| Drug-induced hearing loss refers to the impairment of vestibule and cochlea,especially inner ear sensory hair cells caused by drug ototoxicity,which seriously affects the ordinary life of patients and their families.There are many drugs on the list of drugs released by the World Health Organization that can save lives,such as aminoglycoside(AG)antibiotics used to prevent life-threatening infections and platinum drugs used to treat various cancers,however,both of them have serious ototoxicity.So far,the US FDA has not approved any relevant drug about prevention and treatment.Although many drugs for the prevention and treatment of druginduced hearing loss have entered the preclinical or clinical research stage at this stage,the mechanism of drug-induced ototoxicity has not been clarified.Therefore,the study mainly evaluates whether curcumin(CUR)-loaded nanoparticles have the effect of preventing and treating cisplatin ototoxicity.Using poly(lactic-co-glycolic acid)(PLGA)as the carrier material,chitosan(Chitosan,CS)for surface modification,and loading the drug as curcumin,we used the re-emulsion method to prepare CUR-PLGA NPs and CUR-CS/PLGA NPs nanoparticles.At the same time,we also evaluated the drug-drug conjugates DoNB and DdNB formed by the combination of dexamethasone(Dex)and salvianolic acid B(Sal B),and DoNB formed by their self-assembly Whether NPs and DdNB NPs can antagonize the ototoxicity caused by cisplatin,and explore the mechanism of action of these drugs.Two kinds of nanoparticles,CS/PLGA NPs and PLGA NPs,were prepared by double emulsion method.Their particle size and charge were investigated.The results showed that the two nanoparticles were uniform particle size distribution,but the surface charges of CS/PLGA NPs and PLGA NPs were different,and their charges were about + 9.54 mV and-29.20 mV respectively.The UV-Vis method was used to examine the drug release laws of CUR-PLGA NPs and CUR-CS/PLGA NPs.It was found that the release behavior of these two kinds of nanoparticles was very consistent,and the cumulative release of free drugs within 24 hours had almost stabilized.The drug-loaded nanoparticles did not stabilize until 120 hours,which indicated that both CUR-PLGA NPs and CUR-CS/PLGA NPs had sustained release properties.At the same time,it was shown that the surface-modified chitosan had little effect on the sustained release of PLGA NPs.A confocal laser scanning microscope was used to compare the effects of the two nanoparticles on the uptake and distribution of HEI-OC1 cells in the inner ear tissue.The results showed that the surface-modified CUR-CS/PLGA NPs significantly enhanced fluorescence intensity in HEIOC1 cells and the fluorescence of CUR-CS/PLGA NPs in Corti organs,vascular veins,and spiral ganglia in the inner ear,the fluorescence intensity was significantly higher than that of CUR-PLGA NPs,indicating that chitosan helps the nanoparticles to be taken up into cells.In order to further determine whether CUR-PLGA NPs and CURCS/PLGA NPs have the effect of preventing and treating cisplatin-induced ototoxicity,this study also established a model of sensory hair cell damage induced by cisplatin in vitro and in vivo.The MTT method was used to compare the protective effects of CUR-NPs with different characteristics on the cisplatin-induced injury model of HEI-OC1 cells.The results showed that CUR-NPs could protect HEI-OC1 cells from the toxic and side effects of cisplatin to varying degrees,while blank nano carriers did not show the protective effect.At the same time,the effects of CUR-PLGA NPs and CUR-CS/PLGA NPs on hearing threshold,inner ear hair cells and Caspase-3 receptor expression in the inner ear of cisplatin induced guinea pig hearing loss model were investigated by combining ABR test,immunofluorescence,immunohistochemistry and microscopic image analysis.The results showed that both CUR-PLGA NPs and CURCS/PLGA NPs could significantly reduce the hearing threshold of guinea pig hearing loss model induced by cisplatin,and protect the inner ear hair cells of guinea pigs from the ototoxic effects of cisplatin,no matter whether it was prevention or treatment.In addition,CUR-PLGA NPs and CUR-CS/PLGA NPs might protect inner ear hair cells by regulating the expression of Caspase-3 receptors in the cochlea.The drug-drug conjugates DdNB and DoNB were two prodrugs with dual properties of salvianolic acid B and dexamethasone,which were prepared in the early stage,they were self-assembled to form DdNB NPs and DoNB NPs respectively.Previous studies have found that the two prodrugs,DdNB and DoNB,and their nanoparticles could protect HEIOC1 cells to reduce the ototoxicity of cisplatin or other ototoxic drugs.In order to confirm whether the two drug-drug conjugates and their nanoparticles have the ability to protect inner ear hair cells in animal models,we combined the techniques of ABR test,immunofluorescence,immunohistochemistry and microscopic image analysis to investigate the preventive effects of these drugs on the ototoxicity of cisplatin after local or systemic administration.The results showed that in the guinea pig hearing loss model induced by cisplatin,different concentrations of DdNB,DoNB,Dd NB NPs and DoNB NPs had different protective effects on inner ear hair cells after local administration.Immunohistochemistry showed that these drug-drug conjugates and their nanoparticles might play a protective role by regulating the expression of GR receptors in the inner ear tissue.In order to investigate whether the way of administration affected the effect of the drug,the effects of 1.75 mg/mL DdNB,0.7 mg/mL DoNB,0.70 mg/mL Dd NB NPs and 1.75 mg/mL DoNB NPs on the inner ear sensory hair cells of guinea pig hearing loss model induced by cisplatin were observed by confocal laser scanning microscope,The results showed that the effects of these drugs after systemic administration are slightly worse than those of local administration,but they also had varying degrees of protection.In order to investigate the biological safety of PLGA NPs,CS/PLGA NPs delivered via the Transtympanic,HEI-OC1 cells were used as in vitro model and guinea pigs as in vivo model to investigate the cytocompatibility and biocompatibility of these drugs.The results showed that when CS/PLGA NPs with a concentration gradient of 0.05-5 mg/mL was treated with HEI-OC1 cells for 24 or 48 hours,the survival rate of HEI-OC1 cells was 102%-107% and 100%-83%,respectively.The results showed that CS/PLGA NPs had good biocompatibility,and the drug-cell interaction time was preferably 24 hours.However,when PLGA NPs with a concentration gradient of 0.05-5 mg/mL were treated with cells for 24 or 48 hours,the survival rates of HEI-OC1 cells were 102%-86% and 100%-80%,respectively,which indicated that PLGA NPs had good biocompatibility and the time had little effect on the cell viability of HEIOC1 cells.The previous work of our research group has proved that the drug-drug conjugate and its nanoparticles had good biocompatibility.Guinea pigs were used as experimental subjects,HE staining was used to investigate the safety of different drugs for delivery in inner ear.The results showed that these drugs had good biocompatibility with cochlear tissue.To sum up,the CUR-NPs investigated in this study had the potential to prevent and treat the ototoxicity caused by cisplatin,which the CURCS/PLGA NPs with positive surface charge had a better effect.At the same time,the drug-drug conjugates and nanoparticles prepared by our research group have different degrees of protection after local or systemic administration,which may lay a foundation for the prevention and treatment of drug-induced hearing loss. |
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