| Clostridium perfringens is a Gram-positive bacterium widely distributed in nature,primarily inhabiting the intestinal tract of animals,making it a typical zoonotic pathogen.More than 17 types of exotoxins produced by C.perfringens have been identified,with Clostridium perfringens ε-toxin(ETX)being one of the most lethal.ETX is a major pathogenic factor produced by type B and type D C.perfringens,and is also the primary toxin responsible for enterotoxemia in sheep and goats.Diseases caused by ETX are characterized by rapid onset,lack of specific drugs,and fast fatality,resulting in significant economic losses for the livestock industry.Furthermore,due to the high toxicity of ETX,it is considered a potential biological weapon and biological terrorism agent.In China,it is included in the list of dual-use bioproducts for import and export control.Therefore,research on drugs targeting ETX holds great value for biosecurity and social stability.In recent years,rapid development of nanotechnology has led to increasing applications in the medical field.Among these applications,membrane-camouflaged biomimetic nanoparticles(MNPs)directly use naturally derived cell membranes to provide nanoscale particles with an enhanced biological interface.The surface of the nanoparticle is wrapped with cell membrane,which replicates most of the biological information and functions from the source cell surface to the nanoparticle surface.This approach not only enhances the biocompatibility of the nanoparticles and allows them to circulate in the body for longer periods of time,but also endows them with most of the biological functions of the source cell membrane,enabling them to have more extensive and complex diagnostic and therapeutic functions.In this study,PLGA nanoparticles were wrapped in the cell membrane of ETXsensitive cells to prepare MNPs using the characteristic of ETX toxin to form pores in the cell membrane.The MNPs act as long-circulating drugs to neutralize ETX toxins in animal bodies,thereby being used for the treatment of fatal ETX toxin poisoning.In this study,recombinant ETX toxin with a GST tag at the N-terminus(GST-ETX)was expressed using the E.coli expression system.The purified protein was obtained through GST tag purification columns and then concentrated by ultrafiltration.The purity of the purified protein was confirmed using SDS-PAGE,and its concentration was determined using the BCA method.After dilution to an appropriate concentration,the protein was stored at 4°C or-20°C for further use.In this study,PLGA nanoparticles were prepared using the emulsion solvent evaporation method,and nanoparticles with a diameter of around 100 nm were obtained by optimizing the component ratio.The particles were separated by differential centrifugation and then rapidly frozen at-80℃ before being lyophilized using a freeze dryer to remove water and stored at-20℃ for later use.Cell membranes from Madin-Darby canine kidney(MDCK)cells and human red blood cells(RBC)were extracted and mechanically wrapped onto the surface of the PLGA nanoparticles to prepare the MNPs.The resulting MNPs were stored at 4℃ for later use.The physical properties of the nanoparticles and the membrane-camouflaged nanoparticles were characterized using techniques such as Flow Nano Analyzer,dynamic light scattering(DLS),and transmission electron microscopy(TEM).Next,the cytotoxicity of GST-ETX,polymeric nanoparticles(NPs),and two types of MNPs was evaluated using the MTS assay and red blood cell hemolysis assay in vitro,with MDCK cell models and RBC models.In addition,the ability of the two types of MNPs to neutralize GST-ETX and protect sensitive cells was evaluated in vitro using MDCK cell models and RBC models.The ability of the two types of MNPs to protect MDCK cells in vitro was also examined using a high-content imaging system.Finally,to evaluate the in vivo toxicity of GST-ETX,NPs,and the two types of MNPs,BALB/c mice were used as animal models,and safe MNPs were selected based on their safety.Subsequently,therapeutic experiments,blood biochemistry analysis,and histopathological methods were employed to evaluate the ability of MNPs to treat ETX poisoning in vivo.In vitro imaging experiments on animal tissues were conducted to verify the metabolism and interaction between MNPs and GST-ETX in vivo.Furthermore,the therapeutic effects of intravenous injection and pulmonary delivery of MNPs on mice with inhalational ETX poisoning were evaluated using similar experimental methods,as well as the short-term preventive ability of pre-injection or inhalation of MNPs in mice.In this study,a large amount of GST-ETX with toxicity equivalent to natural ETX was successfully expressed and purified for subsequent experiments.PLGA nanoparticles with a diameter of about 100 nm were prepared and used to produce a large amount of PLGA nanoparticles in subsequent experiments by optimizing the preparation protocol.Two different MNPs were prepared by physically wrapping cell membranes from different sources,and their physical properties and ability to neutralize ETX in vitro were evaluated.The results of the in vitro evaluation showed that MNPs were capable of neutralizing ETX in the environment,thus protecting cells in completely lethal concentrations of ETX,and their protective ability was positively correlated with the sensitivity of the membrane source cells to ETX.In this study,highly pure GST-ETX was successfully expressed and obtained,which exhibited toxicity equivalent to natural ETX.Through optimization of the preparation protocol,PLGA nanoparticles with a diameter of approximately 100 nm and uniform size distribution were successfully prepared.Two different types of MNPs were prepared by encapsulating PLGA particles with cell membranes from different sources,and their physical characteristics were characterized.The in vitro evaluation demonstrated that both RBC membrane-camouflaged nanoparticles(RBC-NPs)and MDCK cell membrane-camouflaged nanoparticles(MDCK-NPs)could effectively neutralize ETX in the environment,providing protection against lethal concentrations of ETX.The protection efficacy of MDCK-NPs was found to be superior to that of RBC-NPs,which was positively correlated with the sensitivity of the cell membrane source to ETX.The in vivo safety assessment of the two types of MNPs showed that those with cell membranes from simpler cells,such as RBCs,did not cause any immune response in animals,making them safer for use compared to MNPs with cell membranes from nucleated cells,such as MDCK cells.In animal treatment evaluations,it was confirmed that both intravenous and pulmonary delivery of MNPs were safe and effective in treating ETX toxicity at non-lethal doses.Furthermore,it was found that MNPs could alter the biological distribution of ETX in vivo,capturing it and safely transporting it to the liver and spleen for degradation,rather than allowing it to accumulate in sensitive organs such as the kidneys and brain.This provides insight into the specific mechanism by which MNPs treat toxin poisoning and confirms their effectiveness as a treatment for toxin poisoning.Finally,the therapeutic effects of MNPs administered through intravenous and pulmonary delivery were shown to last up to three days in animals,supporting the hypothesis that MNPs,as long-circulating drugs,can provide shortterm prevention of ETX toxicity or long-term treatment.In conclusion,the membrane-camouflaged biomimetic nanoparticles platform shows great potential for the development of a novel therapeutic drug against ETX poisoning.MNPs have been demonstrated to be safe and effective for both treatment and prevention of ETX toxicity via intravenous injection and inhalation.This study provides new insights into the selection of cell membrane sources for MNPs and treatment administration routes,and also presents new evidence for the use of nanoparticles in providing long-term protection against toxin poisoning. |
