Fibrin-Targeted Nanoparticles for Finding, Visualizing and Characterizing Blood Clots in Acute Ischemic Stroke

Abstract

Recanalization of the occluded artery is the gold standard treatment for acute ischemic stroke, which includes enzymatic fibrinolytic treatment with the use of recombinant tissue plasminogen activators (rtPAs) to disrupt the occluding clot, the use of mechanical thrombectomy to physically remove the clot, or a combination of both. Fibrin is one of the main components of blood clots causing ischemic stroke and is the target of rtPA upon activation of plasminogen in the clot. In addition, fibrin content also influences the efficacy of mechanical thrombectomy. Current imaging methods can successfully identify occlusions in large vessels; however, there is still a need for contrast agents capable of visualizing small thrombi in ischemic stroke patients. In this work, we describe the synthesis and the in vitro characterization of a new diagnostic nanoparticle, as well as the in vivo evaluation in an animal model of thromboembolic stroke. Gd-labeled KCREKA peptides were synthesized and attached onto the surface of PEGylated superparamagnetic nanoparticles. Magnetic resonance imaging (MRI) of blood clots was performed in vitro and in vivo in animal models of thromboembolic stroke. KCREKA-NPs were synthesized by attaching the peptide to the amino (N) termini of the PEG-NPs. The sizes of the nanoparticles, measured via DLS, were similar for both KCREKA-NPs and PEG-NPs (23 ± 4 nm, PDI = 0.11 and 25 ± 8 nm, PDI = 0.24, respectively). In the same line, r2 relaxivities were also similar for the nanoparticles (149 ± 2 mM Fe s−1 and 151 ± 5 mM Fe s−1), whereas the r1 relaxivity was higher for KCREKA-NPs (1.68 ± 0.29 mM Fe s−1 vs. 0.69 ± 0.3 mM Fe s−1). In vitro studies showed that blood clots with low coagulation times were disrupted by rtPA, whereas aged clots were almost insensitive to the presence of rtPA. MRI in vitro studies showed a sharp decrease in the T1 × T2 signals measured for aged clots incubated with KCREKA-NPs compared with fresh clots (47% [22, 80] to 26% [15, 51]). Furthermore, the control blood showed a higher value of the T1 × T2 signal (39% [20, 61]), being the blood clots with low coagulation times the samples with the lowest values measured by MRI. In vivo studies showed a significant T1 × T2 signal loss in the clot region of 24% after i.v. injection of KCREKA-NPs. The thrombus age (2.5% ± 6.1% vs. 81.3% ± 19.8%, p < 0.01) confirmed our ability to identify in vivo fresh blood clots. In this study, we developed and tested a dual MRI nanoparticle, acting as T1 and T2 contrast agents in MRI analyses. The developed KCREKA-NPs showed affinity for the fibrin content of blood clots, and the MRI signals provided by the nanoparticles showed significant differences depending on the clot age. The developed KCREKA-NPs could be used as a tool to predict the efficacy of a recanalization treatment and improve the triage of ischemic stroke patients.

Nanocarrier Drug Release and Blood-Brain Barrier Penetration at Post-Stroke Microthrombi Monitored by Real-Time Förster Resonance Energy Transfer-Based Detection System (FedEcs)

“…However, in many cases recanalization of large cerebral vessels does not result in reperfusion of the cerebral microcirculation and restoration of cerebral blood flow and may thus allow additional tissue damage to occur 16 . In previous attempts to dissolve microthrombi after stroke researchers attached the main thrombusattaching protein (Cys-Arg-Glu-Lys-Ala) CREKA on the surface of nanocarrier [17][18][19] , however, due to the lack of appropriate imaging techniques it remained elusive whether these particles delivered their cargo into microclots. In the current study, we did not use any active targeting strategy, still our LNDs ended up in large numbers within microclots.…”

Integrative Approaches in Acute Ischemic Stroke: From Symptom Recognition to Future Innovations

“…Additionally, the therapeutic window for rtPA is constrained, typically to 4.5 h post-stroke onset, after which its utility diminishes and risks escalate. Thus, tools to predict patient responses to rtPA could revolutionize ischemic stroke management [200].…”

Current trends and future perspectives of stroke management through integrating health care team and nanodrug delivery strategy

“…Conjugating the MRI imaging with nanoscaled materials has clarified the capability of monitoring non-invasive post-stroke inflammation aspects. Enabling the control and monitoring of the inflammatory reaction allows the identification of the patient subsets, resulting in immunomodulatory therapies ( Debatisse et al, 2020 ; Tian et al, 2021 ; Alonso-Alonso et al, 2022 ). As an example, Sillerud et al (2020) applied novel iron-platinum nanoparticles conjugated with MRI, showing dynamic development monitoring of neuroinflammation during stroke progression in a living rat brain.…”