Detection And Application In Targeting Antitumor Of Ricin

Ricin,a lethal protein toxin derived from the castor bean plant,is a heterodimeric glycoprotein consisting of two polypeptides,the ricin A-chain(RTA)and the ricin B-chain(RTB),linked by a disulfide bond.RTB is a lectin responsible for binding specific eukaryotic cell surface receptors containing glycoproteins and glycolipids to facilitate the internalisation of ricin into cells.While,RTA provides the deadenylase activity that irreversibly depurinates 28 S rRNA and terminates protein synthesis within cells.Ricin presents great potential in cancer treatment,however,it was realized that RTB is non-specific in binding the receptors present on the cell surface,limits the potential application of ricin as an anti-cancer agent.Therefore,it is the focus to not only retain the toxic effects of ricin on tumor cells,but also reduce the side effects.Moreover,ricin is one of the most fascinating poisons due to its high toxicity,and owing to its high cytotoxicity,ease of preparation,lack of medical countermeasures,ricin has been listed as one of the most potential biological warfare agents for terrorist attacks by the Centers for Disease Control(CDC).Therefore,the sensitive analysis and detection of ricin are of great significance both in response to terrorist attacks and in the prevention of poisoning.In this paper,we developed a graphene oxide-based isothermal strand-displacement polymerase reaction(ISDPR)for ricin detection,and combined with the nucleolin aptamer,carbon dots that target the Golgi apparatus and photosensitizer to develop a targeted therapy system of ricin on tumors.The main contents are listed as follows:(1)A simple,rapid,and sensitive RTB detection method using aptamer as recognition element is explored.In this design,the ricin-binding aptamer(RBA)was first hybridized with a short blocker,and the complex was then immobilized on MBs through biotin-streptavidin recognition.Upon the addition of RTB,blocker could be released from RBA because of the formation of RBA/RTB complex.After magnetic separation,the free blocker can trigger the ISDPR process and the hairpin DNA modified with a fluorescent group could generate a new double-stranded DNA.Since the duplex DNA cannot be adsorbed by the graphene oxide and showed a strong fluorescence signal.On the contrary,in the absence of RTB,the hairpin probe keeps its original stem-loop structure and can be adsorbed on the GO surface,resulting in a low background signal.Thus,RTB could be sensitively detected by the significantly increased FL Intensity.The background signal of this method was significantly reduced by the utilization of GO.In addition,the ISDPR was used in our method to amplify signals,which further improved the sensitivity.(2)A recombinant protein toxin is constructed for tumor treatment by coupled a CDs with distinctive capacity for long-term Golgi targeting to RTA.CDs-RTA conjugates were prepared by non-covalent binding.Due to the good biocompatibility of CDs and the excellent targeting properties to Golgi apparatus,CDs-RTA conjugates would easily internalize into cells.The endocytosed CDs-RTA conjugates then preferentially accumulate in the Golgi apparatus.From which,CDs anchor here,while RTA separates from the CDs and is further retrogradely transported into the cytosol route through ER to exert its real toxic action.With the protection of CDs,RTA can be greatly prevented from degradation by lysosome,enhanced the cytotoxicity of RTA.The proposed method increased the endocytosis efficiency and reduced the enzymatic hydrolysis.Besides,the complex could also be precise delivered to Golgi apparatus to further exert its real toxic action,improve the efficiency of treatment.Based on the above strategy,the photosensitizer was further introduced to develop a liposomal drug delivery system for enhanced targeted anticancer Chemo/Photodynamic therapy.The liposomes are prepared firstly by load CDs-RTA conjugates and a photosensitizer pheophorbide a(PPa).Then the nucleolin(NCL)aptamer,which has strong binding affinity to NCL,a protein overexpressed on many types of cancer cells was modified on the surface of the liposomes.When incubated with different cells,the complex can specifically enter the tumor cells while have little influence to normal cells.Under NIR laser irradiation,PPa can induce reactive oxygen species(ROS)generation to perform photodynamic therapy(PDT).Meanwhile,the CDs-RTA complexes released by the liposomes and further exert the chemotherapeutic effect.The dual drug-loaded liposomes display enhanced targeted tumor penetration and increased cytotoxicity.In summary,we developed a simple,rapid,and sensitive RTB detection method using aptamer as recognition element.The background signal in this strategy was significantly reduced by making full use of the GO.And the ISDPR was used to amplify signals,which further improved the sensitivity and accuracy.Meanwhile,we introduced the CDs with eminent Golgi targeting ability and constructed the recombinant plant protein toxin with RTA.Through the use of CDs,we improved the cellular uptake efficiency of RTA and reduced the degradation by lysosome,achieved precise subcellular drug delivery,enhanced the therapeutic efficiency of RTA.Finally,the CDs-RTA complexes and the photosensitizer were encapsulated by liposomes,which further modified the nucleolar aptamer to develop a targeted anticancer Chemo/Photodynamic therapy system.Under laser irradiation,the photodynamic therapy of photosensitizer and chemotherapy effect of RTA is produced,which improves the effect of tumor treatment while have little influence to normal cells,thus reduce the side effects of plant protein toxin for tumor treatment.