Construction And Evaluation Of PLGA Nanoparticles Carrying RtPA Used In The Detection Of Thrombosis And In Targeted Thrombolysis

Objective: We construct Fe3O4-based poly (lactic-co-glycolic acid)(PLGA) nanoparticles carrying recombinant tissue plasminogen activator(rtPA) to use in the detection of thrombi and in targeted thrombolysis usingMRI monitoring, and evaluate their physicochemical properties and theefficiency of targeted thrombolysis ability.Methods: Cyclic arginine-glycine-aspartic peptide (cRGD) wasgrafted onto the chitosan (CS) surface to synthesize a CS-cRGD film usingcarbodiimide-mediated amide bond formation. A double emulsion solventevaporation method (water in oil in water [W/O/W]) was used to constructFe3O4-based PLGA nanoparticles: Fe3O4-PLGA, Fe3O4-PLGA-rtPA,Fe3O4-PLGA-rtPA/CS and Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles.Optical microscope, transmission electron microscope (TEM) and laserparticle size analyzer and were used to determine morphology, size, strctureand zeta potential of the nanoparticles. Laser scanning confocal microscope and flow cytometry were used to confirm the coating of cRGD to thenanoparticles. The carrier rate of Fe3O4, encapsulation efficiency of rtPA andactivity of released rtPA were measured by atomic absorption spectrometer,colorimetric method and chromogenic substrate assay, respectively. In vitroand in vivo experiment were performed to evaluate their targetedthrombolysis ability.Results: The Fe3O4-based nanoparticles were constructed successfullyand have a regular shape, a relatively uniform size. TEM showed the Fe3O4particles were distributed on the surface of the nanoparticles. The averagesize, PDI and zeta potential of Fe3O4-PLGA-rtPA/CS-cRGD nanoparticleswere395.2±12.2nm,0.163±0.024and29.0±1.5mV. The cricoid greenfluorescence was observed on the Fe3O4-PLGA-rtPA/CS-cRGD nano-particles (cRGD labeled by fluorescein isothiocyanate) using laser scanningconfocal microscopy and the carrier rate of cRGD determined by flowcytometry was84.27%. The carrier rate of Fe3O4, encapsulation efficiencyof rtPA were49.3±3.4%and63.7±1.5%. In vitro activity of releasedrtPA in Fe3O4-PLGA-rtPA/CS and Fe3O4-PLGA-rtPA/CS-cRGD nano-particles were higher than that of Fe3O4-PLGA-rtPA nanoparticles. Thepathologically frozen section revealed numerous Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles specifically accumulated on the edge of thethrombus. In the in vitro thrombolysis experiment, the thrombolysis ratesof the Fe3O4-PLGA-rtPA, Fe3O4-PLGA-rtPA/CS, and Fe3O4-PLGA-rtPA/ CS-cRGD nanoparticles improved by1.98,2.41, and3.05times at60mincompared with free rtPA. Fe3O4-based nanoparticles could reduce the T2*signal in MR scanning and there was no significant difference in R2*andSNR between Fe3O4-based nanoparticles and Fe3O4solution with the sameconcentration of Fe3O4.3.0T MR scanner showed Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles could be imaged in in vitro and in vivoexperiments after targeted to the thrombus.Conclusion: The results suggest the potential of theFe3O4-PLGA-rtPA/CS-cRGD nanoparticles as a dual-function tool in theearly detection of a thrombus and in the dynamic monitoring of thethrombolytic efficiency using MRI.