| Classical inner ear disease involves the entire membranous labyrinth and is characterized by the triad of sensorineural hearing loss, tinnitus and vertigo, which will seriously have impact on human hearing and balance. Intratympanic(IT) administration is often used in clinical for the treatment of inner ear disease, in which drugs mainly permeate into the inner ear through the round window membrane(RWM). RWM permeability is influenced by the size and surface properties of the penetration material, penetration enhancer, etc. Poly(lactic-co-glycolic acid)(PLGA) nanoparticles(NPs) as a drug carrier and cell-penetrating peptides as penetration enhancers are capable of promoting drug penetration through the RWM. Therefore, PLGA-NPs with different characteristics,including different sizes and different surface properties were designed in this study, and the NPs mixed cell-penetrating peptides were conducted. Near-infrared imaging system was applied to evaluate on the extent of the NPs into the cochleae. This is expected to build a more effective nano-based drug delivery system of the inner ear.Single-emulsion solvent evaporation method was used to prepare coumarin-6 loaded PLGA-NPs. Single factor of preparation process was conducted to obtain factors that had greater impact on particle size. Three factors with greater influence were revealed: PVA concentration, water-oil ratio and sonication time, while PLGA molecular weight, density and ultrasound power had smaller effect. By changing the organic phase in the preparation of NPs, combining with PVA concentration, water-oil ratio and ultrasonic time and power,different size of PLGA-NPs were prepared, with diameter of <100(79.9), 100-200(157.5)and 200-400(302.7) nm, respectively, corresponding polydispersity index(PDI) were respectively 0.158, 0.117 and 0.198, zeta potentials were-14.74,-13.32 and-7.41 m V,respectively. Storage stability of NPs after lyophilization was investigated. Based on the evaluation of NP appearance, re-solubility, their particle size and PDI after freeze-drying,the results showed mannitol of 2% was the most appropriate lyoprotectant to remain the NP stability. The diameter and PDI of redissolved NPs with different size range were consistent with before when it was stored in desiccator at room temperature, and the stability can be maintained for up to 2 months.Chitosan(CS)-modified PLGA-NPs were prepared by the principle that CS with positively charge was attracted to the negative surface of the PLGA-NPs. IR spectroscopy and X-ray photoelectron spectrogram could determine the presence of amino of CS on CS-PLGA-NPs’ surface, which was proved that CS was successfully modified to the surface of NPs. X-ray diffraction analysis showed that the crystal form for CS was changed into the amorphous state when CS was adsorbed on the surface of NPs. Zeta potential of CS-PLGA-NPs with different size range of 100-200(173.9) and 200-400(276.5 and 313.7)nm possessed positive zeta potential of 18.47, 10.70 and 1.37 m V, respectively. The absorption quantity of CS measured by ninhydrin test was 0.047 ± 0.007, 0.110 ± 0.072 and0.106 ± 0.012 mg·mg-1(n = 3), respectively.Poloxamer 407(P407)-modified PLGA-NPs were prepared by the principle that hydrophobic interaction between hydrophobic end of polypropylene oxide of P407 and the hydrophobic surface of the PLGA-NPs. By changing the preparation conditions, the size of NPs modified with P407 can be controlled in different range that were <100(77.2),100-200(177.4) and 200-400(296.9) nm, respectively, their PDI were-7.33,-15.90 and-3.08 m V, respectively. The absorption quantity of P407 were 34.10±4.78, 32.47±2.67 and28.71±1.10 μg·mg-1(n=3) respectively, which was measured by the colored complex formed by the polyoxyethylene segments in P407 interacting with cobalt thiocyanate reagent. m PEG-modified PLGA-NPs was prepared by using m PEG-PLGA polymers directly. m PEG-PLGA-NPs of different size were prepared with the range of <100(67.0),100-200(142.8) and 200-400(211.9) nm, respectively; their zeta potential were-10.79,-12.36 and-7.58 m V, respectively. All NPs prepared had encapsulation efficiency above80% by refrigerated ultracentrifugation. When being observed under TEM by negative staining, they possessed round shape, evenly distribution without sticky.The fluorescence imaging system was used to evaluate the PLGA-NPs to permeate through the RWM to cochleae. Imaging conditions including cochlear imaging in vivo and in vitro and the selection of fluorescent probes were investigated. The results showed that the image of Di R loaded NPs had less background interference after intratympanic administration than the image of coumarin-6 loaded NPs. Guinea pigs were administrated Di R loaded NPs by IT and intravenous(IV) respectively. The results for imaging of in vivo showed that NPs were accumulated mainly in the liver after IV administration, whereas this phenomenon did not occur after IT administration; results for imaging of ex vivo tissue showed that NPs accumulated mainly in the liver and spleen after IV administration, and NPs accumulated mainly in the cochlea after IT administration. Therefore, comparing to IV administration, IT administration can achieve a higher concentration in the cochlea, avoid removing by the liver and spleen.After IT administration of Di R loaded NPs with different size, at the time point of 0.5h, the amount of fluorescence in cochleae through application of 300 nm NPs was significantly higher than that applying 80 nm(p<0.05), but there was no obviously difference between NPs of 150 nm and 300 nm. At the time point of 24 h, the amount of fluorescence in cochleae after administration of 80 nm NPs was higher and the amount of fluorescence after application of 150 nm and 300 nm NPs had little difference. After IT administration of Di R loaded NPs with different surface decoration at the time point of 0.5h, fluorescence in cochleae through application of CS-PLGA-NPs was significantly stronger than that of unmodified, P407- and m PEG-PLGA-NPs(p<0.001); at the time point of 24 h, fluorescence in cochleae after administration of P407-PLGA-NPs was brighter than other NPs(p<0.001). These results suggested that the quantity of NPs cross RWM into the cochlea can be adjusted by changing the particle size and NP surface properties.Moreover, we used the fluorescence imaging system to investigate the effect of different cell-penetrating peptides(CPP) on NP permeation into the cochlea. The resultsrevealed that the CPP chosen in our study(TAT, Penetratin, LMWP and R8) can promote NPs to enter the cochlea and LMWP had the strongest effect(p <0.05). Then the mixture of LMWP and different NPs(PLGA-NPs and P407-PLGA-NPs) was evaluated. The fluorescence intensity of the cochleae through the administration of P407-PLGA-NPs was significantly higher than the administration of PLGA-NPs. Furthermore, the cochleae after application of mixture of NPs and LMWP had higher fluorescence than that of the PLGA-NPs(p<0.05). This revealed P407-PLGA-NPs as a carrier had stronger ability to cross the RWM than PLGA-NPs, and LMWP could further enhance this ability.Taking together, NPs with different diameter could be prepared by changing the preparation conditions. We can make use of the hydrophobic surface and negative charge of PLGA-NPs to prepare NPs modified by P407 and CS, separately. The results proved that the quantity of NPs across RWM into the cochlea can be adjusted by changing the particle size and surface properties of NPs. At the same time, mixing NPs with CPP further enhanced this effect, which provided experimental data for the construction of a more efficient nano-based delivery system for the inner ear. |
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