STROKE THROMBOLYSIS WITH r-tPA IMPROVED BY CERIUM OXIDE NANOPARTICLES – STRIC-ON

The synthesis of the nanoparticles aimed at degrading the clot consisted in two steps :

- in the first step, the CNP were coated with polyethylenglycol polymers (PEG to 1) avoid their agreggation and 2) to allow the grafting of DNase I on the amine function of the PEG. Characterization of the CNP@PEG was then performed (size, homogeneity, quantity of polymers coating the CNP, number of amines per CNP)

- in the second step, DNase I was added to the CNP@PEG either by direct grafting on the amine fonction or by indirect binding via glutaraldehyde.

Once the CNP were produced, it was verified that both the PEG coating and the DNase I grafting altered neither the antioxidant activity of the nanoparticles nor the ability of the DNase I to cleave DNA:

- Antioxidant capacity: This was assessed directly on oxygen free radicals (H2O2 or superoxide anions) or on mouse endothelial cell cultures. These endothelial cells are part of the blood vessel walls and can therefore be damaged by oxygen free radicals produced during post-stroke reperfusion. Cultured cells were subjected to oxidative stress induced by glutamate—a mediator released in massive quantities during a stroke—and the effect of the CNP@PEG-DNase was studied.

Toxicity and Internalization: We also investigated the toxicity of our CNPs on these endothelial cells. Furthermore, the CNPs were grafted with fluorescent agents to observe their internalization within the endothelial cells.

- To verify that grafted DNase I on CNP@PEG is still able to degrade DNase, we studied its activity firstly on commercial fibrilar DNA then on NETs produced by the stimulation of neutrophils by phorbol-12-myristate-13-acetate (PMA).

Finally we studied the capacity of our modified CNP to improve r-tPA-induced clot lysis :

- the first study was performed on a blood clot formed in a well in the shape of a ring (halo test). The degradation of this clot by the treatments leads to the appearance of a red coloration in the center, the intensity of which can be measured.

- the second study involved working with clots enriched with neutrophils stimulated to produce NETs, and measuring the weight loss of the clot in the presence of the treatments; the percentage of weight loss serves as an indicator of clot degradation.

- the third technique involved a blood flow through microchannels and filming both the formation of a clot and its degradation by rt-PA combined with either free DNase or DNase grafted onto CNPs.

Bare CNPs agregate at physiological pH 7.4 so they can not be administered to patients. The coating of our CNPs by PEG polymers prevent their agregation (Figure 1A-C). The size of the coated CNP (CNP@PEG) ranged from 10 to 25 nm (depending on the PEG used) and was homogeneous (Figure 1D-F).

Glutaraldehyde, as a link between DNase I and the amine of the PEG coated on the CNP, allowed to graft more DNase I; this technique was kept for the subsequent studies.

After successful grafting of DNase, we demonstrated that the coating and grafting impact neither the antioxidant capacities of the CNP nor the capacity of DNase I to cleave DNA :

- CNP@PEG-DNase with their superoxide dismutase and catalase activity still degraded reactive oxygen species (ROS) superoxide anions and H2O2 (Figure 2). On endothelial cell culture, CNP@PEG-DNase were shown to enter the cells (Figure 3) and was devoid of toxicity (Figure 4). Then, oxidative stress was induced by glutamate, a mediator released during stroke. Glutamate ROS production was reduced by the well known antioxidant N-acetylcystein (NAC). CNP@PEG-DNase similarly suppressed the ROS produced by glutamate (Figure 5).

- DNase grafted on CNP@PEG was shown to cleave fibrillar DNA (Figure 6) as well as DNA from NETs produced by stimulation of neutrophils with PMA.

Finally, we tested the effect of CNP@PEG-DNase on r-tPA induced clot degradation. In the 3 tests used, halo test, clots enriched with NETs and studies including blood flow into microchannels, combining CNP@PEG-DNase to r-tPA improved the clot lysis compared to r-tPA alone.

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The CNP@PEG-DNase successfully improve the degradation by r-tPA of human blood clot, while maintening their antioxidant capacity which is thus a very promising strategy. Future works will aim at confirming these results in a in vivo model of stroke consisting in forming a clot into a cerebral artery in mice.In this model we will evaluate the effect of our modified CNP on r-tPA induced clot lysis and reperfusion, as well as the eir effect on cerebral lesion size and neurological deficit. The relevance of our strategy is highlited by recent publications reporting the benefit of DNase I grafted on nanoparticles or nanoclusters for stroke treatment (Zhang Tet al. DNase I-Mediated Chemotactic Nanoparticles for NETs Targeting and Microenvironment Remodeling Treatment of Acute Ischemic Stroke. Adv Sci (Weinh). 2025; 12:e03689;Sun J et al. DNase1 Mimic TMNCs Disrupt Neutrophil Extracellular Traps and Free Radical Circulation for Ischemic Stroke Therapy. Adv Healthc Mater. 2025; 14).

Ischemic stroke results from the thrombotic obstruction of cervical or cerebral arteries leading to cerebral infarction with neurological dysfunction, and represents the second cause of mortality and the first cause of acquired disability in France and Europe. The primary goal of stroke treatment is therefore the on-time restoration of blood flow in the occluded artery. Currently, r-tPA (recombinant tissue plasminogen activator), a thrombolytic agent, is been used for this purpose. However, age-related contraindications or delay to instore the treatment has limited its use to less than 15% of patients. Furthermore, early arterial recanalization with good outcome and reduced mortality in these patients is obtained in only one-third of treated cases. The mechanism of this resistance to thrombolysis by r-tPA remains unknown. A special attention was recently given to the presence of DNA fibers or NETs (neutrophil extracellular traps) entangled with the mesh of fibrin in the clot. Since DNA fibers are resistant to the effect of plasmin their presence in the clot may explain the failure of r-tPA to lyse thrombi. In this project, we aim to evaluate the impact of DNase I, a specific nucleolytic enzyme, on thrombus lysis as adjuvant of r-tPA treatment. Our hypothesis is that concomitant treatment with r-tPA and DNase I specifically targeted to the thrombus will contribute to an efficient and safe thrombolytic strategy to improve stroke outcome. The simultaneous hydrolysis of both DNA and its core proteins by combined proteolytic and nucleolytic activity of serum DNase I and plasmin locally generated seems necessary to disintegrate NETs.
The main objective of our project is to use nanoparticulate carrier-based systems that convey active DNase I and preferentially accumulate on the clot by being equipped with ligands that target the clot fibrin. To tailor new all-in-one nanovectors fulfilling these characteristics, we propose the use of cerium oxide nanoparticles (CeO2 or nanoceria) coated with DNase I. Cerium oxide nanocrystals are non-stoichiometric particles with tri- and tetravalent cations Ce3+ and Ce4+ at their surface. The coexistence of two oxidation states confers to these particles remarkable catalytic properties, their activity being similar to that of catalase, superoxide dismutase or peroxidase enzymes. Catalytic processes involving sub-10 nm nanoceria were shown to lead to the decomposition of reactive oxygen species. In upstream research, nanoceria has been used as a therapeutic agent in the treatment of oxidative stress associated with diseases in animal models including cardiomyopathy, sepsis, multiple sclerosis. Oxidative stress that occurs after a stroke is a well-known and major contributor to neuronal but also vascular lesions, which in turn lead to cerebral hemorrhages particularly deleterious for the patients. Moreover, recanalization after stroke, through oxygen transported by the blood may aggravate oxidative stress. Cerium oxide nanoparticles with their antioxidant capacities may thus protect vessels during recanalization and prevents cerebral hemorrhages. Our approach complements classical r-tPA fibrinolysis by focusing on NETs and oxidative stress using bifunctional cerium oxyde nanoparticles bearing DNase I activity.