Enhancing Thrombolysis Safety in Post-Acute Ischemic Stroke with Tissue Plasminogen Activator-Associated Microparticles

Abstract

Recombinant tissue-type plasminogen activator (tPA) is the only approved thrombolytic drug for acute ischemic stroke, a condition associated with severe disabilities and high mortality. However, when the blood-brain barrier (BBB) is damaged, tPA can exacerbate cerebral injury and increase the risk of hemorrhagic transformation, limiting its use to a small subset of patients. To address this challenge and minimize extravascular accumulation, we combined tPA with micrometer-sized particles (DPN). We then tested their safety and neuroprotective effects. After a 1 h transient occlusion of the middle cerebral artery, free tPA, tPA-DPN, or saline was administered to assess mice survival, neurological behavior, and infarcted area extent. Free-tPA exacerbated brain damage, resulting in a modest 10% survival rate at 24 h post intervention. Conversely, tPA-DPN displayed a far better prognosis, with a 75% survival rate comparable to that of saline. No statistical differences were documented between tPA-DPN and saline for the Activity Score and the Neurological Severity Score. tPA-DPN did not increase lesion volume or BBB permeability, unlike free-tPA, which led to an over 2-fold enlarged lesion volume and 50% higher BBB permeability. The safety profile of tPA-DPN is attributed to the robust conjugation of tPA onto DPN and the lack of DPN extravasation, resulting in negligible cerebrovascular damage of free tPA and glial and neuron impairment. The vascular confinement of tPA linked to microscopic particles reduces drug side effects and represents a valuable strategy for safe and effective tPA delivery, even in the postacute stroke phase.

Keywords: blood–brain barrier; glial cells; nanomedicine; neuroprotection; thrombolytics.

1

Characterization of DPN loaded with tissue-type plasminogen activator (tPA). (A) Schematic representation of DPN loaded with tPA. (B) Synthetic route for the tPA conjugation on PLGA molecules. (C) Representative images of the morphology of tPA-DPN after tPA-conjugation obtained by scanning electronic microscopy (scale bar 1 μm). (C) is 6000× magnification acquisition. The inset shows high magnification detail with a single particle (scale bar 1 μm). (D) Multisizer size distribution and (E) DLS ζ-potential measure of DPN and tPA-DPN (n = 18, ****, p < 0.0001, two-tailed unpaired t-test). (F) tPA bioconjugation efficiency (BE), expressed as the percentage of the tPA amount measured on tPA-DPN compared with the tPA input. Concentration amounts were evaluated through bicinchoninic acid assay (BCA assay) (n = 18). (G) Quantification of the tPA amount per millions of DPN by the BCA assay (n = 18).

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In vitro thrombolytic efficacy of tPA-DPN by halo assay. (A) Schematic representation of the “halo” thrombolytic test. (B) Longitudinal real-time acquisition allows the calculation of additional parameters “activation time” (A _t), CLR _max, and T _0.5.. (C) In vitro thrombolytic plot with free-tPA, DPN, and tPA-DPN clot dissolution profile. (D–F) show the additional parameters evaluated within the test (n = 6, two-tailed unpaired t-test).

3

tPA-DPN neuroprotective evaluation in the preclinical model of transient Middle Cerebral Artery (MCA) occlusion by filament. (A) Schematic of the animal model and description of the experimental end points. Briefly, 24 h before the surgery all animals were tested for behavioral scoring to check the initial status of the mice. Activity and Neurological Severity scoring were repeated 24 h post-occlusion (PS; fMCAO1h) and treatment (saline, tPA 10 mg kg^–1 or tPA-DPN 10 mg kg^–1). (B) Survival rate 24 h PS, after treatment with just vehicle (saline), free-tPA 10 mg kg^–1, and tPA-DPN 10 mg kg^–1. (C) Activity scoring and (D) neurological severity scoring (NSS). Black symbols refer to animals found dead after 24 h. Results are expressed as mean ± SD (n ≥ 6; *p < 0.05, **p < 0.01, respectively; one-way ANOVA, with Tukey correction).

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Histological analysis for lesion size. (A) Representative images from the three different experimental groups, stained with Cresyl Violet, to highlight the lesion area (white), against the undamaged areas (violet). The scalebar, in the lower left of each panel, is 5 mm. (B) Lesion volume, expressed in mm³, was obtained measuring the unstained Cresyl Violet negative brain area over the sections. Black symbols refer to animals found dead. Results are expressed as mean ± SD (n > 4; **p < 0.01; one-way ANOVA, with Tukey correction). (C) Pattern of the ischemic lesion area over the brain and among the different conditions. Each section was localized inside the brain and over the antero-posterior axes, using the Bregma point as center (0). (D) Detail of the posterior part of the brain (from −5 mm to 0 mm from the Bregma point) for the CV^– area distribution. (E) Area Under the Curve (AUC) calculations were performed individually for each sample. AUC measurements were plotted for each experimental group as mean ± SD (n = 6; **p < 0.01, one-way ANOVA, with Tukey correction).

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Histological analysis for blood–brain barrier leakage. (A) Representative IgG-stained images, displaying BBB leakage. IgG presence is visible as green signal, while blue is the counterstaining of the cell nuclei. The scalebar, in the lower left of each panel, is 5 mm. (B) IgG⁺ volume, expressed in mm³, was obtained measuring the brain area over the sections positive to the fluorescent IgG signal (n = 6; *p < 0.05; one-way ANOVA, with Tukey correction). (C) Pattern of the IgG-positive area over the brain and among the different conditions. Each section was localized inside the brain and over the antero-posterior axes, using the Bregma point as center (0). (D) Detail of the posterior part of the brain (from −5 mm to 0 mm from Bregma point) for the IgG area distribution. (E) area under the curve (AUC) calculations for the IgG⁺ profiles, performed individually for each sample. AUC calculation expressed as mean ± SD (n ≥ 5; *p < 0.05, one-way ANOVA, with Tukey correction). (F) 3D image reconstruction from histological samples, with volume overlapping among whole brain (light blue), lesion size (yellow), or BBB leakage (green).

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T1-weighted MRI analysis for blood–brain barrier leakage. (A) Schematic representation of the experimental plan for BBB leakage MRI investigation. 24 h before the surgery, T1-weighted (T1w) imaging was performed to check the initial status of the mice. The contrast agent (ProHance) was systemically injected 15 min before the imaging. MRI T1w imaging with contrast agent was repeated 3 h post-occlusion (PS; fMCAO 1h) and treatment (saline, tPA 10 mg kg^–1 or tPA-DPN 10 mg kg^–1). (B) Representative T1-weighted images after the injection of the contrast agent (CA, ProHance) 3 h PS. Scalebar 5 mm. (C) Comparison of the BBB leakage among experimental groups. Results are expressed as mean ± SD (n = 3; **p < 0.01, ***p < 0.001, respectively; one-way ANOVA, with Tukey correction). (D) 3D image reconstruction from MRI acquisitions, with volume overlapping among whole brain (light blue) and BBB leakage (green).

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In vitro effect of free-tPA and tPA-DPN on primary glial cells. (A), (B), and (C) show the impact on cell metabolisms of 24 h free-tPA treatment at different concentrations. (D) Schematic representation of the in vitro model composed by a mixed co-culture of glial cells in the presence of a porous barrier (Transwell chamber, with 0.4 μm pore-size). The different treatments (free-tPA 50 and 150 μg mL^–1, tPA-DPN 150 μg mL^–1, DPN or complete media) were loaded inside the apical part of the inset. (E) Representative confocal images showing cells morphology change after the different treatments. Here, nuclei were stained with DAPI (blue), while Iba1 and GFAP were in green and red, respectively. Black and white images of each row of the panel show the single marker expression for each area acquired, with the last color image displaying the composite image with all the markers. Images were acquired at 20× magnification (scale bar: 100 nm). (F,G) Immunofluorescence analysis on Iba1 and GFAP positive area inside the region of interest, expressed as percentage. Results are expressed as mean ± SD (n = 3; *p < 0.05; one-way ANOVA, with Tukey Correction).

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Immunofluorescence analysis of microglia and astrocytes response in stroke under the different treatments. (A) Representative images of microglial cells, stained for DAPI (nuclei, blue) and CD11b (green). The two panels represent the situation in the contralateral (Co) and ipsilateral (Ip) hemispheres. Black and white images of each row of the panel show the single marker expression for each area acquired, with the last color image displaying the composite image with all the markers. All images were acquired at 40× magnification (scale bar: 100 μm). (B) Percentage of area positive to CD11b expression, measured in Co and Ip hemispheres. Results are expressed as mean ± SD (n ≥ 3; two-tailed unpaired t-test). (C) Representative images of cerebral astrocytic cells, stained for DAPI (nuclei, blue) and GFAP (red). The two panels represent the situation in the contralateral (Co) and ipsilateral (Ip) hemispheres. Black and white images of each row of the panel show the single marker expression for each area acquired, with the last color image displaying the composite image with all the markers. All images were acquired at 40× magnification (scalebar 100 μm). (D) Percentage of area positive to GFAP expression, measured in Co and Ip hemispheres. Results are expressed as mean ± SD (n ≥ 3; *p < 0.05; two-tailed unpaired t-test).

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Advantages of using micrometric-particles as thrombolytic agents. The intravascular confinement of tPA using tPA-microparticles, like the tPA-DPN, reduces cerebral side effects and improves survival and behavioral outcomes.