Engineered nanoconjugates for simultaneous detection and degradation of stroke-associated microthrombi.

Background

Microthrombi obstructing downstream microcirculation in acute ischemic stroke (AIS) are difficult to treat and visualize with current imaging methods.

Methods

To address this need, a novel theranostic agent, IO@PDA@tPA, was developed by combining iron oxide microparticles (IO) coated with polydopamine (PDA) and conjugated with recombinant tissue-type plasminogen activator (r-tPA). The amidolytic and fibrinolytic capacities of r-tPA grafted on IO@PDA were assessed using the spectrofluorometric test, the clot lysis assay, and the whole blood halo assay. IO@PDA@tPA was then tested in vivo in a preclinical ischemic stroke model induced by thrombin injection into the middle cerebral artery in both non-diabetic and diabetic mice. Two doses equivalent to 2.5 and 5 mg/kg r-tPA were tested. The presence of microthrombi was monitored via molecular MRI. A series of T 2*-weighted sequences for microthrombi imaging and magnetic resonance angiography (MRA) was performed over 45 min. At 24 h, lesion size, vessel patency, and hemorrhagic transformation were assessed with T2 -weighted imaging, MRA, and T2 * -weighted MRI, respectively. A grip test was performed to assess functional recovery one day before stroke (baseline), and at 24 h and five days after stroke. Additionally, inflammatory processes were evaluated five days post-stroke by flow cytometry in the non-diabetic cohort.

Results

This agent exhibited in vitro clot lysis activity. In vivo, administration of IO@PDA@tPA at one-quarter of the standard r-tPA dose enabled both visualization and degradation of microthrombi, as detected by T2 * -weighted MRI. This treatment significantly reduced lesion size and promoted recanalization 24 h after stroke onset. In the hyperglycemic mice cohort, the agent demonstrated efficacy comparable to r-tPA without increasing hemorrhagic risk-a common complication of free r-tPA. Moreover, full functional recovery observed within five days post-stroke. Flow cytometry indicated that IO@PDA@tPA mitigated inflammatory processes.

Conclusion

IO@PDA@tPA represents a promising theranostic agent targeting microthrombi in AIS, reducing the required r-tPA dose and limiting associated side effects.