| Background: Tumor is the serious disease which threatens human health.Removing tumor mass by surgery is the major treatments for cancer therapy.Chemotherapy,radiotherapy,hormone therapy,biological therapy have also made a major contribution in inhibiting rapid growth of malignant cells.Most chemotherapeutic drugs are cytotoxic drugs,it has toxicity lead to poor therapeutic effect.In recent years,studies have shown that ultrasound combined with drug-loaded microbubbles deduced tumor cells apoptosis.But the microbubbles were too large to deliver drug effectly.Nanodroplets are ultrasound contrast agent and its diameter is below 1?m.It can pass through the tumor microvascular endothelial cell gap.The nanodroplets can be vaporized into microbubbles under ultrasound irradiation or in heating.However,the material of nanodroplets should solvent by organic during the preparation that easily causes toxic side effects.Carboxymethyl-hexanoyl chitosan(CHC)is a natural polymer material,soluble in water and nontoxic,biological.Therefore,the purpose of this study is to prepare CHC-PFP-DOX nanodroplets by synthesizing CHC and Doxorubicin(DOX)as model drugs and to study the ultrasound imaging effect of CHC-PFP-DOX nanodroplets,to study the anti-tumor effect of ultrasound combination in CHC-PFP-DOX nanodroplets for providing method basis in the clinical cancer therapy.This study contains of the following three parts:1.Preparation and characterization of CHC-PFP-DOX nanodroplets were studied.2.CHC-PFP-DOX nanodroplets ultrasound imaging were studied in vitro.3.The ultrasound combination of CHC-PFP-DOX nanodroplets for anti tumor was studied in vitro.Part 1: Preparation and characterization of CHC-DOX-PFP nanodropletObjective: To synthesize CHC and prepare CHC-PFP-DOX and characterize its quality.Methods: CHC was synthesized by acylation of carboxymethyl chitosan and hexanoic anhydride.The compounds were identified by infrared spectroscopy and nuclear magnetic resonance spectroscopy.CHC-PFP-DOX nanodroplets with the drug carrier mass ratio of 1: 5,1: 10 and 1: 20 were prepared by ultrasonic emulsification.The particle diameter and electric potential of nanodroplets were measured by Malvern laser particle size analyzer.The morphology was observed by transmission electron microscopy.The encapsulation efficiency of CHC-PFP-DOX nanodroplets were determined by UV spectrophotometry.The concentration of DOX released from CHC-PFP-DOX nanodroplets was measured by HPLC.Results: CHC-PFP-DOX nanodroplets were successfully prepared by CHC.The diameter of CHC-PFP-DOX nanodroplets in the drug carrier mass ratio of 1: 5,1: 10,1: 20 is(458±43)nm,(286±55)nm and(216±21)nm,the drug loading efficiency were(52.06±10.14)%,(38.67±13.43)% and(9.50±5.50)%,the entrapment efficiency were(8.68±1.70)%?(3.21±0.70)% and(0.45±0.26)%.Under the action of ultrasound,the cumulative release rate of CHC-PFP-DOX nanodroplets(the drug carrier mass ratio of 1: 5)with ultrasound was higher than that of the control group((70.28 ± 3.32)% vs(36.07 ± 1.68)%).Conclusion: Carboxymethyl chitosan and hexanoic anhydride can synthesize CHC.The CHC-PFP-DOX nanodroplets have a tight size distribution with desirable entrapment efficiency and drug loading efficiency in the the drug carrier mass ratio of 1: 5.Part 2: Study on the effect of CHC-DOX-PFP nanodroplets for ultrasound imaging in vitroObjective: To investigate the conditions of CHC-PFP-DOX nanodroplets for ultrasound imaging in vitro,for its preparation application in ultrasound imaging.Methods: In vitro ultrasound imaging model was established.The ultrasound image was performed by Philips i U-22 ultrasonic diagnostic instrument.The ultrasound imaging of CHC-PFP-DOX nanodroplets under different temperatures(25?,35?,45?),concentration(2mg/ml,5mg/ml)and mechanism index(MI=0.4,0.5,0.6)was explored.The image intensity of each group was measured by the soft of Image J.Results: The normalized gray value of nanodroplets was 7.52 ± 1.71 at 45 ?,and 12.42 ± 1.51 at 37 ?,the difference is statistically significant between the two groups(P <0.05).5mg / ml nanodroplets was significantly higher than 2mg / ml nanodroplets(P <0.05).The standardized gray value of MI = 0.5 group was significantly higher than MI = 0.4 group(P <0.05),but there was no significant difference(P> 0.05)with normalized gray value of MI = 0.6 group.Under the condition of temperature of 37 ?,MI = 0.5 and the concentration of 5mg / ml.The normalized gray value of naondroplets was significantly higher than the gray scale of Son Vue(11.54 ± 1.41vs8.65 ± 0.95,P <0.05).Conclusion: The nanodroplets obtained good effect under the condition of 37 ?,the mechanical index of 0.5 and the concentration of 5mg / ml,and its effect is better than Son Vue.Part 3: In vitro study of anti-tumor effect of ultrasound combined with CHC-PFP-DOX nanodropletsObjective: To investigate the toxic effects of CHC on normal cells and tumor cells and the antitumor effect of ultrasound combined with CHC-PFP-DOX nanodroplets in vitro.Methods: The toxicity of the human vascular smooth muscle cells and A2780 cells under three concentrations(1 mg / ml,5 mg / ml,10 mg / ml)of CHC were explored.The best ultrasound parameters and the different drug concentration to determine the amount of DOX added were explored.The effects of CHC-PFP-DOX nanodroplets on the ovarian cancer cells were investigated.The experimental groups were followed: US group,CHC-PFP group,CHC-PFP-DOX group,DOX group,US+CHC-PFP group,US+DOX group,US+CHC-PFP-DOX group,The survival rate of each group was detected using CCK-8 reagent.Results: Under the concentration of(1 mg / ml,5 mg / ml and 10 mg / ml)CHC,the cell viability of human vascular smooth muscle cells were(98.95 ± 6.36)%,(98.11 ± 6.49)%,(96.63 ± 3.69).And there was no significant difference between the groups(P> 0.05).The cell viability of A2780 cells were(99.55 ± 8.9)%,(96.32 ± 6.9)% and(93.91 ± 2.81)%.There was no significant difference(P> 0.05).The survival rate of each group(US group,CHC-PFP group,CHC-PFP-DOX group,DOX group,US+CHC-PFP group,US+DOX group,US+CHC-PFP-DOX group)were(92.50±7.21)%,(92.90±2.16)%,(85.69±13.22)%,(49.56±4.64)%,(85.24±2.73)%,(45.62±8.46)%,(34.60±4.11)%?There was significant difference(P< 0.05)between US+CHC-PFP-DOX group and DOX group.There was significant difference(P<0.05)between US+CHC-PFP-DOX group and US+DOX group.Conclusion: CHC has no toxic effect on normal cells and tumor cells.Ultrasound combined with CHC-PFP-DOX nanodroplets can improve the killing effect of drugs on the tumor.This provides a basis for cancer therapy. |
PDF Full Text Request