| Background:With the progress of time, environmental degradation and the development of spectrum of disease, diagnosis and treatment of diseases in the field of modern medicine has been challenged. However, the modern clinical medicine in the treatment is gradually fulled of new charm and new opportunities.Nowadays, traditional Chinese medications and its processed drugs are widely used in clinical practice,but they are same shortcomings such as low bioavailability, low effective concentration, slow onset, short half-life, and penetrating poorly blood-brain barrier,which not only seriously hinder drugs effectiveness, but also restrict the development of modern medication.In recent years, drug-loaded technology of nanoparticles used in the medical field, for example, nano-medicine, nano-inclusion technique, polymer nano-particles carrier technology, liposome nano-particles, solid dispersion technology and other fields, these are still in early stage and many problems need to be solved,including uncertainty efficacy of nano-medicine and possible side effects, difficultly control on active ingredients and instability of nano-medicine.Because of many of poorly soluble active ingredients of traditional Chinese medication, poor oral absorption and low bioavailability,therefore, it is necessary to find the right way to improve the solubility of poorly soluble active ingredients of traditional Chinese medication and its bioavailability, which is one of the main challenges faced by medical workers.The research that the active ingredients of Chinese herbal compound are grafted onto magnetic nano-particles in order to enhance bioavailability and the performance of controlled slowly drugs and the formulation of targeted new drug has not been reported,This paper will start with one of the major drug with ligustrazine (TMP)of Zheng Tian Pill,gradually finding more perfect route of administration of traditional Chinese medication.Integrating magnetic nano-materials to medicine TMP monomers together form a composite nano-spheres that can well improve defects such as low bioavailability, slow onset, low plasma concentration, short half-life, the poor ability to wear blood-brain barrier.As magnetic nano-particles have super-paramagnetic, small size effect, surface effect, quantum tunneling effect,magnetic response,biocompatibility and biodegradability, functional groups and other characteristics, its unique advantages in the field of life sciences show a promising prospect.Objective:Based on the preparation, characterization of magnetic nanoparticles and the comprehensive evaluation for the determination of bovine serum albumin adsorption performance to look for a relatively good magnetic nano-particles with super-paramagnetic, targeting and biocompatibility excellent properties as a carrier of traditional Chinese medicine in order to better improve the low bioavailability of traditional Chinese medication or its processed drugs, slow efficacious, low plasma concentration,the short half-life,badly wearing blood-brain barrier,etc,which can help look for a new dosage of TMP and a new recipe of the treatment of migraine headache.Methods:1.The preparation and characterization of nickel Cobalt ferrite(Ni0.5Co0.5Fe2O4) with magnetic nano-particles and determination of adsorption of bovine serum albumin (BSA).1.1The Nio.5Co0.5Fe2O4nano-composites were synthesized by co-precipitation, using NiCl2·6H2O (98%), CoC4H6O4·4H2O(99.5%), and Fe(NO3)3·9H2O (98.5%) as starting raw materials without further purification. The raw powers were dissolved in deionized water in required stoichiometric proportions.The solutions were then poured together into a beaker and stirred in a magnetic blender at80℃. After2h, aqueous ammonia solution was added to the container, until brown suspension took shape at pH-8.5. After stirring for about30min the suspension was ball-milled for24h with ethanol as a milling medium in order to mix the reactants enough, and then dried in a cabinet dryer at80℃overnight to obtain the precursor samples. The dried powders were finally annealed in N2atmosphere for2h at different temperatures in the range of550～1050℃.1.2The crystalline phase of Ni0.5Co0.5Fe2O4nano-composites was identified by the x-ray diffraction (XRD) technique. X-ray diffraction gram of all the samples from10°to80°at a scanning step of0.02°/2s was recorded using a Rigaku X-ray diffractometer with Cu Ka radiation (1=1.54056A). The magnetic properties were measured using a vibrating sample magnetometer (VSM) at a room temperature under a maximum field of30kOe.1.3The adsorption of bovine serum albumin (BSA) protein on nano-particles was analyzed with a UV spectrophotometer (UV2401pc) at room temperature. The aqueous solution with pH about7.4contained BSA (purity>98%)1.000mg/ml before the adsorption and for each measurement,3.00to15.00mg nano-particles was used as the adsorbent. The adsorbent was stirred ultrasonically in the aqueous BSA solution for1h at room temperature.2.The preparation and characterization of Lanthanum-nickel-manganate(La2NiMnO6) with magnetic nano-particles and determination of adsorption of bovine serum albumin (BSA).2.1The La2NiMnO6(LNMO) nano-composites were synthesized by co-precipitation, using La(NO3)3-5H2O(99.5%), Ni(CH3COO)24H2O (98%) and Mn(CH3COO)4-4H2O(99%) as starting raw materials. The raw powers were dissolved in deionized water in required stoichiometric proportions.The solutions were then poured together into a beaker and stirred in a magnetic blender at80℃. After2h,aqueous ammonia solution was added to the container, until brown suspension took shape at pH-8.5.After stirring for about30min the suspension was ball-milled for24h with ethanol as a milling medium in order to mix the reactants enough, and then dried in a cabinet dryer at80℃overnight to obtain the precursor samples. The dried powders were finally annealed in nitrogen atmosphere for2h at different temperatures in the range of750～1050℃.2.2The crystalline phase of LNMO nano-composites was identified by the X-ray diffraction (XRD)technique. X-ray diffractogram of all the samples from10°to80°at a scanning step of0.02°/s was recorded using a Rigaku X-ray diffractometer with Cu Ka radiation (X,=1.54056A). The magnetic properties were measured using a vibrating sample magnetometer (VSM, Quantum design PPMS-9, USA)at a room temperature under a maximum field of30kOe. The structural defects in LNMO materials were investigated using J EOL4000EX high resolution transmission electron microscope (HR-TEM) operated at400kV.2.3The adsorption of bovine serum albumin (BSA) protein on nano-particles was analyzed with a UV spectrophotometer (UV2401pc) at room temperature. The aqueous solution with pH about7.4contained BSA (purity>99%)1.000mg/ml before the adsorption and for each measurement,La(Ni0.5Mn0.5)O3nano-particles was used as the adsorbent. The adsorbent was stirred ultrasonically in the BSA solution for1h at room temperature,which put in static precipitation condition after12h to be measured.3.The preparation and characterization of nickel-zinc ferrite(Ni0.5Zn0.5Fe2O4) with magnetic nano-particles and determination of adsorption of bovine serum albumin (BSA).3.1The Nio.5Zno.5Fe204nano-particles were synthesized by co-precipitation, using Ni(CH3COO)2·4H2O(98%), Zn(CH3COO)2·2H2O(99%),and Fe(NO3)3·9H2O(99%)as raw solutions in required stoichiometric proportions. The Ni0.5Zn0.5Fe2O4nano-particles were synthesized by co-precipitation, using Ni(CH3COO)2·4H2O(98%), Zn(CH3COO)2·2H2O(99%),and Fe(NO3)3·9H2O(99%)as raw solutions in required stoichiometric proportions. Before both solutions were dissolved in acetic acid (CH3COOH36%), and the latter were dissolved in deionized water, then the former mixed solutions drip into the latter mixed solutions dropwise and stirred meanwhile in a magnetic blender at80℃. After2h, aqueous ammonia solution was added to the last mixed solutions, until brown suspension took shape at pH-8.5. The obtained suspension were filtrated and dried in a cabinet dryer at80℃overnight to obtain the precursor samples, which was ball-milled for24h with ethanol as a milling medium in order to mix the reactants enough after presintering. The dried powders were finally annealed in nitrogen atmosphere for2h at different temperatures in the range of400-800℃.3.2Characterizations of Ni0.5Zn0.5Fe2O4nano-particles. The structure and morphologies of the resultant nano-particles were determined by X-ray diffraction (XRD) and transmission electron microscopy (TEM,HITACHIH-7650). The magnetic properties were measured by using a vibrating sample magnetometer (VSM, Quantum design PPMS-9, USA) at a room temperature under a maximum field of30kOe.3.3The adsorption of bovine serum albumin (BSA (V)) protein on the Nio.5Zno.5Fe204nano-particles was analyzed with a UV spectrophotometer (UV2401pc) at room temperature. The aqueous solution with pH about7.0and3.0contained BSA (purity>99%)1.000mg/ml before the adsorption and for each measurement,6.00to13.00mg of the Ni0.5Zn0.5Fe2O4nano-particles was used as the adsorbent. The adsorbent was stirred ultrasonically in the BSA solution (pH about7.0and3.0) for1h at room temperature, and which the strongest adsorption was stirred ultrasonically in the different time, which put in Static precipitation condition after12h to be measured.4. To measurement on the preparation, characterization and properties composited nano-particles combining modified tween80fluorescently was labeled magnetic nano-particles of and TMP4.1The LaYPO4:Eu powder were synthesized by co-precipitation, using Y2O3, La2O3and Eu2O3as starting raw materials were dissolved in mixed solution of HNO3(69%,10ml) and deionized water(10ml) in required stoichiometric proportions. The mixed solutions were heated to80℃. After2h,(NH4)2HPO4solution was added to the container, until the suspension took shape at pH=9.5. After stirring for about2h the suspension was filtered and then rinsed repeatedly with deionized water. Afterwards dried in a cabinet dryer at80℃overnight to obtain the precursor samples. After grinding and presintering, the dried powders were finally annealed in nitrogen atmosphere for2h at different temperatures in the range of500～800℃.4.220mg Ligustrazine(TMP) add in the bovine serum albumin (BSA(v)25%,2ml).30mg La(Ni0.5Mn0.5)O3(LNMO950℃) nano-particles and15mg LaYPO4:Eu powders join in the mixed solution,then string fully. Compound of after string join in40ml Polysorbate,then the admixture was stirred ultrasonically in the BSA solution for10min at room temperature.100ml heated to120℃to maintain a constant temperature in heater.after the suspensions were ultrasonic stirred dropping in Polysorbate of with maintaining a constant temperature100drop/min speed and vibrate10min.Then was cooled to25degree with ice. With deionized water washing three times, each time with deionized water50ml,centrifuge15min with3000R/min,After centrifugal composite nano-particles to natural evaporation drying,4degrees to save backup.4.3The structure and morphologies of the resultant nanoparticles were determined by X-ray diffraction (XRD) of all the samples from10°to80°at a scanning step of0.02°/2s was recorded.The magnetic properties were measured by using a vibrating sample magnetometer (VSM, quantum design PPMS-9, USA) at a room temperature under a maximum field of30kOe.Dispersed composite nano-particles with ethanol, and then placed in a copper-line, high-powered electron microscope composite nano-particle morphology and particle size.4.4First,use fluorometer to measure the content of TMP in composite nano-particles then,samples of composite nano-particles by1:5(mg/ml) was added at a concentration of5%of dilute hydrochloric acid solution and allowed to stand at8℃after mixing ultrasound for24hours.Last,2000r/min centrifugal10min, the supernatant was used to measure the TMP content by fluorescence photometer.4.5Composite magnetic nano-particles TMP (TMP) in vitro release assay;prepared composite nano-particles by accurately weighed was added the appropriate amount of phosphate buffer solution (containing0.2%sodium azide as an antimicrobial agent,0.1%Tween-80as a wetting agent) using for the release of the medium,which was placed in a water bath shaker at100r/min shaking speed,37℃condition in order to determinate the vitro release rate of composite nano-particles TMP. Respectively, in the set time (0.5,1,2,5,10,15,20,25,30,35, and40h) removed by centrifugation at15000r/min15min, After aspiration of the supernatant was added an equal amount of fresh release medium. The content of TMP using fluorescence spectrometer measurements. TMP in phosphate buffer solution is calculated(pH7.4,37℃) cumulative release percentage and cumulative release percentage plotted against time.4.6Cytotoxicity determination of the composite magnetic nano-particles, detecting the influence of composite magnetic nano-particles on human hepatoma cell line HepG2proliferation activity using the MTT assay.4.7Data analysis:The results of measurement data with mean±standard deviation (x±s) is that the cytotoxicity experiments, the experimental and control groups were compared using ANOVA with SPSS13.0statistical software for analysis, P<0.05set for the difference was statistically significant, using curve fitting fit scatter plot.Result:1. The Ni0.5Co0.5Fe2O4(NCFO) nano-particles have been successfully prepared by the chemical co-precipitation process. XRD analysis showed that the each of sample magnetic nano-particles NCFO were all the typical single-phase cubic spinel structure,with the annealing temperature rises from550to950℃, magnetization Ms from35.95to67.19emu/g, when the annealing temperature was further increased to1050℃, Ms decreased. NCFO nano-particles of Grain size and saturation magnetization were initially increased with increasing annealing temperature, and then reduced with increasing annealing temperature and determined the average particle diameter of magnetic nano-particles in NCFO using transmission electron microscope ending with The average particle diameter of about50nm.NCFO magnetic nano-particles showed a bovine serum albumin had good adsorption properties, when the annealing temperature was750℃particle size of magnetic nano-particles NCFO33.3nm revealed the strongest suction force is71(mg/g) or so.2. La2NiMnO6(LNMO) nano-particles have been successfully prepared by the by the chemical co-precipitation process.The grain size of the LNMO nano-particles are largely influenced by annealing temperature.As the annealing temperature increases from750to1050℃, the average grain size increases from about33.9to39.6nm, On the other hand, the coercivity initially increases, reaching a maximum value of42.3Oe when the average grain size was about37.9nm at950℃, and then reduces. The LNMO nano-particles showed good adsorption performance in bovine serum albumin protein, and the preliminary optimized adsorption was obtained for the LNMO nano-particles annealed at850℃.These LNMO nano-particles were a potential carrier for large biomolecules, which will be widely used in the biomedical field.3.Successfully prepared by chemical magnetic nano-particles Ni0.5Zn0.5Fe2O4(ZNFO) by co-precipitation method,Characterized by XRD technique, NZFO powder formed a single cubic spinel structure, and do not produce other miscellaneous phase.Annealing temperature on the grain size of magnetic nanoparticles and magnetic powder NZFO had some impacts. With increasing annealing temperature, the saturation magnetization was enhanced. When bovine serum albumin solution at a pH of7.0environment, the annealing temperature was600℃, magnetic nano-particles NZFO samples had the strongest adsorption performance of bovine serum protein, that is, the adsorption value of about38.35mg/g.4. After modification of Tween80, europium-doped lanthanum phosphate (LaPO4: Eu) phosphor powder labeled composite magnetic nano-particles (TMP monomer, bovine serum albumin V,La(Ni0.5Mn0.5)O3(LNMO-850), LaPO4:Eu-800, Tween-80composed of composite) successfully prepared using the principle of elevated temperature protein. The size of composite nano-pheres was about0.5μm and obtain higher drug loading and encapsulation efficiency by high-powered electron microscope. In vitro by measuring the release of the composite magnetic nano-particles and found that TMP modified by Tween80and magnetic nano-particle entrapment showed a significant slow-release properties. Data by curve fitting, the fit is0.56476. The results showed that the half-life of the composite magnetic nano-particles was about25h. Composite magnetic nano-particles to detect the impact of human hepatoma cell line HepG2proliferation activity by MTT assay showed that the composite magnetic nano-particles are safe. After statistical analysis, both nano-drug group and the control group showed no significant difference in HepG2human hepatoma cell line proliferation activity.Conclusion:the preparation and characterization of three magnetic nano-particles such as Iron-nickel-cobalt(Ni0.5Co0.5Fe2O4), nickel lanthanum manganite (La2NiMnO6), nickel-zinc ferrite(Ni0.5Zn0.5Fe2O4), and the measurement and evaluation on bovine serum albumin the of adsorption performance. Alternatively magnetic nano-particles selected samples in a nitrogen atmosphere, lanthanum-nickel-manganese annealing temperature of850℃as the preparation of the composite nanoparticles.After modification of Tween80, europium-doped lanthanum phosphate (LaPO4:Eu) phosphor powder labeled composite magnetic nano-particles (TMP monomer, bovine serum albumin, La (Ni0.5Mn0.5)O3(LNMO-850),LaPO4:Eu-800, Tween-80composed of composite) successfully prepared using the principle of elevated temperature protein.Experimental results showed that the TMP has a higher rate of loaded drug and encapsulation of magnetic nano-particles in the composite, and the modification by Tween80and the encapsulation of magnetic nano-particles showed a significant slow-release properties. Detecting the effect of composite magnetic nano-particles on human hepatoma cell line HepG2proliferation activity using the MTT assay, the results were displayed composite magnetic nano-particles were safe in the appropriate concentration rangeIn this study, the use of magnetic nano-particles as drug carriers of TMP that provided it for a new dosage form of TCM and has laid a solid foundation, bringing a new dawn for the treatment of migraine. |
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