고실내 투여 후 쥐 내이에서 리포솜 나노운반체의 생체적합성

Methods

Materials

Sphingosine (Sph), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (ammonium salt) [DSPE-PEG-2000] were purchased from Avanti polar lipids (Alabaster, USA). DiI (Vybrant DiI cell-labeling solution, 1 mM in solvent) and N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-hosphoethanolamine, triethylammonium salt (TRITC-DHPE) were purchased from Thermo Fisher Scientific (Waltham, USA). Gd-DOTA (DOTAREM) was from Guerbet, Cedex, France. Hepes and EDTA were from Sigma. The purity of the lipids was evaluated using thin-layer chromatography on silicic acid-coated plates (Merck, Darmstadt, Germany) developed with a chloroform/methanol/water mixture (65:25:4, v/v/v). An examination of the plates after iodine staining and, when appropriate, upon UV illumination revealed no impurities. The lipid concentrations were determined gravimetrically with SuperG (Kibron, Espoo, Finland); a high-precision microbalance. The polyvinylpyrrolidone-stabilized AgNPs were supplied by Colorobbia (Firenze, Italy). The AgNPs were dispersed in deionized water (370.7 mM), and scanning electron microscopy showed that the AgNPs are spheroids with a particle size of around 100 nm. Dynamic light scattering (DLS) showed a mean hydrodynamic size of 117 ± 24 nm and a mean zeta potential of −20 ± 9 mV.

In the visualization of nanocarrier uptake in the inner ear and the evaluation of biological barrier function, 5 male Sprague Dawley rats, weighing between 334 and 348 g, were provided by the Biomedicum Helsinki, Laboratory Animal Centre, University of Helsinki, Finland (this is the defined animal center that provides animals for MRI experiments in Biomedicum); in the ABR and histological studies, 18 Sprague Dawley rats, weighing between 300 and 400 g, were provided by the Experimental Animal Unit of the University of Tampere School of Medicine in Finland. Animal assignments in each study were shown in Table 2. All animal experiments were approved by the Ethical Committee of University of Tampere (permission number:ESAVI/3033/04.10.03/2011). Animal care and experimental procedures were conducted in accordance with European legislation. Animals in the Gd-MRI study were anesthetized with isoflurane with 5% isoflurane–oxygen mixture (flow-rate 1.0 L/min) for induction and 3% for maintenance via a facemask. Animals for the ABR and histological studies were anesthetized with a mixture of 0.5 mg/kg medetomidine hydrochloride (Domitor ^® , Orion, Espoo, Finland) and 75 mg/kg ketamine hydrochloride (Ketalar ^® , Pfizer, Helsinki, Finland) via intraperitoneal injection followed by an intramuscular injection of enrofloxacin (Baytril ^® vet, Orion, Turku, Finland) at a dose of 10 mg/kg to prevent potential infection. The animal’s eyes were protected by Viscotears® (Novartis Healthcare A/S, Copenhagen, Denmark).

Preparation and Characterization of LPNs with and without Gd

Preparation of Gd-containing LPNs

LPNs of unilamellar vesicles with an apparent hydrodynamic particle diameter (Z _av ) of 110 ± 15 nm that contain Gd-DOTA were prepared according to the previously published method [6]. A concentration of 1 mM Gd-DOTA-containing LPN (LPN + Gd-DOTA) refers to 1 mM liposomes encapsulating 500 mmol/L of Gd-DOTA.

Preparation of Blank LPNs

Blank LPNs of unilamellar vesicles with Z _av of 115 ± 10 nm were prepared according to a previous publication [6].

Administration of LPNs

Under general anesthesia with isoflurane with 5% isoflurane–oxygen mixture (flow-rate 1.0 L/min), 50 μl of either LPNs or LPN + Gd-DOTA were injected into the left middle ear cavity through the tympanic membrane penetration under an operating microscope (OPMI1-F, Carl Zeiss, Jena, Germany). The same amount of deionized water (H₂ O) was injected transtympanically in rats that were assigned to the negative group. After the injection, the animals were kept in the lateral position with the injected ear oriented upward for 15 min before further measurements.

Evaluation on Biological Barrier Function Using Gd-MRI

One animal receiving transtympanic injection of LPN + Gd-DOTA was selected to demonstrate distributions of LPN in the inner ear using MRI without contrast agent. Two animals receiving transtympanic injection of blank LPNs were engaged in MRI study for evaluation of the biological barrier function. Two animals receiving transtympanic injection of AgNPs (370.7 mM, 40 μl) were used as positive control. The contralateral ear without any injection was used as negative control in all studies. A 4.7T MR scanner with a bore diameter of 155 mm (PharmaScan, Bruker BioSpin, Ettlingen, Germany) was utilized. The maximum gradient strength was 300 mT/m with an 80-μs rise time. A gadolinium-tetraazacyclododecane-tetraacetic acid (Gd-DOTA, 500 mM, DOTAREM, Guerbet, Cedex, France) solution was injected into the tail vein (0.725 mM/kg) 2 h before the MRI measurements. The imaging protocol and rapid acquisition with relaxation enhancement (RARE) sequences were applied according to a previous publication [10]. MRI scanning commenced at several time points after the transtympanic injection. The first MRI time of around 5 h was determined by taking the penetration time of liposome nanoparticles from the middle ear to the inner ear as a reference [1, 3, 6]. The final imaging time of 8 d was selected according to the course of potential acute inflammation and the availability of the scanner. ParaVision PV 4.0 (Bruker, MA, USA) software was used for the post-processing and quantification of MR images.

ABR Measurement

The auditory function of animals receiving injections of both blank and Gd-containing LPNs were evaluated using ABR measurements using BioSig32 (Tucker Davis Technologies, FL, USA) in a custom made, soundproof chamber. The ABR thresholds upon click and tone burst stimuli were recorded before and at a certain time point post-administration of LPNs. The first ABR measurement was followed on 2 days post-administration of AgNPs, allowing the animals to recover from the general anesthesia during the injection and to ensure the injected solution to be entirely cleared from the middle ear cavity. The second follow-up time of 4 days post-injection was chosen because it is close to the peak time of potential mitochondrial impairment-induced cell death in the cochlea [22]. The third follow-up time of 7 days is the time point when temporary threshold shifts remained significantly approved in an animal model of mitochondrial toxin-induced hearing loss [28]. The ABR recording procedure followed the previous report [11].

Glycosaminoglycan Staining in Rat Cochlea After Administration of LPNs

Hematoxylin-eosin staining to assess potential inflammatory infiltration and periodic acid Schiff’s staining to evaluate potential glycosaminoglycan accumulation in the cochlea after administration of LPNs were performed according to a previous publication after ABR measurements over 7 days [11] The slices were observed and digital images were acquired under a light microscope (Leica DM2000 microscope equipped with an Olympus DP25 camera) for further analysis.

Immunofluorescence Staining for Hyaluronic Acid and Receptors

Immunofluorescence staining for hyaluronic acid, CD44, and TLR2 were performed according to a previous publication after ABR measurements over 7 days [11, 21].

Cell Death Detection

Potential nuclear DNA fragmentation in the cochlea was investigated using terminal transferase (TdT) to label the free 3′OH breaks in the DNA strands of apoptotic cells with TMR-dUTP (TUNEL staining) following the reported procedure [11]. Slices exposed to recombinant DNase I (Fermentas, Vantaa, Finland, 100 U/ml in 50 mM Tris/HCl, pH 7.5, 1 mg/ml bovine serum albumin) at 37 °C for 10 min, which induced DNA strand breaks prior to the labeling procedures, were utilized as positive controls. The samples were observed under a confocal microscope.

Confocal Microscopy

The samples were observed under a Nikon inverted microscope (ECLIPSE Ti) combined with an Andor confocal system installed with Andor iQ 2.8 software (Andor Technology, Belfast, UK). The excitation lasers were 488 nm (green excitation) and 568 nm (red excitation) from an Andor laser combiner system, and the corresponding emission filters were 525/50 (Alexa Fluor-488) and 607/45 nm (Cy ^TM 3 and TMR Red). DAPI was excited with light at 405 nm generated from a light-emitting diode and was detected using a 450–465 nm filter.

Analysis and Statistics

ImageJ (1.45S, National Institutes of Health, Bethesda, USA) software was used for signal intensity measurements. For light microscopy of periodic acid Schiff’s staining, the region of interests (ROIs) including spiral ligament, spiral limbus, and stria vascularis were selected using freehand selections button. The “measure” function was used to obtain the mean gray scale value of the ROI, which was inversely correlated with the staining intensity. For confocal microscopy of immunofluorescence staining, the ROIs including stria basal cells, spiral ganglion cells, spiral ligament, and spiral limbus were extracted using photoshop CS6 (version 13.0, Adobe Systems Software Ireland Ltd, Dublin, Ireland) program and were imported into ImageJ program. The images were split into individual channel, and the green (corresponded to CD44 and TLR2) and red (corresponded to hyaluronic acid) channels were selected for further quantifications. The “Threshold” was adjusted using the “set” button in the “Image” menu, and “Limit to Threshold” option should be selected and “Direct to” should be defined to the corresponding channel in the “Analyze” menu. Then the gray scale value, which was inversely correlated with the staining intensity, was obtained using the “Measure” function in the same menu.

Statistical analyses were performed using the IBM ^® SPSS ^® Statistics Version 20 software package (SPSS Inc., Chicago, USA). A one-way ANOVA and Kruskal-Wallis test were used to compare ABR threshold shifts and signal intensities (grayscale) of staining for glycosaminoglycan and hyaluronic acid secretions, and TLR2 and CD44 staining between the LPN injected-ear and saline injected-ear groups in the different cochlear structures among various turns. Least significant difference (LSD) test was used as post hoc analysis. Higher numbers in the grayscale analysis correlate with lower signal intensities of the staining. 피  < 0.05 was accepted as statistically significant.