| Objective:Folate-chitosan-stearic acid(FA-CSO-SA)nanomicelles containing Tetrandrine(TET)were prepared,and the size,potential,encapsulation rate,drug loading,stability,in vitro release,anti-inflammatory activity,in vitro cell uptake and pharmacokinetics of the nanomicelles were investigated.In order to improve the uploading rate and targeting of drugs,so as to achieve the purpose of improving the therapeutic effect of drugs and reducing the side effects of drugs.Methods:1.Folate-chitosan-stearic acid(FA-CSO-SA)conjugate was synthesized,and the preparation process of FA-CSO-SA was optimized.The structure of the conjugate was identified by infrared spectroscopy(FTIR)and hydrogen nuclear magnetic resonance spectroscopy(~1H-NMR).The microstructure of FA-CSO-SA conjugate was observed under transmission electron microscope(TEM).The particle size and Zeta potential were measured by laser particle size analyzer.The critical micelle concentration(CMC)was determined.2.A high performance liquid chromatography(HPLC)analysis method for TET was established,and FA-CSO-SA/TET nanomicelles were prepared by ultrasonic method.The encapsulation rate,drug loading and particle size were respectively used as indicators.X-ray diffraction(XRD)and differential scanning calorimetry(DSC)were used to identify the structure of micelles loaded with drugs,and to investigate their stability and release of micelles and TET in vitro under different conditions.3.The toxicity of blank carrier on RAW264.7 mouse macrophages was investigated,and the inhibitory effects of TET raw material and FA-CSO SA/TET on proliferation of RAW264.7 mouse macrophages were compared to evaluate the anti-inflammatory effects of drug-loaded nanomicelles in vitro.Doxorubicin(DOX)was used as a marker to investigate the uptake of nanomicelles by macrophages of RAW264.7 mice.A method was established for the determination of TET concentration in plasma of Wistar rats by UPLC-MS/MS.The rats were given the same concentration of TET raw material and FA-CSO-SA/TET by a single intrabitoneal injection,and the blood concentrations at different time points were measured,and the blood concentration-time curves were drawn to investigate the corresponding pharmacokinetic parameters.Results:1.The optimal preparation conditions of FA-CSO-SA were as follows:Molar specific folate(FA):CSO-SA=3:1,reaction temperature 45℃,rotation speed 400 r/min,hydration volume 30 m L,reaction time 48 h,dialysis time 24 h.The structural identification results showed that FA had been combined with CSO-SA.The physical and chemical properties of FA-CSO-SA conjugate were characterized.The particle sizes of CSO-SA and FA-CSO-SA were(113.0±2.0)nm and(143.4±3.5)nm(n=3),and the average PDI values were 0.125 and 0.220,respectively.The Zeta potentials of CSO-SA and FA-CSO-SA were(28.8±0.6)m V and(28.8±0.5)m V(n=3),respectively.The FA-CSO-SA couplings were almost circular,distributed evenly,and there was no adhesion between particles.The critical micelle concentrations of CSO-SA and FA-CSO-SA were determined to be(20.28±2.97)μg/m L and(11.24±0.31)μg/m L(n=3),respectively,indicating that nanomicelles could be formed at lower concentrations.2.The optimal process of drug carrying micelle was obtained by single factor and orthogonal test screening as follows:carrier to drug mass ratio of 8:4,ultrasonic power of 200 W,and ultrasonic frequency of 200times.The encapsulation rate and drug loading of drug-loaded nanmicelles were(98.86±0.30)%,the drug loading was(28.57±0.34)%,the particle size was(227.0±9.4)nm(n=3),the average PDI was 0.421,and the Zeta potential was(12.6±2.3)m V(n=3).The results of XRD and DSC show that TET has been loaded into the nanometer micellar carrier.The stability test results showed that the drug-loaded nanomicelles had good storage stability.In vitro release test showed that drug-loaded nanomicelles had the ability to slow release drugs and reduce the side effects of drugs.3.The toxicity of FA-CSO-SA on macrophages of RAW264.7 mice was investigated:After treating RAW264.7 cells with different concentrations of FA-CSO-SA for 24 h,the cell survival rate was still higher than 80%when the carrier concentration reached 1000μg/m L,indicating that FA-CSO-SA prepared in this study could be used as a drug carrier material with low toxicity and side effects.The results of in vitro anti-inflammatory test showed that the proliferation inhibition rate of different concentrations of TET on RAW264.7 cells was lower than that of FA-CSO SA/TET,indicating that TET could enhance the anti-inflammatory effect after being prepared into nanomicelles.The cell uptake test showed that the FA modification could promote the uptake of nanomicelles by activated macrophages.The concentration of TET in rat plasma samples was determined by UPLC-MS/MS method using theophylline as internal standard.The measured range of TET was 2-1000 ng/m L,the lower limit of quantitation(LLOQ)was 2 ng/m L,and the methodological investigation was in accordance with the requirements of the 2020 edition of Chinese Pharmacopoeia.The results of pharmacokinetic tests showed that the preparation of TET into drug-carrying nanomicelles could prolong the circulation time of drugs in vivo and improve their bioavailability.Conclusions:In this study,a novel TET-loaded nanomicelle was prepared,which has good targeted anti-inflammatory effect in vitro and has significant advantages in dissolution and bioavailability,and FA-CSO-SA also has good biosafety,indicating that FA-CSO-SA/TET nanomicelle is expected to become a new type of anti-inflammatory drug carrier. |
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