| As a new therapeutic gas,hydrogen has many advantages in terms of safety and effectiveness.Hydrogen has small molecular weight,strong diffusion ability,easy to pass through tissues and cells,and play a therapeutic role by removing strong oxidizing substances.Compared with other gases such as nitric oxide,hydrogen sulfide,carbon dioxide,hydrogen has the characteristics of non-toxicity.Aminoglycoside antibiotics are the first-line clinical drugs for the treatment of bacterial infections,but when they play a role,they can induce the production of reactive oxygen species and cause serious ototoxicity.The ototoxicity caused by these drugs is usually irreversible,and hydrogen-saturated normal saline can be used to antagonize the ototoxicity caused by aminoglycosides.Due to the low dissolution of hydrogen,the application of hydrogen in medicine is greatly limited.Hydrogen is not only difficult to reach the target site to take effect,but also unable to stay at the target site for a long time.Therefore,it is very important to study how hydrogen can take effect on the target site more efficiently and permanently.In recent years,the Microparticle Drug Delivery Systems(MDDS)have been deeply studied and become an important platform for the treatment of diseases.With the development of microparticle delivery systems,a number of unique gas microparticle delivery system have been developed to meet the needs of different diseases.The small size of these platforms allows them to effectively target the site of the disease while releasing gas to either direct treatment or enhance treatment.The appearance of gas microparticle delivery system provides a theoretical basis for the effective action of hydrogen for the treatment of drug-induced hearing loss.In this paper,we studied the microparticle drug delivery system containing ammonia borane(AB)which produce hydrogen to study its protective effect against gentamicin-induced ototoxicity,hoping to provide reference and the application of hydrogen microparticle system in the treatment of deafness.Perfluocarbon-ammonia borane-poly lactic-co-glycolic acid microparticles(PFC-AB-PLGA MPS)were successfully prepared by W/O/W Multiple emulsion method and solvent volatilization method.Ammonia borane was the prodrug for releasing hydrogen which can release hydrogen under acidic conditions,in the meanwhile perfluorocarbons have ultrasonic response characteristics.Single factors such as the amount of PLGA,volume of aqueous phase,ultrasonic conditions,homogenization time and amount of ammonia borane were investigated.After optimizing the process,the hydrogen carrying capacity of the particles was greatly increased from 0.2 m M to 1.87 m M.Then,the morphology and structure of the particles were characterized by scanning electron microscopy and Fourier transform infrared respectively.The results showed that the particles were spherical particles with smooth surface and were coated with perfluorohexane and ammonia borane.Finally,the stability of the particles was investigated.The results showed that the particles had good stability in normal saline.Poloxam temperature-sensitive gel was prepared by cold method.The PFC-AB-PLGA gel with p H and temperature responsiveness can be obtained by adding poloxamer 188 and poloxamer 407 to the microparticle solution.The gelation temperature of gel was determined to be 37℃ by the inversion method.The results of scanning electron microscopy indicated that the gel was a three-dimensional loose network structure.Meanwhile,in vitro release experiments were showed that gel can be maintained for a long time in acidic medium,which provided a guarantee for slow delivery of hydrogen to reducing the ototoxicity caused by gentamicin.In order to establish a platform for hydrogen content detection,methylene blue-platinum reagent for hydrogen content detection was prepared.The release curves of the microparticles at different p H values were measured by methylene blue-platinum reagent.The particles almost completely released hydrogen within 1 hour.Due to the slow release of the gel under acidic conditions,the perfluorohexane was incorporated into the gel to regulate the release of hydrogen by taking advantage of the characteristics of ultrasonic response of perfluorohexane.The gel coated with perfluorohexane can be completely dissolved and hydrogen can be released after 3 minutes of stimulation under 4MHz focused ultrasound.The results show that the hydrogen release from PFC-AB-PLGA gel can be controlled effectively by ultrasound.Secondly,in order to prove the antagonistic effect of PFC-AB-PLGA microparticles and gels,gentamicin-induced HEI-OC1 cell injury model and gentamicin-induced zebrafish hair cell injury model were investigated.The uptake of particles by cells was first investigated,and it was observed through a fluorescence microscope that the particles containing coumarin-6 could be well taken up by cells.However,microparticles and gels have no obvious preventive effect in the gentamicin-induced HEI-OC1 cell injury model.In the experiment of zebrafish,it was found that under the condition of continuous hydrogen,it has a certain preventive effect on hair cell damage caused by gentamicin.At the same time,it is proved that PFCAB-PLGA microparticles can effectively reduce the damage of zebrafish hair cells induced by gentamicin at 3.125 μg/m L.PFC-AB-PLGA gel had a protective effect on hair cell injury of zebrafish after 24 h administration at the concentration of 3.125 μg/m L.Compared with the microparticle and gel groups,the 1.5626 μg/m L PFC-AB-PLGA gel showed the best antagonistic effect at 1 hour after ultrasound..Finally,HEI-OC1 cells and zebrafish were used as the research objects.The toxicity of PFC-AB-PLGA particles and gel to cells was first investigated by MTT method,and then the drug was co-incubated with juvenile zebrafish to evaluate the colliculus hair cells,and the embryo toxicity of the drug to zebrafish was further investigated.The results showed that,compared with the normal control group,There were no significant differences between PFC-AB-PLGA particles and gel in survival rate,hatching rate,malformation rate and body length,which indicated that PFC-AB-PLGA particles and gel had good biocompatibility.In conclusion,the preparation of PFC-AB-PLGA particles and gel was completed in this project,and PFC-AB-PLGA particles can produce a large amount of hydrogen in order to meet the needs of clinical treatment of diseases.PFC-AB-PLGA gel not only has the ability to release hydrogen slowly,but also can release hydrogen rapidly by ultrasound,which provides a new strategy for the clinical use of PFC-AB-PLGA particles and gel to antagonize the ototoxicity of gentamicin. |
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