Bovine serum albumin nanoparticles as controlled release carrier for local drug delivery to the inner ear
© Yu et al.; licensee Springer. 2014
Received: 1 January 2014
Accepted: 26 June 2014
Published: 9 July 2014
Nanoparticles have attracted increasing attention for local drug delivery to the inner ear recently. Bovine serum albumin (BSA) nanoparticles were prepared by desolvation method followed by glutaraldehyde fixation or heat denaturation. The nanoparticles were spherical in shape with an average diameter of 492 nm. The heat-denatured nanoparticles had good cytocompatibility. The nanoparticles could adhere on and penetrate through the round window membrane of guinea pigs. The nanoparticles were analyzed as drug carriers to investigate the loading capacity and release behaviors. Rhodamine B was used as a model drug in this paper. Rhodamine B-loaded nanoparticles showed a controlled release profile and could be deposited on the osseous spiral lamina. We considered that the bovine serum albumin nanoparticles may have potential applications in the field of local drug delivery in the treatment of inner ear disorders.
KeywordsBovine serum albumin Nanoparticle Controlled release Drug delivery Round window membrane Inner ear
Inner ear disorders, including sensorineural hearing loss (SSHL), commonly occur in clinics. The traditional systemic therapies are almost ineffective due to the blood-labyrinth barrier, which prevents the transport of drugs from the serum. Local drug delivery, especially intratympanic injection, has become popular for two decades because of its efficiency and safety. The round window membrane (RWM) is a semipermeable membrane between the middle and the inner ear, through which particles less than 3 μm in diameter could penetrate.
Local drug delivery to the inner ear by intratympanic injection was first described by Schuknecht in 1956 in the treatment of Ménière's disease. In 2006, Kopke et al. reported a significant hearing improvement of patients with sudden sensorineural hearing loss after methylprednisolone administration locally.
Although intratympanic injection is easy to perform in the clinic, the loss of drug through the Eustachian tube becomes the obstacle to treat inner ear disorders efficiently. Thus, hydrogel- and particle-based vehicles (or carriers) have been investigated recently for sustained and prolonged drug supply. In 1998, Balough et al. described that the local injection of a fibrin-based sustained release vehicle impregnated with gentamicin allowed for a prolonged effect without absorption in the untreated ear or blood. Horie et al. reported that drug-loaded polylactic/glycolic acid (PLGA) microparticles were capable of delivering lidocaine into the cochlea in a sustained manner. The PLGA nanoparticles were found to be distributed throughout the inner ear after application on the RWM of chinchilla. Moreover, Tan et al. demonstrated that brain-derived neurotrophic factor encapsulated in nanoporous poly(l-glutamic acid) particles could be released in a sustained manner with maintained biological activity and efficiently rescue primary auditory neurons in the cochlea of guinea pigs with sensorineural hearing loss. Nowadays, nanoparticles have received much more interest for the treatment of inner ear diseases for their drug loading and sustained release capacity.
Various methods such as desolvation, emulsion, template, microfluidic, mechanical stretching, and self-assembly have been reported to prepare protein-based particles or capsules[7–11]. In this study, we demonstrated that bovine serum albumin (BSA) can form nanospheres by desolvation method and can be used for local drug delivery.
BSA is a natural protein able to form complexes in various shapes. This protein is biocompatible, biodegradable, nontoxic, and nonimmunogenic. Due to these features, albumin particles are a good system for drug and antigen delivery[11–14]. To the best of our knowledge, there have been no reports of local delivery of drug-loaded albumin particles into the inner ear. Here, we illustrate a method for creating sphere-shaped BSA nanoparticles (BSA-NPs) with biocompatibility in high yield. A model drug, rhodamine B (RhB), was loaded onto the BSA-NPs for drug loading capacity, release, and in vivo studies. In vivo biodistribution suggested that the RhB released as well as the RhB-loaded BSA-NPs (RhB-BSA-NPs) tended to accumulate and penetrate through the RWM of guinea pigs. Therefore, the BSA-NPs would be prospectively considered as controlled release carriers for local drug delivery in the treatment of inner ear disorders.
Materials, mice, and cell culture
BSA and RhB were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technology Inc. (Shanghai, People's Republic of China). Ultrapure water used in all experiments was produced by Milli-Q synthesis system (Millipore Corp., Billerica, MA, USA). L929 mouse fibroblast cells (obtained from the Cancer Institute of the Chinese Academy of Medical Sciences, People's Republic of China) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, Thermo Scientific Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS) at 37°C with 5% CO2. Guinea pigs weighing 250 ~ 300 g were purchased from the Tianjin Experimental Animal Center, People's Republic of China, and had free access to food and water. Animal study protocols were approved and performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals.
Preparation of BSA-NPs and RhB-BSA-NPs
BSA-NPs were prepared by the desolvation method. Briefly described, 100 mg of BSA was dissolved in 1 ml of sodium chloride solution (10 mM). Then, 8.0 ml of ethanol was added dropwise into the BSA solution under magnetic stirring (400 rpm) at room temperature. Subsequently, the as-prepared BSA-NPs were cross-linked with 0.2% glutaraldehyde (GA) for 24 h or denatured at 70°C for 30 min. BSA-NPs (50 mg) were incubated with certain amounts (5, 10, 15, 17.5, and 20 mg) of RhB for 2 h in the preparation of RhB-BSA-NPs. The particles were centrifuged and washed with ultrapure water.
Characterization of the BSA-NPs
The morphological characteristics were determined by transmission electron microscopy (TEM, JEOL, JEM-100CXII, Akishima-shi, Japan), scanning electron microscopy (SEM, ZEISS SUPRA 55VP, Oberkochen, Germany), and confocal laser scanning microscopy (CLSM, FV-1000, Olympus Corporation, Shinjuku-ku, Japan). For TEM, a drop of diluted suspension of BSA-NPs was placed on the copper grid and the air-dried specimen was observed. For SEM, a drop of diluted suspension was deposited on a silicon wafer. The air-dried sample was coated with gold and observed. RhB-BSA-NPs were observed by CLSM at an excitation wavelength of 555 nm and an emission wavelength of 580 nm.
The BSA-NPs were dispersed in ultrapure water at a concentration of 0.1 mg/ml. The particle size and zeta potential determinations were performed by using a Malvern particle size analyzer (Zetasizer Nano-ZS, Malvern, UK).
Drug loading capacity and encapsulation efficiency
BSA-NPs (50 mg) were incubated with RhB (5 ~ 20 mg) for 2 h. After washing with ultrapure water, the supernatants were collected and analyzed for residual drug concentration by UV-vis analysis.
Encapsulation efficiency (w / w%) = amount of RhB in BSA-NPs/RhB initially added × 100
In vitro drug release behavior
The assay was evaluated in a standard static diffusion cell at a speed of 100 rpm in a shaker at 37°C. The amount of RhB was evaluated using UV-vis spectrometer (560 nm). The amount of RhB released was evaluated at a series of time points, and the release curve was made accordingly.
Cell biocompatibility assay
Cells were seeded in 96-well plates at a density of 1,000 cells/well. BSA-NPs with GA fixation (NP-GA) or heat denaturation (NP-H) were added to each well for a 24-h incubation. Cell viability was determined by CCK-8 assay. Untreated cells served as the control. The morphology of L929 cells in each group was also observed by using a phase contrast microscope.
In vivo assay
Guinea pigs were killed to sample the acoustic bullae (including the RWM). The acoustic bullae were placed in the solution of BSA-NPs and shaking for 30 min at 37°C. The air-dried specimens were observed by SEM.
The penetration of RhB released from the RhB-BSA-NPs was evaluated by live images and microscopes. Guinea pigs were anaesthetized and the RWMs were exposed. The heat-denatured RhB-BSA-NPs and RhB dispersed in PBS were injected slowly into the bullae of the right and left ear, respectively. The left ear injected with RhB solution was the control. In vivo imaging system (Caliper IVIS imaging system, PerkinElmer, Waltham, MA, USA) was used to trace the particles at time points of 0 and 72 h. The RWM was then imaged by fluorescence microscopy and SEM to observe the distribution of RhB and BSA-NPs.
The statistical data was presented as the mean value and standard deviation. The analysis of t test was used in SPSS 12.0 to determine significant differences between groups, and P values less than 0.05 were considered statistically significant.
Results and discussion
Morphology of BSA-NPs
Drug loading and release study
The BSA-NPs and RhB-BSA-NPs had zeta potential values of -15.4 and +4.98 mV, respectively. The potential difference demonstrated that the positively charged RhB had an interaction with the negatively charged BSA, which also promoted the attachment of RhB to the BSA. The fluorescent image of the RhB-BSA-NPs (Figure 2b) further confirmed that RhB had attached to the BSA-NPs. Thus, the model drug and small molecules could affect certain parameters including size and charge of polymers, which was in agreement with the previous reports[16–19].
The drug loading capacity and encapsulation efficiency of BSA-NPs were also evaluated. The drug loading capacity of BSA was 15.4% for RhB (Figure 2c). The maximum encapsulation efficiency was 40.9% (Figure 2c). It was likely attributed to the electrostatic interaction and hydrophobic interactions between RhB and BSA followed by diffusion of the model drug into the BSA matrix[8, 16]. Nevertheless, the drug cannot diffuse into the matrix more after achieving the kinetic equilibrium state. The results in this report were consistent with the report described by Shi and Goh.
The in vitro drug release profile of RhB from BSA-NPs is shown in Figure 2d. A good sustained release profile is achieved. The cumulative release of RhB over a period of 150 hours was 429.14 μg, indicating a good affinity between the BSA and RhB. This was governed by Fickian diffusion due to the electrostatic interaction, which restricted the release of positively charged RhB from negatively charged BSA in vitro.
In vitro cytocompatibility study
In vivo distribution and drug delivery of BSA-NPs
In summary, BSA-NPs were fabricated via a desolvation method. The heat-denatured BSA-NPs had a great potential application for local drug delivery into the cochlea to treat inner ear diseases due to the tiny size, good biocompatibility, drug loading capacity, and controlled release profile. Further studies will focus on the evaluation of drug-loaded BSA-NPs, including prednisolone. We will evaluate their pharmacokinetics, pharmacodynamics, and delivery mechanism in animal model. The BSA-NPs also shed light in the treatment of human inner ear diseases.
ZY is a professor from the Department of Otorhinolaryngology, The Second Artillery General Hospital of Chinese People's Liberation Army, Beijing, 100088, People's Republic of China, and Center of Otorhinolaryngology, Naval General Hospital of Chinese People's Liberation Army, Beijing, 100037, People's Republic of China. MY is a Ph.D. from the Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, College of Basic Medicine, China Medical University, Shenyang 110001, People's Republic of China. ZZ, GH, and QX are Ph.D. from the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College, The Key Laboratory of Biomedical Material of Tianjin, Tianjin, 300192, People's Republic of China.
bovine serum albumin
bovine serum albumin nanoparticles
round window membrane
cell counting kit-8
BSA-NPs with GA fixation
BSA-NPs with heat denaturation
dynamic light scattering
transmission electron microscope
scanning electron microscopy.
We are grateful for the financial support of the Project in the Eleventh Five-Year Plan of the Second Artillery General Hospital of Chinese People's Liberation Army.
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