| Cisplatin(CDDP)is an extensively used chemotherapeutic agent yet with severe ototoxicity.CDDP-induced ototoxicity generally manifests in the form of irreversible,progressive,and bilateral sensorineural hearing loss,which seriously affect patients’ ability to communicate and their quality of life.There are no recommended regimen or FDA approved drugs for the prevention and treatment of CDDP-induced ototoxicity,so it is urgent to find effective drugs.Dexamethasone(DEX),a synthetic glucocorticoid,is used as therapeutics to counteract hearing loss.However,frequent administration and high doses are required to achieve the desired efficacy,which may lead to severe side effects.The low aqueous solubility of DEX also hindered its applications.In our previous study,salvianolic acid B(SAL),a water-soluble active component of Salvia miltiorrhiza,showed protective activity against drug-induced hearing loss.SAL is water-soluble and convenient to be administered.However,its efficacy is limited by poor biomembrane permeability,resulting in extremely low bioavailability.In the previous study,hydrophilic SAL and lipophilic DEX was conjugated via an ester or amide bond to obtain amphiphilic conjugate.On this basis,the stability of the conjugates,the preparation and characterization of the conjugates assembled into nano self-delivery system,the prevention of CDDP ototoxicity and the biocompatibility of the conjugates were studied.Firstly,the conjugate was synthesized and characterized according to the previous conditions of the research group,and its stability in artificial perilymph and phosphate buffer was investigated.The results of high-resolution mass spectrometry showed that the conjugate and its intermediates were successfully synthesized,and the conjugate could be more stable in both artificial perilymph and phosphate buffer,indicating that the conjugates had better stability.Amphiphilic DEX-SAL conjugates can self-assemble to form nano self-delivery system by solvent evaporation method.The particle size and stability,drug release of self-delivery system was studied and cell uptake in vitro of nanoparticles were investigated by loading coumarin-6.The results showed that the nano self-delivery system had uniform particle size distribution and good stability.The drug release behavior of nano self-delivery system in vitro was consistent and had a certain sustained-release effect.The fluorescence intensity of self-delivery system group in HEI-OC1 cells was significantly higher than that of free coumarin-6 group,indicating that the self-delivery system could be effectively absorbed by cells.In order to investigate whether the conjugate and its self-delivery system can antagonize the ototoxicity of CDDP,a zebrafish hair cell injury model induced by CDDP was established.The results showed that in zebrafish model,compared with DEX,SAL and their physical mixture at the same concentration,the conjugates and nanoparticles had good effect on CDDP-induced ototoxicity.At the same time,the preventive effect of cis/trans isomers on CDDP-induced deafness in guinea pigs was further investigated.The results showed that cis/trans isomers could protect guinea pig inner ear hair cells from the ototoxicity,and had a good preventive effect on CDDP-induced deafness.In view of the good anti-ototoxic effect of conjugates and their self-delivery system,the interaction between conjugates and nanoparticles and glucocorticoid receptors in guinea pig inner ear was further investigated by immunohistochemistry to explore the mechanism.It has been found that the conjugates and nanoparticles can bind to the cochlear glucocorticoid receptor and promote the expression of the receptor without affecting the binding affinity and receptor activation.At the same time,the drug concentration in the perilymph of cochlea was determined by HPLC,which showed that the conjugates and nanoparticles could reach the cochlea tissue.Therefore,it is speculated that the conjugates and self-delivery system may play a pharmacological role in restoring cochlear damage by targeting and activating receptors and affecting specific DNA sequences in the nucleus.The results showed that the conjugate and its self-delivery system could significantly enhance the activity of CDDP on Hep G2 cells,MCF-7cells and A549 cells,prevent the ototoxicity of CDDP,and improve the anticancer effect of CDDP.Finally,the safety of the conjugates and nanoparticles was evaluated by embryo toxicity test of zebrafish,auditory function test of guinea pigs and cochlear histology.The results showed that there were no significant differences in the survival rate,hatching rate,deformity rate,body length and average fluorescence area of hair cells between the conjugates and nanoparticles groups.In addition,after intratympanic injection of the conjugates and nanoparticles,the hearing thresholds of guinea pigs were normal within the measured frequency range.No inflammatory reaction was observed in the cochlea of all experimental groups,and no other adverse reactions were observed,indicating that the conjugates and nanoparticles have good biological safety.In conclusion,the designed and synthesized amphiphilic DEX-SAL conjugate and nano self-delivery system can overcome the defects in the nature of the parent drug,exert the synergistic effect of the two drugs,enhance the curative effect,and have good biocompatibility.It provides a new drug and dosage form for the prevention and treatment of CDDP-induced hearing loss,and a new idea for the research and development of anti-deafness drugs. |
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