Study Of Targeted Drug-loaded Poly (Lactic Acid) Ultrasonic Contrast Microbubbles

Objective In order to develop a new drug delivery system for breast cancer,we prepared and characterized the graphene oxide-mediated and DOX-loaded polylactic acid microbubbles?FA-DOX/GO-DOX/PLA microbubbles?.And then,we investigated the effect of ultrasound imaging for the drug delivery system in vivo and in vitro.Next,we inspected the activity of the drug delivery system on the human breast cancer MCF-7 cells in vitro.Last,we also observed its acute toxicity in mice.Methods The modified Hummer's method was used to prepare graphene oxide?GO?.The conjugate of GO and PVA?GO-PVA?was synthesized and characterized.We regarded DOX as a model drug,and prepared targeted DOX-loaded PLA composite microbubble contrast agent?FA-DOX/GO-DOX/PLA?by double emulsification-solvent evaporation combined with amide reaction.We took PLA concentration,PVA concentration,PVA dosage and isopropanol concentration as the investigate objects,regarded appearance morphology,particle size and stability of microbubbles as the evaluation index and screened the optimal formulation of microbubbles by L9?3⁴?orthogonal test.At the same time,we took ultrasonic power of colostrum,ultrasonic power of rejuvenation,magnetic stirring time and centrifugal speed as the investigate objects,regarded appearance morphology,particle size and stability of microbubbles as the evaluation index and screened the best preparation process of microbubbles by L9?3⁴?orthogonal test.Subsequently,the appearance morphology,particle size,zeta potential and loading rate of DOX and GO of composite microbubbles were characterized.Inhibitory effect of FA-DOX/GO-DOX/PLA composite microbubbles on MCF-7 cells was detected by CCK-8in vitro.Acridine orange staining was used to observe the activity of MCF-7 breast cancer cells after drug treatment.And then,we observed the toxicity and mortality of KM mice by one-time intraperitoneal injection of compound microbubbles and calculated LD₅₀ and95%confidence interval by Kou's method.Doppler color ultrasound imaging system was used to observe the effect of ultrasound microbubbles in vitro and in vivo.Results The optimal formulation of FA-DOX/GO-DOX/PLA composite microbubbles was a PLA concentration of 0.06 g·mL^-1,a PVA concentration of 3.50%,a PVA dosage of40 mL and an isopropanol concentration of 5.50%.The best preparation conditions was as follows:ultrasonic power 120 W was prepared in colostrum,ultrasonic power was 210 W in double emulsion,magnetic stirring time was 2.50 h and centrifugal speed was 2400r·min^-1.FA-DOX/GO-DOX/PLA composite microbubbles prepared by the optimal prescription and the best preparation process are spherical with a round and spherical,distribution with an average particle size of about 600 nm and a zeta potential of?-37.50?10.00?mV.The DOX loading rate in composite microbubbles was?7.42?0.62?%,and the GO loading rate was?19.56?0.27?%.Cell experiments in vitro showed that under the same drug concentration,the different drug groups were dose-dependent on the tumor cells,and FA-DOX/GO-DOX/PLA microbubbles,DOX/GO-DOX/PLA microbubbles,DOX/PLA microbubbles,DOX/GO-PLA microbubbles and free DOX groups on the proliferation of MCF-7 cells decreased gradually,with statistical significance.The results of fluorescent staining with acridine orange?AO?showed that the inhibitory effects of different drug groups on MCF-7 cells but the inhibitory effect of FA-DOX/GO-DOX/PLA composite microbubbles on MCF-7 cells was higher than other groups.In acute toxicity test,the LD₅₀ of compound microbubbles injected intraperitoneally was 87.53 mg·kg^-1 and the 95%confidence interval was?74.25¹03.19?mg·kg^-1,which were calculated by Kovacs method.The results of ultrasound imaging in vivo and in vitro,composite microbubble ultrasound contrast agent was significantly better than the control group in vitro and the agent in New Zealand rabbit major organs had good ultrasound imaging and circulated in vivo safely.Conclusions It is excellent that the particle size,zeta potential,entrapment efficiency,drug loading and stability of FA-DOX/GO-DOX/PLA composite microbubbles prepared by the double emulsification-solvent evaporation method.Cell experiments in vitro showed that the modification of FA can greatly enhance the cytotoxicity of doxorubicin to breast cancer MCF-7 cells by specifically binding to FA receptors of the cell surface,compared with other drug groups.After a one-time intraperitoneal injection of composite microbubbles,it has no obvious toxicity to the mental state and behavior of KM mice and is biologically safe.Ultrasound imaging of composite microbubbles significantly enhanced in vitro and in vivo.Therefore,this project initially confirmed that FA-DOX/GO-DOX/PLA composite microbubble is a promising drug delivery system.