Preparation And Evaluation Of Dual PH-sensitive Charge-conversion Nanoparticle Loaded DOX Prodrug

Doxorubicin?DOX?is an anthracycline broad-spectrum antitumor drug which achieve therapeutic goals by interacting with DNA molecules to interfere with nucleic acid synthesis.DOX was extensively used in the treatment of ovarian cancer,cervical cancer,lung cancer,liver cancer,gastric cancer,malignant lymphoma and acute leukemia and other types of tumors in clinaical application.However,DOX has more serious side effects,including cardiotoxicity,myelosuppressive toxicity,nephrotoxicity and gastrointestinal reactions,which severely limit its wide clinical application.In order to achieve the targeted ability of DOX,enhance the treatment effect and reduce the side effect,this program prepared a dual pH-sensitive charge-conversion prodrug nanoparticles with DOX prodrug?CAD?,chitosan modified Pluronic copolymer?F127-CS?and folic acid modified gelatin polymer?Gel-FA?and carried out a series of in vitro and vivo experiments,the main research contents and results were detailed as follows:1.Synthesis and structure identification of produg CAD and carrier material Gel-FANegative charged small molecule prodrug?CAD?was synthesized by DOX and cis-aconitic anhydride.The structure of CAD was identified by high performance liquid chromatography?HPLC?,nuclear magnetic resonance spectroscopy?1H-NMR?,fourier transform infrared spectroscopy?FT-IR?and electrospray mass spectrometry?ESI-MS?.Moreover,the method of determination of doxorubicin contents in different pH buffer medium by HPLC was established in this research and the acid hydrolysis experiment of CAD was investigated.The results showed that CAD converted into DOX under acid conditions and the degree and speed of hydrolysis increased as the value of pH.The polymer Gel-FA was synthesized by amide reaction.The structure of Gel-FA was identified by 1H-NMR and FT-IR.The isoelectric point of Gel-FA?pI=6-6.5?was confirmed by determining the Zeta potential in different pH conditions.2.Preparation and Investigation of Physical and Chemical Properties of DOX-loaded NanoparticlesCAD/F127-CS/Gel-FA was prepared through electrostatic interaction force by thin film ultrasonic dispersion method under alkaline conditions.Through the experiment of single factor screening the optimal preparation process of nanoparticles was selected as the total material quality?20mg?,drug quality?4mg?,materials rate?F127-CS/Gel-FA=1:1?w/w??,the total volume?6mL?,the total ultrasonic time?6min?,temperature?40 ??.The research results of physicochemical properties of DOX prodrug nanoparticles were Zeta potential?-11.872±0.12mV?,average particle size?179.6±5.06nm?,the most morphology form into a ball and no adhesion,particle size distribution is homogeneous.The well physicochemical properties make sure that the nanoparticle exhibit enhanced permeability and retention?EPR?effect to achieve passive targeted ability.The research evaluated the drug charge-conversion acid release profile through observing the morphology and determining the Zeta potential of DOX loaded nanoparticles under different pH value environment by transmission electron microscopy?TEM?and Zeta potential analyzer.Results revealed that Gel-FA obtained the pH-sensitive charge-conversion property,and the nanoparticle system was just formed under weak base condition.3.In vitro Evaluation of DOX-loaded nanoparticlesThe in vitro release experiment was carried out in sodium hydrogen phosphate-citric acid buffer solution with different pH value,which simulate the pH of the different physiological conditions from normal blood to tumor cells in body?pH = 7.4,6.8,6.0 and 5.0?.The result demonstrated that DOX exhibit a significant pH-responsive release profile.As the value of pH decrease,the release quantity and speed of DOX increase.In vitro cell anti-tumor activity of CAD/F127-CS/Gel-FA nanoparticles was evaluated by Hela cells and HepG2 cells.The results showed that CAD/F127-CS/Gel-FA nanoparticles hold strong cell toxicity,and the cell toxicity of folic acid positive Hela cells was stronger than the folic acid negative HepG2 cells.The celluar uptake experiment was conducted with Hela cells and the result proved the CAD/F127-CS/Gel-FA nanoparticles could be successfully uptaken by tumor cells and the uptaken quantity were consistant with the results of cell toxicity.4.In vivo Evaluation of DOX-loaded nanoparticlesThe last part of this research was the in vivo evaluation of CAD/F127-CS/Gel-FA nanoparticles.Wistar rats were used as animal models to study the pharmacokinetic characteristics of CAD/F127-CS/Gel-FA nanoparticles in animals by tail vein injection.The results revealed that AUC?0-??value of the DOX solution was increased by nearly 20 times,and the MRT and half-life were increased by more than 6 times.As a result,CAD/F127-CS/Gel-FA nanoparticles could significantly improve the in vivo distribution and improve the bioavailability of DOX.At the same time,the acute toxicity test was carried out by using the LD₅₀,death rate and the histopathological observation as the major parameters.The in vivo safety of the nanoparticles was investigated and evaluated.The results showed that the LD₅₀ of the drug-loaded nanoparticles was much higher than that of the DOX solution(LD₅₀ = 13.134mg/kg).And the pathological study of the main tissues and organs can prove that the drug-loaded nanoparticle system greatly reduced the DOX various toxicities and side effects.The CAD/F127-CS/Gel-FA nanoparticles could be considered non-toxic to the main tissues and organs in a certain dose range.In general,dual pH-sensitive prodrug nanoparticles which was designed and prepared in the subject is a promising nanopesticide delivery system.The CAD/F127-CS/Gel-FA nanoparticles achieved the experimental idea including charge reversal of acid-sensitive targeted release and folic acid initiative targeted ability,improving the body distribution and the bioavailability of drugs as well as significantly reducing the toxic side effects of DOX.