Research Of Inner Ear Delivery Based On MPEG-PLGA Nanocarrier For Protein

Various reasons such as noise exposure or ototoxic drug application could cause damage of cochlear cells and (or) tissue structure. Generally, the damage process start from the pathological changes of the cochlear hair cells (including the outer hair cells and inner hair cells), consequently the organ of Corti’s. Several studies have confirmed that fundamental method of hearing loss treatment is hair cells regeneration or repearment of hair cell in specific pathological periods.Biological treatment is considered as the most potential way in the various research of deafness treatment. At the same time, the internal fluid structure in the inner ear and relatively small independent space, also makes the cochlea can be used as a ideal biological treatment system of study. During more than half a century of researches, the scientists have explored the pathogenesis of deafness and development of auditory nerve cell, and also has found a series of key molecular which can protect or restore the hearing function, such as nucleic acids, peptides and small molecules. These molecules can lead to the repairment or regeneration of inner ear sensory cells, and finally the protection or restoration of auditory function. Select an ideal delivery vector which can deliver the intervention molecular into the inner ear tissue safely and efficiently is a key step in the biological treatment of deafness.Common vectors divided into two categories:viral and non viral vectors. The last century ninety’s, the researchers have used viral vectors to introduce foreign genes into the inner ear for the first time, which opens the development and application of vector-technology in the field of protection or regeneration of hair cell of inner ear. However, due to the defects of viral vectors, or even lethal reports, researchers have begun to consider the safety problem of viral vector. In recent years, nano vectors are becoming a hotspot in the field of biomedical research involving the study of selection and application of alternative vectors gradually. And nano vectors have been proved that process stable and safe characteristic, as well as cell specific. These advantages would provide a new strategy for the traditional intervention and therapy of ear diseases.Poly lactic acid glycolic acid (poly (D, L-lactide-co-gly-colide), PLGA) has recently emerged as the most representative nano carrier system. The PLGA copolymer consists of lactide (LA) and and glycolide (GA), and has well biocompatibility and biodegradability. The PLGA nanoparticles modified with PEG have well-controllable characteristic, and provide hydrophobic core which enclosure multiple polypeptides, proteins drugs at physiological pH conditions. Moreover, these nanoparticles would offer complex surface of PEG to bind and interact with negatively charged cell membrane. Consequently, with the protection of the mPEG-PLGA, polypeptide and protein drugs would be delivered into cells through endocytosis with no significant degradation. And finally achieve the therapeutic effect.Our present study contains four parts:firstly, the mPEG-PLGA nanoparticles were obtained by surface modification of PLGA with PEG. Meanwhile, nanoparticles connected with BSA protein through the double nipple encapsulation method. And particle morphology and hydrodynamic diameter were examined by transmission electron microscopy (TEM) and Dynamic light scattering (DLS). The protein release rate of nanoparticles was measured as well. The mPEG-PLGA-BSA-FITC-NPs have shown to be a sustained release delivery vehicle and the loading rate is51.2%. Secondly, we investigated the delivery efficiency and cytotoxicity of mPEG-PLGA-BSA-FITC-NPs with cultured Hela cells in vitro. At the same time, the efficiency and cytotoxicity of mPEG-PLGA- BSA-FITC-NPs nanoparticles in vivo were evaluated via inner ear delivery in guinea pigs. And FITC positive cells were observed by fluorescence confocal fluorescence microscopy. The mPEG-PLGA-BSA-FITC-NPs as a potential application vector was verificated and highlighted in the regeneration of hair cells of inner ear. In the third part of the study, we have established a mini pig animal modle of noise-induced deafness through noise exposure using mini pigs by electric spark pulse noise meter firstly. After hearing assessment and cochlear pathological study, we have abtained a miniature pig animal model of noise-induced deafness based on large mammals successfully. Finally, we attempt to apply the mPEG-PLGA-BSA-FITC-NPs nanoparticles in noise-induced deafness miniature pig model through an intact round window membrane way which can permeate molecular into the inner ear. Finally, our studies would provide the important application data of pre-clinical studies using mPEG-PLGA-BSA-FITC-NPs nanoparticles in the field of deafness therapy.The innovative point of the study:1. We have modified PLGA nanoparticles using PEG, and successfully obtained mPEG-PLGA-BSA-FITC-NPs with a mean particle size of232+2.6nm through encapsulation of BSA protein via double nipple method. The mPEG-PLGA-BSA-FITC-NPs have shownd to be an ideal protein delivery system with good dispersibility and sustained release characteristics.2. We have measured the efficiency and cytotoxicity ofmPEG-PLGA-BSA-FITC-NPs of with cultuled Hela cells in vitro, and get higher safety and effectiveness. At the same time, for the first time in the inner ear of guinea pig mPEG-PLGA-BSA-FITC-NPs delivery, and presents the obvious time dependence.3. The mini pig animal modle of noise-induced deafness was established through noise exposure using mini pigs successfully.4. We attempt to apply the mPEG-PLGA-BSA-FITC-NPs nanoparticles into the established mini pigs of noise-induced deafness animal model firstly, and provide a research platform for future necessary hearing intervention study of large mammals.