Construction and Evaluation of Fe₃O₄-Based PLGA Nanoparticles Carrying rtPA Used in the Detection of Thrombosis and in Targeted Thrombolysis

Abstract

Thrombotic disease is extremely harmful to human health, but early detection and treatment can help improve prognoses and reduce mortality. To date, few studies have used MR molecular imaging in the early detection of thrombi and in the dynamic monitoring of the thrombolytic efficiency. In this article, we construct Fe3O4-based poly(lactic-co-glycolic acid) (PLGA) nanoparticles to use in the detection of thrombi and in targeted thrombolysis using MRI monitoring. Cyclic arginine-glycine-aspartic peptide (cRGD) was grafted onto the chitosan (CS) surface to synthesize a CS-cRGD film using carbodiimide-mediated amide bond formation. A double emulsion solvent evaporation method (water in oil in water [W/O/W]) was used to construct Fe3O4-based PLGA nanoparticles carrying recombinant tissue plasminogen activator (rtPA) (Fe3O4-PLGA-rtPA/CS-cRGD). Fe3O4-PLGA, Fe3O4-PLGA-rtPA, and Fe3O4-PLGA-rtPA/CS nanoparticles were constructed using the same W/O/W method. The results showed that the Fe3O4-based nanoparticles were constructed successfully and have a regular shape, a relatively uniform size, a high carrier rate of Fe3O4 and encapsulation efficiency of rtPA, and a relatively high activity of released rtPA. Transmission electron microscope (TEM) images revealed that the iron oxide particles were relatively uniformly distributed in the nano-spherical shell. The Fe3O4-based nanoparticles could be imaged using a clinical MRI scanner, and there were no significant differences in the transverse relaxation rate (R2*) or in the signal-to-noise ratio (SNR) values between the Fe3O4-based nanoparticles and an Fe3O4 solution with the same concentration of Fe3O4. In vitro and in vivo experiments confirmed that the Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles specifically accumulated on the edge of the thrombus and that they had a significant effect on the thrombolysis compared with the Fe3O4-PLGA, Fe3O4-PLGA-rtPA, and Fe3O4-PLGA-rtPA/CS nanoparticles and with free rtPA solution. These results suggest the potential of the Fe3O4-PLGA-rtPA/CS-cRGD nanoparticles as a dual-function tool in the early detection of a thrombus and in the dynamic monitoring of the thrombolytic efficiency using MRI.

Fe₃O₄-based PLGA nanoparticles as MR contrast agents for the detection of thrombosis

“…First, to increase their targeting ability, a cyclic Arg-Gly-Asp (cRGD) peptide was used in this study instead of the Arg-Gly-Asp-Ser (RGDS) peptide. In addition, the constructed particles were smaller than those in our previous studies 24,25 to prevent the accumulation of the majority of NPs in the liver and spleen. Furthermore, we dynamically visualized the targeting procedures by 7.0-T MRI using an abdominal aorta model of thrombosis in rats because of its possible application to the more complicated in vivo situation.…”

“…[24][25][26][27] PLGA was used as the core of the drug carrier, Fe 3 O 4 was embedded in the layer of PLGA, and cRGD was coated on the surface of the PLGA-Fe 3 O 4 layer. A schematic representation of the Fe 3 O 4 -PLGA-cRGD NPs is shown in Figure 1.…”

“…The remaining steps in the production of PLGA-Fe 3 O 4 NPs were identical to those in our previous study. 25 Fe 3 O 4 -PLGA-cRGD was prepared using carbodiimide-mediated amide bond formation. First, appropriate amounts of EDC and NHS were added at a molar ratio of 1.5:1-0.1 M MES buffer solution (pH 6) to activate the carboxyl of Fe 3 O 4 -PLGA.…”

“…17,22,23 Similarly, in our previous studies, we synthesized novel types of thrombus-targeting particles and confirmed their targeting ability and contrast-enhancing effects in vitro. 24,25 Although further validation of their targeting ability, biodistribution, and toxicity in vivo are required, there have been few studies of MRI contrast agent behavior, particularly in vivo, which limits their potential translation to clinical use.…”

“…All instruments and measurements were provided in the "Methods" section of our previous study. 25 In vivo toxicity study Eight SD rats were used for the in vivo toxicity study. These rats were raised in stainless cages in ventilated animal rooms with a typical environment (23°C±1°C, 55%±15% humidity, and 12 h light/dark cycle) and had free access to water and food.…”