| IntroductionZoledronic acid as a hydrophilic bioactive agent is a kind of bisphosphonates. It have been widely used in the treatment of bone diseases associated with in-creased bone resorption, such as postmenopausal oste-oporosis and Paget's disease. Its administration by intravenous or oral application is not con-venient for the patients because some cases under-went orbital inflammatory disease after intravenous infusion of zoledronic acid and oral route often suffers from low bioavailability, and may even in-duce side effects such as osteonecrosis of the jaws 8.So direct administration in focus site is a desired selection.During the past few decades, considerable efforts have been made to develop drug delivery systems (DDSs) by electrospinning because they have some unique features such as a high surface area to volume ratio, flexibility of morphological designability, and an extracellular matrix-like structure. Hydrophobic agents can be conveniently loaded into polyester nanofibers through direct electrospinning, and sustained release can be achieved, however, incorp-oration and sustained release of hydrophilic agents by conventional electros-pinning techniques remain challenging.Emulsion electrospinning is a current method to load water-soluble agent into polymer fibers. Some research entrapped human nerve growth factor (NGF) into poly (phosphoester) fibers via emulsion electrospinning. The loading efficiency of NGF in the fibers was extremely low due to the possible aggregation of the protein aqueous droplets suspended in poly(phosphoester) organic solution during electrospinning process. Coaxial electrospinning is another important method to prepare fibers loading water-soluble agents. It achieved a core-shell structure and proved its function as drug delivery device. However, a special apparatus and careful selection of operation parameters must be needed to ensure de-sired results. Further researches about hydrophilic agents release remain continue.We attempt to control zoledronic acid release utilizing sandwich structure-like multilayer nanofiber meshes. Vitro drug release experiment of the structure shows a desired and controllable zoledronic acid release profile. Compared with other DDSs, the main advantages of the sandwich structure-like fiber meshes are facile preparation condition and the generality, whether hydrophobic or hydrophilic pharmaceuticals can get help from this structure to control their release.Materials and methods(1) MaterialsPoly (L-lactic acid) (PLA Mw=50,000) and Poly-ethylene oxide (PEO, Mw=80,000) were gifts from Changchun Institute of Applied Chemistry Chinese Academy of Sciences. Zoledronic acid and Rifampicin were purchased from Jinan Wedo Industrial Co., Ltd (Shandong, China). A biogel was purchased from Beijing Fuaile Science and Technology Development Co., Ltd (Beijing, China). The biogel consists of three components. The other chemicals were obtained commercially, and were of analytical grade.(2) Preparation of electrospun solutionsPLA was dissolved at 5 wt% in chloroform.In order to obtaining homogeneous nanofiber,5 wt% of Benzyltrie- thylammonium chloride (with respect to PLA) was added to chloroform as a surfactant. PEO was dissolved at 12wt% in 2:1 (w/w) ratio of distilled water and ethanol,2wt% Zoledronic acid (with re-spect to PEO) and 5wt% Sodium dodecyl sulfate (SDS, with respect to PEO) were added to the mix-ture solvent as a model and a surfactant, respectively.(3) ElectrospinningElectrospinning was performed using the following device:a high-voltage power supply, an infusion pump, a glass syringe equipped with a stainless steel blunt-ended needle (inner diameter=0.8mm), and a stainless steel collector (S=15×15cm2). The PLA solution was delivered from the needle to the collector at a flow rate (5ml/h), with an air gap of 15-20cm between the collector and the needle tip. The driving voltage was 2.5-3.0kV. The PEO solution was delivered at a constant flow rate (0.8-1 ml/h).To eliminate remained organic solvent, all the experiments were conducted at a temperature about30?. All the elec-trospinning parameters were kept constantly.(4) Fabrication of Sandwich structure-like nanofiber meshes Sandwich structure-like nanofiber meshes were fabricated via sequential electrospinning. First, PLA solution was electrospun as bottom layer of the structure. Then PEO solution was electrospun as middle layer. Finally, same PLA solution was electrospun as top layer. PLA nanofiber was in external layer of the construction as barrier mesh and PEO nanofiber containing zoledronic acid was in internal layer as drug-loaded mesh. Different samples were fabricated by adjusting the thicknesses of the external layer and the internal layer. A biogel or PLA nanofiber electrospun toward cross-section was used to seal cross-section of the structure edge..(5) Morphological analysesAn environmental scanning electron microscope (ESEM, Model XL 30 ESEM FEG from Micro FEI Philips) was applied to observe the microscopic structures of the electrospun nanofibers. Accelerating voltage of ESM was 20 kV. Samples were mounted on metal stubs using an adhesive tape and vacuumcoated with a platinum layer before examination. Morphology of electrospun nanofibers was re-corded.(6) In vitro release experimentsThe medicated multilayer nanofiber meshes were cut into 6.0cm×6.0cm pieces. Each sample was soaked in a glass vial with 20.0ml of PBS (pH=7.4) at 37?and kept under rotational shaking (100 rpm). At various time points, lml supernatant was retrieved from the vial and replaced with an equal volume of fresh medium. The concentration of zoledronic acid in the supernatant was then determined via an Agilent 1100 HPLC (Agilent Technologies). The column was a Prevail C18 (Agilent Technologies). The mobile phase consisted of 0.03 tetrabuty-ammonium hydroxide in distilled water (pH=2.54) and acetonitrile (97/3 vol.%) at a flow rate ofl.0 ml/min. Concentration determination was performed using UV detection at a wavelength of 218 nm.A standard calibration plot of zoledronic acid in the concentration range of 0.04-0.3mg/ml was used to determine the concentration of the released drug. The detected area of zoledronic acid was converted to its concentration according to calibration plot.Results(1) Sandwich structure-like multilayer meshes and Fibers characterizationsHydrophilic Zoledronic acid was hardly incorporated in hydrophobic polymer such as PLA, or the burst release would happen. To date, it is seldom that a hydrophilic polymer is used solely as drug-loaded fibers for controllable drug release due to its high water solubility. To solve the problem, we tried to fabricate sandwich structure-like device for controlled release of Zoledronic acid by combining water-soluble PEO and hydrophobic PLA as inner layer and outer layer, respectivelyTo observing the sandwich structure obviously, Rifampicin was added into PEO solution so that PEO fibers were tinged with the color of orange. Rifampicin did not be added in zoledronic acid release study. Result can be seen that whether the pure PLA or drug-loaded PEO fibers, their surfaces are smooth, the beads are not formed, and diameter is also even. zoledronic acid disperse in PEO fibers uniformly. When a ratio of distill water and ethanol is 2:1(w/w), the effect is satisfactory.(2) In vitro zoledronic acid release studyBecause of PEO fibers high water solubility, the junction of each fiber layer in the sandwich structure-like meshes is easy to depart in PBS without any limbic cross-sectional management. Biogel has been used to strengthen friable tissues safely and effectively in medical researches 10.And then the cross-section sealed with a biogel and electrospun PLA were observed respectively. Release behavior of zoledronic acid from the meshes is demonstrated.The sample B shows extent of initial release (ca.72%) with a sustained release pattern during 12 hours, while there is higher extent of initial release (ca.88%) with less controlled release during same period for the sample A. The release behavior of the sample A results from parting between layers, which have been observed in our experiment. It could be the electrospun PLA seal was not good enough to prevent departing. Though the release behavior of the sample B is unsatisfactory, it keeps the structure complete. So we select the biogel management as unique method to fabricate our DDS. The reason why the sample B is unsatisfactory could be that thickness of barrier mesh is too thin to control drug release effectively. It will be discussed the effect of thickness on drug release in the following section. The difference between Sample B and sample C is thickness change of barrier mesh, while thickness of drug-loaded mesh is identical. The drug release profiles from sample B and sample C are evaluated. Sample B presents a pronounced initial burst release (72.1%), which is followed by a gradual release during the rest time. By contrast, sample C shows a satisfactory release behavior. The first rapid release phase of sample C is prolonged to 3hours with about 70.1% release. As expected, with an increase in the thickness of barrier mesh, the release rate significantly decreases in first rapid release phase. These results indicate that the drug release behaviors are closely related to the thickness of barrier mesh. The effect of the barrier mesh thickness on release has been also reported by Tatsuya Okuda.Drug release from polymer devices occur by drug diffusion through the polymer, matrix erosion or a combination of both the mechanisms, and is also affected by other factors.With an increase in thickness of barrier mesh, there is an increase distance of drug diffusion through the polymer that leads to a decrease release profile. PEO matrix erosion could be related to drug diffusion. With an increase in thickness of barrier mesh, the distance of erosive PEO diffusion through the polymer will be increased, so the diffusion of the erosive drug-loaded PEO is delayed. Moreover, due to very high surface area of the PLA fibers, it is speculated that some of the drug is absorbed on the surface of the fibers during the drug diffusion through PLA fibers.To figure out the effect of inner layer variation on drug release. The profiles of sample C and sample D are all composed of a first rapid release phase and a gradual release phase. It is surprising that the drug content released from DDs in sample D is double of what sample C does. For sample D, the release percent reaches to 31.7% at the burst re-lease phase about 2 hours. For sample C, however, about 70.6% is released out during the same stage (about 3 hours). The release percentage of sample D and sample C during the whole period is 49% and 95%, respectively. With an increase in the thickness of inner drug-loaded mesh, there are significantly decreases of release rate in first rapid release phase and release percentage during the whole period. These results indicate that the thickness of drug-loaded mesh play an important role in the drug release behavior. After polymer molecule saturation, further addition of surfactant promotes micelle formation in pure water. Space is limited at the interior of multilayer nanofiber meshes, which lead to PEO molecule saturation in PBS.So SDS in the middle of drug-loaded mesh promotes PEO micelle formation. With zoledronic acid and PEO molecules diffuse out of barrier mesh, PEO micelles are absorbed gradually on the surface of barrier mesh, and eventually, PEO micelles block the gaps and pores of barrier mesh, which result in the great decrease in the release rate. Meanwhile, water infusion into drug- loaded mesh is retarded, which lead to further de-crease in the release rate. This also suggests that biogel can hold structure stable and prevent drug diffusion from cross-section.ConclusionsSandwich structure-like nanofiber meshes were fabricated successfully and characterized with controllable drug release. Vitro release experiments revealed that the sealed cross-section of the structure with a biogel prevent departing between barrier mesh and drug-loaded mesh, with increase in thickness of electrospun barrier mesh and drug-loaded mesh, the drug release rate and initial burst release decreased. Compared with other drug delivery systems, the main advantages of the sandwich structure-like meshes are facile preparation condition and the generality for hydrophobic and hydrophilic pharmacyeuticals. Such release system fabricated via multilayered electrospinning open a new path for hydrophilic or hydrophilic bioactive agent delivery. |
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