Design And Evaluation Of Liposomes Of MC Guided By SS31

Hearing loss induced by aminoglycoside antibiotics has become an important issue that affects human health all over the world.Currently,there are no effective therapeutic strategies in clinical.Finding some drugs to treat drug-induced hearing loss is one of the hot issues in current research.The key of induced ototoxicity is the apoptosis of inner ear hair cells.Current candidates for the treatment of hearing loss commonly suffer from low efficacy that can not meet the clinical requirements.In this paper,a liposome?Lp?drug delivery system was designed,loading with water-soluble drug minocycline hydrochloride?MC?,and modified liposomes with peptide SS31?D-Arg-Dmt-Lys-Phe-NH2?on its surface.It was expected to reduce cochlear hair cell damage induced by the aminoglycoside antibiotic gentamicin?GT?,to realize the treatment of drug-induced hearing loss.In the pre-prescription study,the UV absorption spectroscopy of MC in different dispersion medium was investigated,selecting isoabsorption point as the optimal detection wavelength for content determination in vitro,and then establishing the analysis method of minocycline hydrochloride in vitro.To eliminate their effects,the isoabsorption point of MC at 380 nm and pH range of 4.36 to 6.00,358 nm and pH range of 6.80 to 8.73,357 nm in HBS buffer containing different concentrations of Ca²⁺and 350 nm in HBS buffer containing different concentrations of Mg²⁺was determined.The linearity,precision and recovery of MC in different pH and methanol solution met the requirements.The solution stability of minocycline hydrochloride was investigated,and the result showed that the stability of MC solution is not obviously affected by pH and light at the range of 5.50 to 7.40 of pH.While,as the temperature increased,the degradation rate of MC gradually accelerated.The preparation of Lp/MC was performed by active drug loading method with Ca²⁺gradient.A method was established with using Sephadex G-50 mini-column to determine the entrapment efficiency%?EE%?and drug loading efficiency%?LE%?of minocycline hydrochloride.Taking the EE%and LE%as reference,the influence of the prescription factors such as Ca²⁺concentration,DSPE-PEG2000,cholesterol content,phospholipid concentration,drug-lipid ratio and prrocess factors such as drug loading temperature or time on liposomes were investigated.The optimal formulation of minocycline hydrochloride liposome was determined as HSPC/Chol/DSPE-PEG2000=70:24.4:5.6?molar ratio?,hydration medium with calcium concentration of 120 mM,phospholipid concentration of 8.7 mg/ml,drug-lipid ratio of 1:10?Wt/Wt?,drug loading temperature of 60?and drug loading time of 15 min.Under this condition,the EE%and LE%of Lp/MC were 97.8%and8.9%,respectively.The preparation of SS31-Lp/MC was performed by using the click chemical method?SS31-Cys reacting with DSPE-PEG2000-MAL?with 82.0%±4.5%of the maleimide end group successfully conjugated with SS31 determined by HPLC method.The EE%and LE%of SS31-Lp/MC were 93.4%and 8.5%,respectively.The particle size of the optimal formulation of Lp/MC and SS31-Lp/MC were143.5 nm and 149.9 nm,respectively,and the Zeta potentials were-35.0 mV and-25.8 mV,respectively.The stability results of the two liposomes showed that the particle size,drug content did not change significantly in the dark at 4?for 8 weeks,which indicating that the modification of SS31 had no significant affect on the physicochemical properties of the liposome.HBS buffer was used as a medium to investigate the release kinetics of the two liposomes under different pH conditions and different concentrations of bovine serum albumin?BSA?.The results showed that the pH and BSA concentrations did not affect the release of minocycline hydrochloride from liposomes.Compared with Lp/MC,minocycline released from SS31-Lp/MC in all investigated media displayed a slightly rapid profile.The zebrafish model of gentamicin?GT?induced ototoxicity was established.Investigating the toxicity of MC,blank Lp,SS31-Lp and gentamycin on zebrafish lateral hair cells,as well as the protective effects of MC,blank modified SS31-Lp,Lp/MC and SS31-Lp/MC pre-treatment on hair cells.The effect of liposomes on hair cell Mechanotransduction?MET?channels and mitochondrial membrane potential?MMP?were studied.The results showed the damage of hair cells by MC and gentamicin was dose-dependent.The two blank liposomes had no effect on hair cells.In the chronic exposure,the protective effect of SS31-Lp/MC on hair cells was enhanced.The result of the two scoring system of DASPEI and DiOC2?3?were significant different with that of gentamicin toxicity treatment group.And the liposomes did not affect the function of the hair cell MET channels and did not alter gentamicin uptake.