| The human physiological barrier composed of various biofilms together constitutes a tight defense system of the human body,and plays an important physiological function of preventing "invasion" by foreign invaders,but it is also an obstacle that needs to be broken to deliver drugs to the lesion.There are membrane barriers that seriously affect the bioavailability of the drug in local administration,such as the tear film barrier,corneal barrier during ocular administration,and the cuticle barrier during transdermal administration.For clinical administration of the inner ear,the tympanic membrane(TM)and the round window membrane(RWM)are the two main membrane physiological barriers to be overcome.The current methods to overcome the physiological barrier of the ear membrane can be divided into four categories:biological methods,chemical methods,physical methods,and pharmaceutics improvement methods.Among them,the physical method has attracted the attention of many researchers in recent years due to its high efficiency,simple and convenient method of administration.Common physical methods include opening the physiological barrier with ultrasound,transdermal drug delivery via microneedles,electroporation,and magnetic induction.However,the physiological barriers opened by physical methods cannot be closed timely,it will not only cause damage to the body,but also hidden a greater potential risk.At the same time,in view of the particularity of the dual physiological barrier structure of the ear,ordinary physical methods cannot achieve the sequentially overcoming of the outer ear/middle ear/inner ear dual barriers,and there are disadvantages such as poor patient tolerance.Therefore,it is of great significance to seek a drug delivery method that can sequentially overcome the outer/middle ear/inner ear dual barriers and achieve a safe and effective way to directly deliver therapeutic substances to the inner ear lesions.In this study,a microscale device,microrobots,capable of converting chemical energy into kinetic energy was constructed.The device body is a hollow chocolate rod-shaped microtube with a special structure,which can be loaded with two perfluorocarbon emulsion with different acoustic droplet evaporation(ADV)characteristics.With the help of an ultrasound system control platform capable of generating an ultrasonic trigger signal with spatiotemporal control capabilities,two emulsions could generate energy under different ultrasound treatment to propel the autonomous movement of microrobots and ejection of payloads in the device.Different magnetic field was used to control the direction of microrobotic movement and ejection.The study provides the possibility for therapeutic drugs to actively and sequentially overcome the outer ear/middle ear/inner ear dual barrier and directly deliver to the inner ear lesions.The surface sol-gel method is used to prepare microrobotic structure system.First,the silicon tetrachloride solution is hydrolyzed,condensed,aged,and dried to generate asymmetric hollow silica microtubules.Second,the magnetic iron trioxide nanoparticles were added through the layer-by-layer self-assembly method to obtain magnetic sandwich chocolate rod-shaped microtubes,and the inner surface of the microtubes was functionalized by 3-aminopropyltriethoxysilane.Finally,the functionalized magnetic structural system of the microrobotic drug delivery device was acquired.Coumarin-6-containing perfluorohexane emulsion and perfluoro-nonane emulsion were prepared by phacoemulsification method.The zeta potentials of the two emulsions were-60mV and-40mV,respectively,which illustrate that they had good physical stability.The confocal laser scanning microscope(CLSM)was used to investigate the electrostatic force adsorption and stability of the perfluorohexane emulsion on the inner surface of the structural system-MT(microtubes).At the same time,the optimal loading parameters of temperature-sensitive gel and payload were investigated.The results showed that the mixture of perfluoro-nonane emulsion,temperature-sensitive gel and nanoparticles at a ratio of 1:2:1 was successfully loaded into the MT at suitable loading depth by injecting gas for 15s at room temperature.Through a large number of literature investigations,the ultrasound transmission platform of microrobotic system was successfully established.The ultrasound transmitting platform consists of a PXI signal generator,a radio frequency amplifier,an impedance matcher,and an ultrasonic transducer.A device for in vivo and in vitro experiment is set up.A program for the ultrasound generator is established using NI Lab VIEW software to set appropriate signal.The acoustic performance of the ultrasound transmitting platform was examined using a fiber optic hydrophone test system.The ultrasonic trigger parameters of two different emulsions were determined through theoretical calculations and literature review.The primary ultrasonic parameters were 2.25 MHz center frequency,2V voltage and 20 ?s action time,and the secondary ultrasonic parameters were 4 MHz center frequency,2V voltage and 10 ms action time.3D imaging of the auditory vesicles of the guinea pig was performed using Micro CT to determine the relative position between the probes and guinea pigs during in vivo and in vitro experiments.High-performance liquid chromatography(HPLC)was used to verify the effectiveness of the ultrasound parameters.It was shown that the first-level ultrasound parameters,while propelling the movement of the microrobotic drug delivery device,would not cause leakage of the payload.The second-level ultrasound parameters could be successfully releasing the payload from the device.Using a temperature-sensitive gel as a model,through layer-by-layer scanning by CLSM,the device is shown to have the ability to move and launch payload in vitro.Finally,the device was applied to the acoustic bullae in vitro.The CLSM results showed that the device has an independent ability to move across the TM and the ability to launch a payload across the RWM.Two fluorescent substances,Nile Red and Coumarin 6,were used as markers of the success of trans-TM delivery and trans-RWM delivery.HPLC was used to detect the contents of two fluorescent substances in external ear canal lavage fluid,middle ear cavity lavage fluid,and lymphatic fluid.CLSM was used to observe the movement traces of microrobots in the TM and the distribution of chitosan nanoparticles emitted from microrobots in the RWM.Finally,the biological safety of the microrobotic drug delivery device was investigated in terms of cytotoxicity and single/multiple dose toxicity in animals.Cytotoxicity experiments showed that the substances delivered by the microrobotic drug delivery device and itself would not inhibit the viability of HEI-OC1 cells and L929 cells within a certain concentration range.After single and multiple administrations of the guinea pigs through the microrobotic drug delivery device,the middle ear cavity mucosa not have any qualitative damage or inflammation.The inner ear basement membrane hair cells had good morphology,normal blood routine indicators,and blood biochemical indicators.It was suggested that no matter whether multiple administrations or single administrations will not cause changes to the physiological indicators of the animals,important organ tissue sections show no renal toxicity,liver toxicity and cardiotoxicity.In summary,the microrobotic device constructed in this paper can overcome the dual ear physiological barriers of the TM and RWM and delivering the payload to the inner ear lymph fluid from external ear canal,greatly improving the delivery efficiency of nanoparticles.The design of the device provides a new delivery platform for the treatment of middle ear and inner ear diseases,and provides a new idea for clinically overcoming physiological barriers in the body. |
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