Novel Drug Delivery Model For Compound Multi-component Via Inner Ear Administration: Nanoparticles And Nanoparticles-loaded Temperaturesensitive Hydrogels Delivery Systems

According to the World Health Organization,approximately 360 million people,5% of the world’s population,are suffering from disabiling hearing impairment in 2013.Nearly two-third of adults aged 70 years or old develop significant age-related hearing loss（AHL）,a condition that can lead to social isolation and major communication difficulties,seriously affecting the patient’s normal life.In recent years,local intratympanic（IT）injection has become one of the new methods of treatment of inner ear diseases.IT administration,a convenient,safe and minimally invasive route,can reduce the administration dosage and side effects of drugs.However,due to the large volume of the middle ear cavity and the existence of eustachian tube,the natural diarrhea channel,the actual amount of drug that contact with the round window membrane（RWM）and through the RWM into the inner ear is very limited or not very accurate.The temperature sensitive in situ gel that can change phase with the change of temperature,can achieve injection implantation and long-term release,widely used in ocular,nasal and injection administration,and plays an important role in the field of pharmacy,which has become a hotspot in recent years.Drugs loaded in the thermo-sensitive gels have obvious sustained-release characteristics,which is expected to become a new type of inner ear topical administration system.With the study of thermosensitive hydrogel,some research work has combined thermo-sensitive hydrogels with nanoparticles,liposomes or microspheres to maximize the advantages of both preparations and to overcome the deficiency of single preparation.And the composite drug delivery system can improve the solubility and stability of the drug,and achieve a better sustained and controlled release effect.Salvia miltiorrhiza and Panax notoginseng,as one of commonly used traditional Chinese medicine pairs,both have the effect of promoting blood circulation and removing blood stasis.Compatible using can achieve complementary,so widely used in the treatment of sudden deafness,noise hearing loss and other inner ear diseases.In this study,the effective components of Salvia miltiorrhiza and Panax notoginseng,notoginsenoside R₁（R₁）,and ginsenoside Rb₁（Rb₁）,Rg₁（Rg₁）,Salvianolic acid B（Sal B）and tanshinone IIA（Ts IIA）,were used as model drugs.PLGA nanoparticles were prepared by double emulsion-solvent evaporation technique（W/O/W）with biocompatible PLGA.The size,particle size distribution（PDI）,entrapment efficiency and drug loading of nanoparticles were used as the evaluation indexes,and the prescription was optimized by single factor method.In order to improve the solubility of TS IIA,hydroxypropyl-β-cyclodextrin inclusion complex of tanshinone IIA（TS IIA-HP-β-CyD）was prepared by evaporated method,then incubated with nanoparticles to obtain core-shell PLGA nanoparticles（HP-β-CyD-PLGA NPs）.The average particle size determined by laser particle size analyzer was 234.12±4.83 nm,with the polydispersity index（PDI）of 0.12±0.02,and Zeta potential was 44.42±4.13 mV.The entrapment rate of R₁、Rg₁、Rb₁、Sal B and TS IIA was 78.97±6.39%,85.22±5.95%,85.64±8.23%,76.59±9.19% and 94.70±2.56%,respectively.And the drug loading efficiency was 0.37±7.24%,0.27±5.86%,0.54±9.12%,0.33±12.39% and 0.23±8.34%,respectively.Transmission electron microscopy（TEM）showed that HP-β-CyD-PLGA NPs had significant core-shell structure,which was spherical and uniform distribution.The in vitro release of TS IIA-HP-β-CyD-PLGA NPs was evaluated by dialysis bag method.The results showed that the release of R₁,Rb₁ and Sal B was consistent with the first-order release kinetics model.The release process accord with the Higuchi model and the TS IIA release process accord with the Ritger-Peppas model.The TS IIA-HP-β-CyD-PLGA NPs were loaded into two kinds of thermosensitive gels to synthesis nanoparticles-gel two-phase release system,using poloxamer and chitosan as the main matrix materials.The tube inversion method showed that the two thermosensitive gels containing nanoparticles had good temperature-sensitive properties at 37℃.In vitro drug release studies of HP-β-CyD-PLGA-NP-P-gel showed that each component have a significant sustained release effect,and the release of R₁,Rb₁ and TS IIA in accordance with the Ritger-Peppas model,the release of Rg₁ and Sal B conforms to the Higuchi model.And the release mechanisms for all components were the common effect of dissolution and diffusion.Scanning electron microscopy（SEM）showed that the chitosan gel（HP-β-CyD-PLGA-NPs-CS/GP-gel）possess three-dimensional network structure,and the nanoparticles were successfully encapsulated.In vitro release studies showed that the release of each component in HP-β-CyD-PLGA-NPs-CS/GP-gel also conformed to the dissolution-diffusion kinetics model.The release of Rg₁ was in accordance with the Higuchi model and the other four components were in accordance with Ritger-Peppas model.In addition,in the presence of lysozyme in PBS solution,the gel has good degradation properties.In the guinea pig experiment,The distribution of nanoparticles,which co-loaded rhodamine B and coumarin 6（C-6-HP-β-CyD-RB-PLGA NPs）,nanoparticles-poloxamer gel（C-6-HP-β-CyD-RB-PLGA-P-gel）and nanoparticle-chitosan gel（C-6-HP-β-CyD-RB-PLGA-NPs-CS/GP-gel）in the organ of Corti（OC）,spiral ganglion cells（SGNs）and vascular veins（SV）in the cochlea were investigated.And the drug distribution and pharmacokinetics of salvia miltiorrhiza-Panax notoginseng effective compounds in the guinea pig perilymphal（PL）following IT injection were also investigated.The results showed that the nanoparticles,nanoparticles-poloxamer gel and nanoparticle-chitosan gel drug delivery systems could successfully delivery drugs into the cochlear and distribution in OC,SGNs and SV.The nanoparticle-gel composite systems prolonged the release time of the drugs for 96 h,and the pharmacokinetic parameters showed that they prolonged the mean residence time（MRT）of the drugs in guinea pig PL and increased the bioavailability of TS IIA.In summary,the nanoparticles-gel two-phase release system of Chinese medicine compound combined the properties of nanoparticles and gel.In the form of solution following IT administration,it can occur solution-gel transition in the middle ear cavity,so that extending the contact time of drug with RWM to achieve sustained release effect,and improve local bioavailability.Meanwhile,it can reduce the injection frequency,overcoming the defects of liquid preparations required repeated administration,is a novel drug delivery model for sustained release with great potential for clinical application in the inner ear.