Associate Professor University of Michigan-Dearborn, United States
Introduction: Protein-mediated drug delivery systems offer significant advantages over conventional methods due to their ability to achieve targeted and controlled release of therapeutic agents and minimize potential negative effects. This study aims to develop a novel protein-based plasmin formulation to enhance the treatment of ischemic stroke. This approach leverages the biocompatibility and functional versatility of Bovine Serum Albumin (BSA) aggregates, with and without Perfluoropentane(PFP) microbubbles, to create an efficient and responsive delivery system. The formulation is designed to have specific characteristics that respond to ultrasound stimuli, optimizing delivery at the site of ischemic injury. BSA was selected as the base protein due to its well-documented biocompatibility and ability to form stable nanoaggregates. PFP microbubbles were integrated into the aggregates to exploit their phase-changing properties under physiological conditions, which could enhance the delivery mechanism through ultrasound responsiveness. The aggregates were surface-modified with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC) and N-Hydroxysuccinimide(NHS) and coated with plasmin to facilitate targeted fibrinolytic activity. The prepared samples were characterized using dynamic light scattering (DLS) and spectroscopy to confirm their size, stability, and functional properties. Preliminary experiments indicate that the plasmin-coated BSA aggregates show significant activity in the presence of antiplasmin compared to free plasmin, demonstrating promising potential for targeted delivery, with the capability to break down clots. Current and future studies focus on testing the ability of the particles to break down fibrin clots, testing whether PFP could promote plasmin efficacy when treated with ultrasound, toxicity, and oxidative stress effects of the particles on brain endothelial cells(bEnd.3).
Materials and
Methods: All chemicals and reagents used in this study were purchased from Aldrich except PFP from Strem Chemicals. BSA aggregates were prepared by incubating BSA solutions at a concentration of 25mg/mL in Deionized(DI) water at 50°C. Two subsets of samples were made, one with and one without 2% PFP. Samples were incubated for 24 hours before being centrifuged at 13,300 RPM for 30 minutes at 4°C. Samples were reconstituted in 200µl of cold DI water. The aggregates, with and without PFP, were then modified using EDC and NHS. 1µl of 100mM EDC was added to 100µl of both BSA samples, waiting 5 minutes, then 1µl of 100mM NHS and waiting another 15 minutes before the addition of 5µl of 1 mg/mL Plasmin and was reacted at 4°C for 24 hours. Samples were centrifuged at the same conditions and were reconstituted in either cold 0.01M PBS or pH 7.4 HEPES Buffer. Size measurements were characterized by DLS. BSA aggregation was determined after the initial incubation at 50°C using 100µM Thioflavin T(ThT). Ultrasound responsiveness of the particle was tested using an ultrasonicator 740 from Mettler Electronic Corp (intensity 1.6 W/cm2, 1 cm2 applicator) in the laboratory. Sonication was applied for 3 minutes in 30-second intervals, putting the sample on ice between. Plasmin activity and coating efficiency were quantified using plasmin substrate reading at 370/442 nm fluorescent wavelength, as well as antiplasmin inhibition of the samples.
Results, Conclusions, and Discussions: Results
Both BSA samples, with and without PFP, exhibited aggregation after 24 hours, as observed from initial characterization(Figure 1). DLS measurements revealed a reduction in aggregate size following ultrasound treatment. The PFP-containing sample showed a significant size reduction of 62.47% (from z-avg. 238.2nm to 89.4nm), In contrast, the sample without PFP exhibited a decrease of only 10.24% (from z-avg. 170nm to 152.6nm)(Figures 2 and 3). The coating efficiency of plasmin on BSA particles was determined to be 25%(Figure 4). Antiplasmin inhibition assays were measured as a percentage of the fluorescence intensity relative to the same sample without antiplasmin. At the 15-minute mark, BSA samples that were incubated with antiplasmin showed significantly higher plasmin activity compared to free plasmin. 1.25µg of free plasmin showed 51.23% plasmin activity while BSA with and without 2% PFP showed 75.15% and 77.6% plasmin activity respectively(Figure 5). These results indicate that plasmin coated on BSA shows enhanced functional activity and less inhibition by antiplasmin. The t-test and one-way ANOVA was used to analyze the data and statistical significance was shown as **** p < 0.0001.
Conclusions
The development of a protein-mediated drug delivery system utilizing BSA aggregates, with and without PFP microbubbles, demonstrates promising results for delivering plasmin. The study successfully showed that the BSA aggregates could be effectively surface-modified and coated with plasmin, achieving a coating efficiency of 25%. Both types of aggregates exhibited significant aggregation after 24 hours, with the addition of PFP resulting in a more substantial size reduction upon ultrasound treatment. The antiplasmin inhibition assays revealed that plasmin-coated BSA particles displayed reduced inhibition by antiplasmin and increased functional activity over time. These findings suggest that incorporating PFP enhances the effectiveness of the delivery system, making it a viable candidate for ultrasound-mediated targeted drug delivery in ischemic stroke therapy.
Discussions
This study demonstrates the potential of BSA aggregates, with and without PFP microbubbles, as an effective protein-mediated drug delivery. Incorporating PFP improved ultrasound responsiveness and reduced antiplasmin inhibition, enhancing plasmin activity. These findings suggest that PFP-containing BSA aggregates provide a promising approach for targeted drug delivery systems for ischemic stroke treatment.
Acknowledgements (Optional): We would like to thank NIH R15GM135766 and the University of Michigan-Dearborn for funding.
