Preparation Of Folic Acid Modified Chitosan Loaded Tetrandrine Nanoparticles And Antitumor Effect In Vitro

Objective:In this research,Chitosan(Chitosan,CSO)was used as a carrier,and Folic acid or folate(FA)was chemically modified to obtain a folic acid-chitosan(FA-CSO)conjugate;then the antitumor drug Tetrandrine(TET)was wrapped to prepare folic acid-modified chitosan-loaded tetrandrine nanoparticles(TET/FA-CSO-NPs),and determined its particle size,potential,drug release characteristics and other properties.We expect to obtain a dosage form with strong anti-tumor activity,good targeting and slow and controlled release characteristics.Methods:1.Synthesized the FA-CSO conjugate as a blank carrier,we select the best preparation process and study its physical and chemical properties.We use infrared spectroscopy and hydrogen nuclear magnetic resonance spectroscopy to identify the structure of the composed FA-CSO conjugate.Discovered its morphological appearance characteristics by transmission electron microscope(TEM).Determined its size and Zeta potential by laser particle size analyzer.2.Established an in vitro HPLC analysis method for TET.Prepared TET/FA-CSO-NPs by ionic cross-linking technique.Determined it encapsulation efficiency and drug loading by ultracentrifugation.Carried out the most important factors by single-factor study method,and encapsulation efficiency and drug loading were used as indicators.Selected particle size,encapsulation efficiency and drug loading as indicators respectively,optimized the best preparation method by three-level orthogonal design experiment.3.Prepared TET/FA-CSO-NPs according to the best process selected and examined the process.Investigated the physical and chemical properties of drug-loaded nanoparticles by infrared spectroscopy,X-ray diffraction and differential scanning calorimetry.The changes of diameter,potential,encapsulation efficiency and drug loading are used as indicators to investigate the stability of drug-loaded nanoparticles within 30 days.Investigate the levels of TET nanoparticles and free TET under different p H conditions(5.0,6.8and 7.4)release in vitro by dynamic dialysis method.4.Selected human liver cancer HepG2 cells as a model,observed the cellular uptake of FA-CSO blank nanoparticles with fluorescence microscope.Investigated the anti-tumor activity of FA-CSO blank nanoparticles,and compare the anti-tumor effects of TET nanoparticles and free TET in vitro.Results:1.The identification results of infrared spectroscopy and hydrogen nuclear magnetic resonance spectroscopy show that FA has been successfully coupled to the CSO structure.The particle size of the prepared FA-CSO conjugate is(148.3±1.4)nm(n=3),Zeta potential is(12.0±2.9)m V(n=3).TEM results show that the FA-CSO conjugate is spherical,and round in shape.2.The results of single-factor investigation experiments showed that FA-CSO concentration,TPP concentration and drug loading mass ratio have a greater impact on the preparation of nanoparticles.Orthogonal experiment results showed that the optimal combination of conditions for preparing process is: FA-CSO concentration is 1.5 mg/m L,TPP concentration is 2.5mg/m L,and drug loading ratio is 1:1.3.After optimizing the process,the encapsulation rate of the drug-loaded nanoparticles is(89.49±1.21)%,the drug loading is(24.41±0.33)%,and the average particle size is(214.9±2.1)nm(n=3).TEM results showed that the drug-loaded nanoparticles have a complete spherical appearance,with good dispersibility,and no adhesion.The analysis results of infrared spectroscopy,X-ray diffraction and differential scanning calorimetry showed that TET has been successfully loaded into the nanoparticle structure medium.The results of stability investigation showed that the encapsulation efficiency and drug loading of the drug-loaded nanoparticle suspension did not change significantly within 30 days,the particle size increased slightly,and the Zeta potential decreased slightly.In vitro release experiments showed that the nanoparticles were relative to free TET shows slow-release characteristics.4.The results of in vitro cell uptake experiments of FA-CSO conjugates showed that HepG2 cells can recognize nanoparticles with FA ligands and take them into cells.The results of cytotoxicity experiments of FA-CSO blank nanoparticles are shown in them.At a concentration of 800 μg/m L,the survival rate of HepG2 cells is about 83%.It can be considered that the synthesized FA-CSO blank nanoparticles can be used as a safe and low-toxic drug delivery carrier material.In vitro anti-tumor studies showed that at the same different concentrations,TET-loaded nanoparticles showed stronger cytotoxicity than TET bulk drugs.Conclusions:1.FA-CSO conjugates with higher affinity for folate receptors were prepared as carrier materials,and the optimal preparation process was screened out.The results of structural characterization and physical and chemical properties showed that FA was successfully bound to the CSO structure,and the conjugate had a round appearance and a small particle size.2.The establishment of TET HPLC analysis method is simple,specific,linearity,precision,and recovery rate are in line with the detection requirements of the methodology.TET/FA-CSO-NPs were prepared by ion cross-linking method,and drug-loaded nanoparticles with stable properties,small particle size,high encapsulation efficiency and drug loading were obtained.In vitro drug release studies showed that their release has sustained release properties.3.The MTT method was used to investigate the anti-tumor effect of FA-CSO blank nanoparticles,and the results showed that FA-CSO-NPs had a higher survival rate after acting on HepG2 cells,indicating that its safety is high and its toxicity is low.The inhibition rate of TET/FA-CSO-NPs on the proliferation of HepG2 cells is higher than that of TET,indicating that the prepared nanoparticles have enhanced anti-tumor activity compared with TET.HepG2 cells have a high affinity for FA-CSO-NPs,and the uptake process is time-dependent.