157 patients with B-cell lymphoma in complete remission following autologous transplant to treatment with anti-B4-bR or observation. With a median follow-up time for patients of 5.8 years, the median event-free survival for protocol treatment and observation are 2.1 and 2.9 years, respectively (p = 0.275). The median overall survival for treatment and observation are 6.1 years and not reached, respectively (p = 0.063). Therefore, no differences were found in event-free survival and overall survival between protocol treatment and observation, although there was a trend toward improved survival with observation. These data fail to support a role for anti-B4-bR as consolidative therapy after bone marrow transplant in patients with B-cell lymphoma.

Ricin toxicokinetics and its sensitive detection in mouse sera or feces using immuno-PCR.

BACKGROUND: Ricin (also called RCA-II or RCA(60)), one of the most potent toxins and documented bioweapons, is derived from castor beans of Ricinus communis. Several in vitro methods have been designed for ricin detection in complex food matrices in the event of intentional contamination. Recently, a novel Immuno-PCR (IPCR) assay was developed with a limit of detection of 10 fg/ml in a buffer matrix and about 10-1000-fold greater sensitivity than other methods in various food matrices. METHODS AND FINDINGS: In order to devise a better diagnostic test for ricin, the IPCR assay was adapted for the detection of ricin in biological samples collected from mice after intoxication. The limit of detection in both mouse sera and feces was as low as 1 pg/ml. Using the mouse intravenous (iv) model for ricin intoxication, a biphasic half-life of ricin, with a rapid t(1/2)α of 4 min and a slower t(1/2)ß of 86 min were observed. The molecular biodistribution time for ricin following oral ingestion was estimated using an antibody neutralization assay. Ricin was detected in the blood stream starting at approximately 6-7 h post- oral intoxication. Whole animal histopathological analysis was performed on mice treated orally or systemically with ricin. Severe lesions were observed in the pancreas, spleen and intestinal mesenteric lymph nodes, but no severe pathology in other major organs was observed. CONCLUSIONS: The determination of in vivo toxicokinetics and pathological effects of ricin following systemic and oral intoxication provide a better understanding of the etiology of intoxication and will help in the future design of more effective diagnostic and therapeutic methods.

Protein toxins from plants and bacteria: probes for intracellular transport and tools in medicine.

A number of protein toxins produced by bacteria and plants enter eukaryotic cells and inhibit protein synthesis enzymatically. These toxins include the plant toxin ricin and the bacterial toxin Shiga toxin, which we will focus on in this article. Although a threat to human health, toxins are valuable tools to discover and characterize cellular processes such as endocytosis and intracellular transport. Bacterial infections associated with toxin production are a problem worldwide. Increased knowledge about toxins is important to prevent and treat these diseases in an optimal way. Interestingly, toxins can be used for diagnosis and treatment of cancer.

Detection of residual toxin in tissues of ricin-poisoned mice by sandwich enzyme-linked immunosorbent assay and immunoprecipitation.

This work aimed to evaluate a method to detect the residual ricin in animal tissues. Immunoprecipitation and sandwich enzyme-linked immunosorbent assay (ELISA) were used to detect ricin in the tissues of intoxicated mice. The monoclonal antibodies (Mabs) 4C13 and 3D74 were used to assay the whole ricin molecules via sandwich ELISA. Mab 4C13 was conjugated with Sepharose 4B to capture ricin or ricin A chain by immunoprecipitation. Mice injected intravenously with ricin at the dosage of 5 microg/mouse were killed at different time points after intoxication. The serum, liver, kidney, lung, and intestine were harvested. High levels of ricin were found in serum and liver samples at each poisoning time point by sandwich ELISA, suggesting the possibility of determining ricin intoxication by detecting residual ricin in the serum. However, this method turned out to be ineffective for examining ricin in the kidney, lung, and intestine of poisoned mice. Although the same tissue samples of intoxicated mice were analyzed by immunoprecipitation, positive bands were found. This indicated that some components in the kidney, lung, and intestine could bind with ricin and interfere in its binding activity with the coated antibody. Immunoprecipitation could be used to measure the existence of ricin in these samples.

Identification and characterization of small molecules that inhibit intracellular toxin transport.

Shiga toxin (Stx), cholera toxin (Ctx), and the plant toxin ricin are among several toxins that reach their intracellular destinations via a complex route. Following endocytosis, these toxins travel in a retrograde direction through the endosomal system to the trans-Golgi network, Golgi apparatus, and endoplasmic reticulum (ER). There the toxins are transported across the ER membrane to the cytosol, where they carry out their toxic effects. Transport via the ER from the cell surface to the cytosol is apparently unique to pathogenic toxins, raising the possibility that various stages in the transport pathway can be therapeutically targeted. We have applied a luciferase-based high-throughput screen to a chemical library of small-molecule compounds in order to identify inhibitors of Stx. We report two novel compounds that protect against Stx and ricin inhibition of protein synthesis, and we demonstrate that these compounds reversibly inhibit bacterial transport at various stages in the endocytic pathway. One compound (compound 75) inhibited transport at an early stage of Stx and Ctx transport and also provided protection against diphtheria toxin, which enters the cytosol from early endosomes. In contrast, compound 134 inhibited transport from recycling endosomes through the Golgi apparatus and protected only against toxins that access the ER. Small-molecule compounds such as these will provide insight into the mechanism of toxin transport and lead to the identification of compounds with therapeutic potential against toxins routed through the ER.

Transport of ricin from endosomes to the Golgi apparatus is regulated by Rab6A and Rab6A'.

Ricin is transported from early endosomes and/or the recycling compartment to the trans-Golgi network (TGN) and subsequently to the endoplasmic recticulum (ER) before it enters the cytosol and intoxicates cells. We have investigated the role of the Rab6 isoforms in retrograde transport of ricin using both oligo- and vector-based RNAi assays. Ricin transport to the TGN was inhibited by the depletion of Rab6A when the Rab6A messenger RNA (mRNA) levels were reduced by more than 40% and less than 75%. However, when Rab6A mRNA was reduced by more than 75% and Rab6A' mRNA was simultaneously up-regulated, the inhibition of ricin sulfation was abolished, indicating that the up-regulation of Rab6A' may compensate for the loss of Rab6A function. In addition, we found that a near complete depletion of Rab6A' gave approximately 40% reduction in ricin sulfation. The up-regulation of Rab6A mRNA levels did not seem to compensate for the loss of Rab6A' function. The depletion of both Rab6A and Rab6A' gave a stronger inhibition of ricin sulfation than what was observed knocking down the two isoforms separately. In conclusion, both Rab6A and Rab6A' seem to be involved in the transport of ricin from endosomes to the Golgi apparatus.

Retrospective identification of ricin in animal tissues following administration by pulmonary and oral routes.

A previously characterised amplified ELISA for ricin (sensitivity limit approximately 200 pgmL(-1)) has been employed to quantify ricin following a novel recovery method from selected tissues. Tissue samples from rats dosed by pulmonary instillation or orally with ricin were homogenised and treated with an elution buffer to extract ricin. This is the first time that ex vivo recovery of ricin post exposure following pulmonary or oral challenge has been achieved using clinically acceptable sampling methods, with promise in terms of diagnosis for the timely implementation of therapy. The toxin was detected and quantified using the ELISA in conjunction with pure ricin standards. Extracts from tissues sampled, including lung, blood, liver and spleen tested positive for ricin with maximum yield in lung associated fractions for pulmonary dosing and liver tissue for oral administration. This indicates the potential of lavage and blood sampling for timely diagnosis of ricin poisoning by pulmonary and oral routes, respectively. Time course analysis at 24 and 48 h also indicated the progression of ricin from surfaces of the lung into the lung tissue. Inter-subject variation was observed in the case of oral dosing, with data for ricin-treated and vehicle control tissues not statistically different in all samples. In addition the oral toxicity of the crude ricin administered was found to be higher than expected in the rat, based upon published information and an unpublished in house murine study.

The generation of immunotoxins using chimeric anti-CD22 antibodies containing mutations which alter their serum half-life.

Murine and chimeric RFB4 (anti-human CD22) monoclonal antibodies (MAbs) with mutations in their Fc portions were conjugated to recombinant ricin toxin A chain to generate immunotoxins. The resulting immunotoxins (ITs) constructed with chimeric RFB4 MAbs were designed to have longer or shorter half-lives but similar binding and cytotoxic properties. These ITs can now be evaluated in vivo for improved therapeutic indices. The characteristics of these ITs are the subject of this report.