| The combined delivery of multiple drugs for anti-cancer therapy is a hot spot in the field of pharmacy research.Arginine deiminase?ADI?is a broad-spectrum anticancer enzyme that can induce tumor cell stress by depleting arginine at the tumor site,further enhancing its sensitivity to chemotherapy drugs.However,ADI belongs to one kind of protein drugs,which has shortcomings such as poor stability and low bioavailability.Evodiamine?EVO?is an active ingredient of natural medicine and has better antitumor activity.However,EVO belongs to the BSC IV class of drugs,and has shortcomings such as poor water solubility and low bioavailability.Carboxylated graphene?CG?is a new type of carbon nanomaterials that can convert laser energy into thermal energy,which is widely used in the research of photothermal therapy.CG can also be used as a carrier material to directly deliver drugs.This study designed a mitochondrial targeting nanoparticle that can simultaneously deliver ADI,EVO and CG?AEGMN?,hoping to improve the stability of ADI and improve the bioavailability of ADI and EVO.The first part is the synthesis of HA-HPCD.Objective:To synthesize HA-HPCD and provide carrier materials for the preparation of AEGMN.Methods:HA-HPCD was synthesized according to the chemical synthesis method reported in the literature,and the product was identified by infrared spectroscopy.Results:A white flocculent product was synthesized.The infrared spectrum showed a characteristic peak of ester bond at 1720 cm-1,which can be attributed to the connection of HPCD and HA.Conclusion:HA-HPCD was successfully synthesized.The second part is the preparation and physicochemical characterization of AEGMN.Objective:To prepare AEGMN and investigate the physicochemical properties of AEGMN.Methods:AEGMN was prepared by thin film dispersion method and stirring method.Observed the morphology of AEGMN by electron microscope,and determined the physicochemical parameters of AEGMN such as particle size,zeta potential,refractive index,surface tension coefficient,viscosity coefficient,optimum temperature,optimum pH and Km.Results:AEGMN was prepared with a yellow-brown suspension appearance,and uniformly distributed spherical nanoparticles under transmission electron microscope.The particle size of AEGMN is 99.31?0.43 nm,and the average zeta potential is-1.49?0.30 mV.The refractive index of AEGMN at 25?is1.3392?0.0002,the liquid surface tension coefficient is 49.0845?0.6312mN·m-1,and the dynamic viscosity is 1.3980?0.0033 mPa·s.The optimal temperature of AEGMN is 37?,and the optimal pH is 6.5.The Km of AEGMN is about 0.36?0.02 mmol·L-1,and the Vmax is about 33.78?0.07?mol·L-1·min-1.Conclusion:AEGMN was successfully prepared.AEGMN has suitable physical and chemical properties.The third part is the in vitro stability study of AEGMN.Objective:To investigate the stability of AEGMN under different conditions in vitro.Methods:Place AEGMN at different temperature?4?and 55??,different pH?5.59.0?,proteolytic enzyme and plasma,and measure the change of enzyme activity after treatment for a certain period of time.Results:The enzyme activity of AEGMN at different temperatures?4?and 55??,different pH?5.59.0?,proteolytic enzymes and plasma was higher than that of free ADI.Conclusion:AEGMN can improve the in vitro stability and catalytic activity of free ADI.The fourth part is the mechanism research of AEGMN stability enhancement.Objective:To preliminary study the mechanism of enhanced stability of AEGMN in vitro.Methods:The fluorescence spectra of AEGMN and free ADI,the interaction of ADI and B-AEGMN membrane,and the change of AEGMN fluorescence intensity before and after heat treatment were investigated by fluorescence experiment;the gel electrophoresis experiment was used to investigate the conformation of AEGMN before and after heat treatment.Results:Compared with free ADI,the fluorescence intensity of AEGMN was lower and the spectral peak was slightly blue-shifted.There may be a binding between B-AEGMN and ADI,which reduces the binding sites of ADI and FITC,resulting in a decrease in fluorescence intensity.The thermal stability of AEGMN is better than that of free ADI.Before and after heat treatment,the dimer structure of AEGMN and ADI did not change significantly.Conclusion:The mechanism of the enhanced stability of AEGMN in vitro may be that:there is an interaction between ADI and B-AEGMN membrane and B-AEGMN membrane has an encapsulation protection effect on ADI.These effects can maintain and promote the effective conformation of ADI and improve its stability and catalysis activety as a result.The fifth part is the preliminary study on the pharmacokinetics of AEGMN.Objective:To preliminary study the pharmacokinetics of AEGMN in rats.Methods:AEGMN,free ADI and free EVO were intravenously administered to eighteen male SD rats at the same dose.Blood samples were collected at preset time points and ADI activity or EVO concentration was measured.Pharmacokinetic parameters were calculated by DAS software,and the differences in pharmacokinetic behavior of AEGMN and ADI or EVO in rats were analyzed.Results:Non-compartmental model analysis showed that the AUC?0-t?and MRT?0-t?of AEGMN is about 1.28 times and 1.06 times to that of free ADI,respectively.Besides,the AUC?0-t?and Cmax of AEGMN are about 2.89times and 3.50 times to that of free EVO,respectively.The Cl of AEGMN is significantly lower than that of free EVO.Conclusion:AEGMN improves the bioavailability of ADI and EVO,and improves the pharmacokinetic behavior of ADI and EVO. |
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