Preparation Of A Novel Folate-modified Nanobubbles And Its Improved Targeting Ability To Tumor Cells In Vitro Experiment

PART I Preparation and characteristics of novel nanoscaled ultrasound contrast agentsObjective To prepare lipid nanoscaled ultrasound contrast agents(UCAs),and to explore their physiochemical properties.Methods Orthogonal test was used to screen out the optimal condition for preparation aiming at three mainly factors.Amalgamator was applied to fabricate the lipid nanoscaled UCAs.The morphology,diameter,polydispersity index(P.I),zeta potential and stability of nanoscaled UCAs were assessed.The enhancement effect of nanoscaled UCAs in vitro was also detected.Results The results of orthogonal test indicated that the oscillation time had a significant effect on the diameter of nanoscaled UCAs(P<0.05).The optimal condition of preparation was as follows:the amount of DPPA was 0.5 mL,oscillation time was 90 s,and centrifugation time was 3 min.The mean diameter and P.I of the nanoscaled UCAs fabricated under the optimal condition was(313.20±6.90)nm and 0.15,respectively.The Zeta potential was-12.40 mV.There was no significant difference in the enhancement effect between the nanoscaled UCAs and SonoVue in vitro(t=2.02,P>0.05).The basic physical characteristics of the nanoscaled UCAs remained unchanged after being stored in 4? refrigerator for 20 hours.Conclusion Nanoscaled UCAs with smaller diameter and good stability were successfully fabricated.All these characteristics indicate a promising future in targeted molecular imaging and drug or gene delivery.PART ? Development of a novel folate-modified nanobubbles with improved targeting ability to tumor cellsObjective Conjugation of folate(FOL)to nanobubbles could enhance the selective targeting to tumors expressing high levels of folate receptor(FR).To further improve the selective targeting ability of FOL-modified nanobubbles,a novel FOL-targeted nanobubble((FOL)2-NB)with increasing FOL content(accomplished by linking two FOL molecules per DSPE-PEG2000 chain)was synthesized.Methods(FOL)2-NB was prepared through the methods of mechanical shaking and low-speed centrifugation based on lipid-stabilized perfluoropropane.The bubble size and distribution range were measured by dynamic light scattering(DLS).Enhanced imaging ability was evaluated using a custom-made agarose mold with a clinical US imaging system at mechanical indices of up to 0.12 at a center frequency of 9.0MHz.Targeted ability was also carried out in human breast cancer MCF-7 cells,which over-express the FR,by fluorescence activated cell sorting(FACS)and fluorescence microscopy,respectively.Results(FOL)2-NB with a particle size of 286.87±22.96nm were successfully prepared,and they exhibited superior contrast imaging effect.FACS and fluorescence microscopy studies showed greater cellular targeting ability in the group of(FOL)2-NB than in their control group of Non-targeted-NB(no FOL targeted nanobubbles)and FOL-NB(one FOL molecule per DSPE-PEG2000 chain).Conclusions These results suggest that a new type of stronger targeted nanobubble was successfully prepared by increasing the FOL content per DSPE-PEG2000 chain.This novel(FOL)2-NBs are potentially useful for ultrasound molecular imaging and treatment of FR-positive tumors and are worthy for further investigation.