| Background:Flavonoids(FLAs)are naturally occurring polyphenols widely found in plant foods(e.g.,fruits,vegetables,and teas)and in most medicinal plants.Over the past few decades,more than 10 000 FLAs have been characterized as having a broad range of biological activities.High antiviral,osteogenesis,antibacterial,antioxidant,and other physiological activities have been found to exist in a great number of plant extracts,as well as the corresponding isolated individual compounds,such as luteolin,genistein(GEN),icariin(ICA),and quercetin.Many preclinical and epidemiological data showing the effectiveness of FLAs as botanical drugs to increase bone formation.Unfortunately,the effective concentrations of FLAs for physiological activities found through in vitro experiments are usually an order of magnitude higher than those found through in vivo experiments.Therefore,one of the problems of using Flavonoids to bone reconstruction is their potentially low bioavailability and short half-life.Most FLA-loaded delivery systems,particularly nanosized ones,have been used as alternatives to free compounds.Indeed,numerous nano-drug-delivery systems loaded with FLA systems have been synthesized,including liposomes,poly(lactic-co-glycolic acid)(PLGA),PLA,chitosan,and silica.All these materials can enhance FLAs bioavailability in the body.However,more stable and target-specific nanoparticles are desired.Microsphere systems that are stable and able to sustain drug release for a long time have been reported,but FLAs-loaded micro-drugdelivery systems are rare.The use of conventional single-polymer microspheres is severely undermined by several limitations.Moreover,most FLAs are crystalline,and the dispersion of crystalline substances in a carrier system is less homogeneous than that of amorphous ones,resulting in higher initial burst release of the former.Ideal sustained-release drug-delivery systems should be able to(i)encapsulate drugs as much as possible to minimize loss unless otherwise required,(ii)keep the initial burst of the system to a minimum,and(iii)deliver the necessary drug to target tissues at a therapeutic level without any detriment to the biological activity of the drug.Nanoparticle-in-microparticle controlled-release devices are new and may be classified as ideal sustained-release drug-delivery systems.Several cases have already described,such as PLGA,liposome,and chitosan nanoparticles encapsulated in PLGA microspheres.However,nanoparticle-in-microparticle controlled-release devices as a carrier of FLA have not been reported.In the present work,we developed a delivery platform through a nanocomplex encapsulation technique consisting of a soft gel-like interior and a surrounding PLGA shell.Gelatin-based nanoparticles have been proven to be a relatively safe and effective nonviral gene-delivery vehicles with long circulating time and high accumulation rate at the tumor site.Thus,our method was based on a preparation involving the adsorption of gelatin nanoparticles(GNPs)followed by the coating of nanoparticles with GEN or ICA through hydrogen bonding.The coated nanocomplex was compatible with biodegradable controlled-release polymers and can be easily entrapped in poly(lactideco-glycolide)(PLGA)microspheres.We also reported the design of this delivery platform,as well as the physicochemical,pharmaceutical,and cytobiology characteristics of the resulting microspheres.Methods: 1.Preparation of GNPs The classical two-step dissolution method was used to prepare GNPs,then to characterized them.2 FLA adsorption onto nanoparticles The loading of GEN or ICA into GNPs by FLA adsorption from methanol solution was compared at different concentration of the GNPs,times,temperatures,and p H values of the solution.Typically,GEN or ICA was mixed with 1 m L of nanoparticle suspension at six different temperatures.p H was adjusted to the value under investigation by using HCl or Na OH.After,FLA-loaded nanoparticles were separated from the supernatant by centrifugation,and then the loaded nanoparticles were dried at room temperature until used. 3 Microsphere preparation A specified amount of PLGA was dissolved in methylene chloride(DCM),and loaded nanoparticles with a mean particle size of 120 nm were dispersed in the polymer solution under sonication for 5 min in an ice–water bath formation of S/O mixed phase.Then,the drug–polymer suspension was obtained and added dropwise to an aqueous solution containing 2%(w/v)PVA under specified magnetic stirring for 4 h until the methylene chloride completely evaporated.After centrifugation,the microspheres were collected and washed three times with distilled water to remove traces of residual solvent and PVA.The resulting microspheres were finally freeze dried and stored at 4°C until further used.4 Characterization of nanoparticles and FLA?GNPs@PLGA composite microsphere SEM images were taken using a Hitachi S-4800 scanning electron microscope(SEM).The particle-size distribution of FLA?GNPs@PLGA was calculated with Nano Measurer 1.2.At least 300 microspheres were measured for every sample.To enhance image quality,sample surfaces were spattered with a 5nm layer of iridium.AFM images were obtained using VEECO Dimension icon instrument.TEM images were obtained using a JEM-1011 transmission electron microscope.The composite microspheres were embedded in epoxy resin.The internal structure of the microspheres was observed using a transmission electron microscope through ultrathin-sectioning technique.Samples were prepared as above on a freshly cleaved silicon chip.Measurements of nanoparticle hydrodynamic diameter and potential were carried out on a Zeta Plus instrument in water.hree independent runs were performed,and the results were averaged.The loading of GEN or ICA into GNPs by FLA adsorption from methanol solution was compared at different concentration of the GNPs,times,temperatures,and p H values of the solution.The prepared suspension was divided into 2 ml aliquots,and then GNPs were separated from the supernatant by centrifugation at 6500 rpm for 20 min and washed with 75% aqueous acetone three times.Finally,to concentrate the sample,the content of each tube was resuspended in 1 ml aqueous solution.5 In vitro release of GEN and ICA The in vitro release of GEN and ICA from the FLA?GNPs@PLGA composite microspheres were carried out in PBS.Then,the concentration of GEN or ICA in the medium was determined by measuring the maximum absorbance,respectively.To confirm that GEN or ICA was released intactly from the microspheres,the release medium was measured with HPLC using the same HPLC conditions as before mentioned.Result: The concentration of GNPs,temperature and adsorption time were closely correlated with the concentration of FLA adsorbed.At >3.0 mg/ml,the temperature exceeds 25℃ or time beyond 24 hours,FLA adsorption capacity exceeded 90%,The highest ICA absorption reached 68% of the weight of nanoparticle solid material,and the loading of GEN was lower(about 43%).The microspheres effectively loaded genistein and icariin,and the highest drug loading rates were 12.1%±1.14% and 13.7%±1.21%,respectively.Moreover,both formulated microspheres exhibited minute initial burst of drug release the subsequent release of drugs for more than 40 days.Conclusions: 1.The preparation of the flavonoid gelatin nanocomposites by two-step desolvation method which was a simple and easy preparation process.The composite has a uniform size,high encapsulation efficiency(EE),the excellent structural stability enables it to be stored at room temperature for more than half a year.2.The effect of various parameters on the amount of adsorption was quantified,the cone shaped structure of GNPs was verified.3.PLGA sustained release system was prepared using s/o/w emulsion solventevaporation technique,the PLGA microspheres with homogeneous particle size distribution,the preparation process is simple and easy,and the surface of the microspheres is smooth and round,and the size is controllable;4.The effects of different conditions,such as the PLGA:GNPs mass ratio and the total quality of ICA added were observed to optimize the preparation process,the burst release and pulse release of FLA were significantly reduced,high EE and drug loading without agglomeration can be acquired under the optimized conditions... |
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