Anti-oxidative Injury To Ear By Nano-particles Loaded With Melatonin

Deafness is a common disease which cruelly impairs the health of people and there are about one hundred million people suffered from deafness across world now. Nevertheless, deaf cases are increasing by one million each year. Recently, some diseases, such as blood insufficiency, exposure to noise and side-effects by hearing-toxic drugs, etc.. will impose ischemia and oxygen deficiency to the cochlea. Accordingly, production of oxygen species (ROS) in cochlea will be increased strikingly, as such it will result in the generation of oxygen free radical (OFR). OFR is the incomplete metabolized product of oxygen molecule and has the ability to react with unsaturated fatty acid RH on cell membrane. This will cause direct injury to cell membrane and results in a series of dysfunction and trauma of cell. Deafness will occur.Melatonin(MLT), an important substance releasing from conarium of human brain, is the most powerful substance that has been found to eliminate OFR of endogenesis. MLT has little side-effects and will be not reduced once being oxygenized, which avoids generation of OFR from itself oxidation. MLT is highly lipophilic with little degree of hydrophilia. MLT plays roles of antioxidation and scavenge OFR efficiently in cell membrane, cytoplasam and nucleus due to the abilities of high lipophilia, little hydrophilia and high diffusion. MLT can also inhibit increase of lipid peroxide induced by H₂O₂ and influence the activity of antioxidant enzyme, such as antioxidant enzyme. GSH — PX, etc. It can protect biological molecules, viz lipid, DNA. from damage by OFR because of the above mentioned features of high lipophilia and accumulation in nuculeus. Oral administration of MLT can prevent injury to cochlea via scavenge of OFR. However, the present forms of MLT substance can't cure the chronic trauma of inner ear imposed by oxidative injurydue to short half life time approximately about 45 min, rapid metablization and diffcult access to target organ.The present study will utilize the technology of nano-carrier loaded drugs with strong penetrability that can release drug slowly, prolong the time of drug function and directly gain access to the target organ. This approach has many advantages, such as increased stability, easy store, protection of nucleotides from degradation by nucleotide enzyme, localization following transfection of cells, etc, as such will reduce dosage but not at the expense of action and thus diminish adverse effects of drugs. With aim to pass the barrier of labyrinth and overcome the defects of currently used medicine, the present investigation employed biodegradable materials like PLA, CS, gelatin as dispersive agent, span-80 and tween-80 as emulsive agents. The nanoparticles loaded MLT (MLT-NPs) was prepared by combination of emulsion and solvent. The best regimen was decided through inspection of shapes, sizes and ability of dispersion with atomic force microscope probe (AFM). Under various circumstance of body fluid, sizes and stirring velocity, the nature of releasing ability was characterized by spectrophotometer of ultraviolet radiation (DCM). Additionally, volume of drug loaded and efficiency of encapsulation was evaluated as well.Following injection of MLT-NPs derived from optimized method into the cavy of inner ear, the content of MLT in the inner ear ear lymph fluid was measured by DCM and the degree of audition nerve injury was determined by ABR. The pathological status was examined with nitroblue tetrazolium chloride (NBT) and HE staining approach. The outcome of MLT-NPs releasing and protective effects was analyzed so as to offer rationales for clinical application of nano-medicine.This article consists of the following three parts.Part â…  Preparation and design of nanoparticles loaded with MelatoninAccording to the L₉(3⁴) designing instructions, the biologically-degradable materials PLA and CS were selected as carrier on the base of none-toxicity and none-side-effects, gelatin as dispersive agent. MLT-NPs was prepared with emulsionof sapn-80 and tween-80 and under fixed stirring velocity, fixed reaction temperature, fixed ratio of drug and ingredients and magnetic force. In addition, the characteristics of morphology and distribution of MLT-NPs was viewed under AFM in order to accomplish the ideal MLT-NPs. Effect-response curve was made followed by statistical analysis. With SPSS 10.0. / test was adopted to complete the statistical analysis Result:1. MLT-NPs were prepared under the following conditions, temperature of 30°C, stirring velocity of 800r/mi^ the ration of MLT to PLA of 1: 3 and 0.25% CS. Grades of surface morphology was classified as 9.5 on the basis of data, average sizes of MLT-NPs of 45.84±1.262nm. volume of drug loading of 12.35% and encapsule efficiency of MLT-NPs of 38.33%.2. Under drug concentration of 50mg/L, 40mg/L, 30mg/L. 20mg/L and lOmg/L determined by DCM, the standard curve of MLT is Y=-0.0007 +0.01847X. The absorption was correlated to MLT-NPs pronouncedly with r as 0.9999. The recycle rate is 99.76%±0.87%.3. The maxim S with weighing analysis is 141.79 under the following conditions, 30°C as temperature of solvent volatilization, 1:3 as the ration of MLT: PLA, 800 r/min as stirring velocity and 45 min asstirring time for original and duplicate emulsion.4. Distribution chart of MLT-NPs shows that the size for 54% MLT-NPs centered on 37.45-54.23 nm under optimized design instruction.Part II Analysis of desorption by nanoparticles loaded withIWelatonin in vitroAccording to dialysis methods under constant temperature and stirring velocity, the features and releasing velocity was determined. The parameters consisted of various diameter of MLT-NPs. various body fluid circumstance and various stirring velocity.Results:1. The rapid desorption period and remarkable increase of MLT was revealed when the sizes of MLT-NPs less than 45.84 nm in the first 25h followed by a slow and stable desorption period. When the sizes of MLT-NPs more than45.84nm, only slow and stable desorption period was observed. The expression of dynamic desorption by MLT-NPs was established with regression analysis using Higuchi equation.2. Under man-made gastric juice (pH=1.4), the rate of MLT-NPs desorption is 75.56% at 170h and the efficiency of cumulative desorption agreed with Q=0.0112+0.05709tl/2(r=0.9876) with t,/2=75h. Within 40h, the desorption of drug was 45.25% in manmade arterial plasm (pH=7.4) and concurred with Higuchi equation of Q =0.0334+0.07413tl/2(r=0.9978) with tl/2 = 40h.3. Under 0.9% NaCl, the cumulative desorptions were 85.29%. 90.99%. 92.71% respectively when the stirring velocity were 50r/min, 75r/min, lOOr/min.Part IH Experimental study of nanoparticles loaded with Melatonin against oxidative injury of earThe adult and healthy guinea-pigs with normal hearing were chosen randomly and were divided into experimental group, drug-supplemented control group and placebo-supplemented control group. The model of oxidative injury to ear was prepared by injection of 3%H^O2 to induce injury. After oral administration of MLT-NPs derived from above designed, we examined the following parameters, slow and stable desorption of MLT-NPs in lymph fluid of inner ear under UV spectrophotometer, responses of audition nerve by ABR, pathological alterations of hairy cells and corti's organ after SDH and HE staining, smearing slides of cochlea basal membrane. The slow and stable desorption. antioxidative effects and advantages of nano-carrier of MLT-NPs was validated, which will benefit the current management of prevention and curing of deafness. Results:1. Data from analysis of UV spectrophometer indicated that MLT-NPs treated group showed marked features, such as slow and stable desorption and passing thebarrier of labyrinth, etc. The maxim releasing time in inner lymph fluid for MLT-NPs reached 165h after drug supplemented. The desorption curve correlated with Q=0.01103+0.07696t"2 with t,/2 is 40h and r=0.9931.2. The experimental group showed that the hearing was improved at day of 1, 3, 5 and 7 and the hearing was normal at 7th day after MLT-NPs treatment following oxidative injury when compared with control group.3. The hairy cells of cochlea and corrit's organ were improved as well at 7lh day with SDH and HE staining, smear slides of cochlea basal membrane compared with control group. Conclusions:1 MLT-NPs was prepared by compound of emulsification and solvent evaporation technique.1. We are the first to report here that MLT-NPs with slow and stable releasing characteristics were prepared with physical method.2. The optimum factors for this protocols comprises 30 °C as reaction temperature. 800 rpm as stirring velocity, the ratio of MLT and PLA mixture 1:3, the ration of Tween-80 and Span-80 mixture 5:1 and 0.25% as CS concentration.3. Under a variety of ratio between Tween-80 and Span-80 mixture, the present protocol will provide reference data for other similar regiment after the efficiency of sphericity and encapsule. content of drugs loaded were measured.4. The main factors influencing this protocol consists of stirring speed, temperature of volatilization of solvent, ratio of PLA and MLT, concentration of CS.5. Clinical requirements were met the degree of surface smooth and dispersion inspected under AFM (atomic force microscope probe).II Determination of desorption by MLT-NPs in vitro1. The profiles of desorption for MLT-NPs aligns with clinical application.2. The ability of slow and stable releasing drug from MLT-NPs was correlated with particle size negatively.3. pH can affect the releasing velocity of MLT-NPs.4. Releasing velocity of MLT-NPs was hardly imposed by mechanical force.Ill Observation of inner ear of guinea-pig1. We are the first to report the slow and stable desorption by MLT-NPs in inner ear lymph fluid, which shows the ability to pass labyrinth barrier of inner ear.2. MLT is capable of act against oxidative injury to inner ear and ameliorate the chronic injury.1III Little container of 250ml was determined as the container of first choice to prepare MLT-NPs after 52 initial experiments because more precipitation, low efficiency of encapsule and small volume of drug loaded was found in bigger container like 1000ml. With regard to the sphericity of drug, encapsule efficiency, loading volume of drug and large-scale production of MLT-NPs warrants in-depth investigation.