Polymersomes preventing brain infiltration of CD177^+ neutrophils to mitigate hemorrhagic transformation post-tPA thrombolysis

Domain: biology
Published: 2026-03-25
Editorial outcome: major_revision

Abstract
As an intravenous thrombolytic agent, tissue plasminogen activator (tPA) is limited by hemorrhagic
transformation (HT) and a narrow therapeutic time window. Here we develop ROS-triggered
polyermersomes conjugated with fibrin-targeting CREKA peptide (CP) for tPA encapsulation.
CP@tPA achieves efficient thrombosis targeting and enhanced thrombolytic efficacy, however, it has not been able
to avert the occurrence of hemorrhage subsequent to thrombolysis. Building from our clinical discovery
that stroke patients suffer from tPA-induced HT featured strong expression of CD177, recombinant
CD177 protein was loaded into CP polymersomes (CP@rCD177) as nanomedicine and administrated
prior to CP@tPA thrombolysis. rCD177 can be released in response to elevated ROS within the obstructed
vessels, which binds to endothelial cells through interacting with CD31 and efficiently prevents the
migration of CD177+ neutrophils into the brain parenchyma. The reduced CD177+ neutrophils suppress
the generation of neutrophil extracellular traps (NETs), thereby dampening the inflammatory activation
of microglia and ultimately improving the prognosis of HT. This innovative strategy presents a promising
avenue for attenuating hemorrhagic complications post-tPA thrombolysis.

Introduction
Ischemic stroke, a leading cause of long-term disability and death globally, is characterized by the
occlusion of cerebral blood vessels1-3. The rapid restoration of blood flow through thrombolytic therapy
is crucial for salvaging ischemic penumbra and minimizing neurological deficits. Tissue plasminogen
activator (tPA) has been the only U.S. Food and Drug Administration (FDA)-approved thrombolytic drug
for the treatment of acute ischemic stroke4,5. By intravenous injection, tPA catalyzes the conversion of
plasminogen to plasmin, which in turn breaks down the fibrin mesh of the clot, thereby restoring blood
flow to the ischemic area6. Despite its proven efficacy in thrombolysis, tPA therapy still remains
limitations. Its short half-life necessitates continuous infusion7, and there is a potential risk of hemorrhagic
transformation (HT)8,9, which can exacerbate patient outcomes and augment mortality rates. This risk
necessitates stringent adherence to a 4.5-h time window from symptom onset.

The mechanism underlying tPA-induced HT is complex and multifactorial, involving the disruption
of the blood-brain barrier (BBB), inflammatory responses, and oxidative stress10,11. BBB plays a critical
role in maintaining the homeostasis of the central nervous system, and its integrity is compromised
following ischemic stroke, particularly after reperfusion with tPA12. The disruption of the BBB allows for
the extravasation of blood into the brain parenchyma to cause HT. Additionally, inflammatory processes
are also implicated in tPA-related HT. Post-ischemic inflammation triggered by tPA, involves the
recruitment and activation of neutrophils and other immune cells, which can exacerbate brain injury and
increase the risk of HT13-15. Reactive oxygen species (ROS) generated during reperfusion can further
amplify the inflammatory response and contribute to neuronal damage and BBB dysfunction16,17.
Understanding the intricate mechanisms of tPA-induced HT is crucial for developing strategies to mitigate
this complication.

Our clinical observations have uncovered a significant finding in the context of HT patients who
have undergone tPA therapy. We have noted a robust expression of CD177 in their peripheral blood. As
a cell surface glycoprotein mainly expressed on neutrophils, CD177 engages in a critical interaction with
platelet endothelial cell adhesion molecule-1 (PECAM-1, also known as CD31)18,19. This interaction is
instrumental in modulating the phosphorylation of CD31 and the integrity of endothelial junctions, which
in turn facilitates the transendothelial migration of neutrophils19-21. Moreover, CD177+ neutrophils are
capable of generating elevated levels of ROS, neutrophil extracellular traps (NETs), and myeloperoxidase
(MPO), molecules that are known to disrupt the BBB and contribute to HT22-24. This suggests that CD177+
neutrophils serve as critical predictive biomarkers for thrombolysis-induced hemorrhages, thereby
emphasizing their potential as therapeutic targets to mitigate the risk of HT.

In this study, we develop thrombus-targeting and ROS-triggered polymersomes to separately load
tPA and recombinant CD177 protein (rCD177) for thrombolysis while concurrently inhibiting HT (Fig.
1). Given the abundance of fibrin on the surface of both patient and mouse blood clots (Supplementary
Fig. 1), fibrin-targeting peptide (Cys-Arg-Glu-Lys-Ala, CREKA) was anchored onto the surface of
polymersomes to specifically recognize fibrin3,25. Additionally, phenylboronic acid (PBA) was
incorporated into polyethylene glycol (PEG) and polylactic acid (PLA) as ROS-responsive triggers to
facilitate the structural disintegration of the polymersomes at the thrombus site26,27. This process leads to
a targeted release of tPA, effectively dissolving the thrombus. Additionally, the polymersomes can
prolong the half-life of tPA, thereby achieving sustained thrombolytic effects with a single administration.
Intervention with CP@rCD177 prior to CP@tPA thrombolysis can impede the brain infiltration of
CD177+ neutrophils by binding to CD31, diminishing the generation of NETs and consequently curbing
the inflammatory activation of microglia. Notably, the co-administration of CP@rCD177 with CP@tPA
significantly preserve the integrity of the BBB and mitigate the incidence of tPA-associated hemorrhage,
thereby boosting its therapeutic efficacy in ischemic stroke. Our findings confirm the role of CD177+
neutrophils in hemorrhage following thrombolysis and offer a novel therapeutic approach for the clinical
attenuation of tPA-induced HT.

Results
Preparation and characterization of the polymersomes
To prepare CP polymers, PEG was connected with PBA and subsequently initiated the ring-opening
polymerization of D,L-LA to get PEPLA, which was conjugated with CREKA-peptide to produce the
final product (Supplementary Fig. 2). The chemical structures of CP and the intermediates were confirmed
by 1H nuclear magnetic resonance (1H NMR) (Supplementary Fig. 3). The CP polymersomes were
prepared by solvent evaporation and tPA was encapsulated within the vesicles by electroporation (Fig.
2a). As controls, PEG-PLA (PELA) and PEG-PBE-PLA (PEPLA) were loaded with tPA to prepare
Null@tPA and P@tPA, respectively. Dynamic light scattering (DLS) measurements indicated that the
particle size and zeta potential of CP@tPA are 125.7 nm and -3.43 ± 0.74 mV, respectively
(Supplementary Table1). The drug loading content and encapsulation efficiency of CP@tPA are 7.55%
and 81.73%, respectively.

The phenylboronic moiety undergoes sequential oxidation and hydrolysis when stimulated by
H2O25,28. To confirm the ROS-triggered disruption of boronic ester bonds in polymersomes (Fig. 2a),
transmission electron microscopy (TEM) was performed to examine the morphology changes in
Null@tPA, P@tPA, and CP@tPA. Compared to the polymersomes that maintain a closed vesicular
morphology with a bilayer structure under normal condition, significant structural damage was observed
in the boronic ester-modified P@tPA and CP@tPA after incubation with H2O2 (Fig. 2b). Consistent with
the TEM images, DLS showed size change in the ROS-responsive polymersomes upon H2O2 exposure
(Fig. 2c and Supplementary Fig. 4a). In contrast, the structure and particle size of the non-responsive
Null@tPA remained unchanged (Fig. 2b and Supplementary Fig. 5a). The ROS-induced destruction of
the polymersome structure inevitably accelerates drug release, resulting in a 2.68-fold and 2.3-fold
increase in tPA release from P@tPA and CP@tPA after 24 h of incubation with H2O2 (Fig. 2d and
Supplementary Fig. 4b). No significant change in tPA release was observed when Null were treated with
H2O2 (Supplementary Fig. 5b). Additionally, all three polymersomes exhibited good size stability in PBS,
with slight size changes over a week (Fig. 2e). To ensure in vivo applicability of CP@tPA, we further
evaluated their colloidal stability in fetal bovine serum (FBS) and long-term stability. The results indicated
that CP@tPA exhibited excellent stability within 8 h in FBS (Supplementary Fig. 6a), and maintained
structural integrity for up to 30 days when stored at 4 °C in the dark (Supplementary Fig. 6b). The ROS
scavenging ability of Null is negligible compared to that of P and CP, whereas the responsive
polymersomes reduced H2O2 content by more than 80% (Fig. 2f). Despite the widespread clinical use of
tPA as a thrombolytic agent, its therapeutic application is constrained by the rapid blood clearance due to
its short half-life29. The in vitro cytotoxicity indicated that CP@tPA possessed good biocompatibility
when incubated with bEnd.3 cells for 24 h, showing cell vitality over 85% (Supplementary Fig. 7). The
hemolysis rate of CP@tPA was below 5%, and the blood clotting index (BCI) index was comparable to
that of the saline group (Supplementary Fig. 8), demonstrating good blood compatibility. To investigate
whether the polymersome encapsulation can prolong the blood circulation of tPA, the pharmacokinetics
of CP@tPA was evaluated. We found that CP polymersomes significantly extended the half-life of tPA
from 6.99 min to 65.75 min (Supplementary Fig. 9 and Supplementary Table 2). After 2 h post-
administration, CP@tPA maintained 31.79% of its initial tPA content.

CP facilitates thrombus targeting
We first explored the binding affinity of CREKA-modified polymersomes to fibrin using in vitro clots.
After treating the thrombi with ICG-loaded polymersomes, CP@ICG group displayed remarkably
stronger fluorescence than Null and P groups at each time point (Supplementary Fig. 10a), indicating
enhanced targeting property mediated by CREKA peptide. Specifically, the fluorescence intensity of the
CP@ICG group was 2.03 times and 2.06 times higher than that of the Null@ICG and P@ICG groups,
respectively (Supplementary Fig. 10b). Additionally, the distribution of polymersomes on the thrombus
was observed through SEM. The images revealed that CP were abundantly anchored to fibrin on the
thrombus surface, whereas significantly fewer thrombus adhesions were observed in Null and P groups
(Fig. 2g).

The thrombus-targeting capability of the polymersomes was subsequently assessed in stroke mice.
As shown in Fig. 2h, CP@ICG exhibited a significantly stronger fluorescence signal in the thrombus-
occluded area compared to the non-targeted groups. Quantitative analysis of the average radiance
efficiency at 2 h post-injection indicated that the fluorescence intensity of CP@ICG in the infarct brain
was 5.14-fold and 4.9-fold higher than that of the Null@ICG and P@ICG groups, respectively
(Supplementary Fig. 11). Ex vivo fluorescence analysis further confirmed that CP@ICG possessed a
strong signal in the ischemic area (Supplementary Fig. 12a). Specifically, the CP@ICG intensity in the
infarct brain was 2.7-fold and 3.77-fold higher than that in the Null@ICG and P@ICG groups,
respectively (Supplementary Fig. 12b). Furthermore, immunofluorescence staining with antifibrin
antibody demonstrated that CP@ICG-treated brain tissues exhibited a remarkably high degree of ICG
signal colocalization with fibrin (Fig. 2i), indicating specific targeting to fibrin-rich thrombi. These results
proved that CP polymersomes could effectively accumulated at the thrombus site, thereby enhancing tPA
concentration at the lesion site and contributing to thrombolytic effect. Histopathological and serum
biochemical analyses revealed no significant damage to major organs or impairment of hepatic and renal
functions following CP@tPA treatment (Supplementary Fig. 13a and 13b), suggesting good
biocompatibility and biosafety of these nanodrugs in vivo.

CP@tPA promotes thrombolysis
CP@tPA exerts thrombolytic activity and restores blood flow through CREKA-mediated thrombus
targeting and ROS-triggered payload release (Fig. 3a). We systematically evaluated its thrombolytic
performance across three distinct thrombus models, including carotid artery thrombus, lower extremity
arterial thrombus, and middle cerebral artery occlusion (MCAO). In the lower extremity arterial thrombus
model, free tPA failed to achieve complete clot dissolution within 2 h (Fig. 3b). In contrast, CP@tPA
demonstrated progressive thrombus resolution, evidenced by gradual clot lightening and complete
restoration of blood flow within the same timeframe. Thrombus-induced vascular obstruction creates a
hypoxic environment, promoting ROS production and leading to platelet activation and aggregation30,31.
Using two-photon microscopy, we observed persistent platelet aggregation in the lower extremity arterial
thrombosis, which exacerbates vascular occlusion (Fig. 3c). CP@tPA treatment accelerated platelet signal
reduction (vs. free tPA) and restored functional perfusion after 1 h. Mechanistically, CP@tPA attenuated
thrombogenic inflammation by reducing soluble CD40 ligand (sCD40L) levels – critical mediators of
endothelial activation32,33. The plasma levels of sCD40L in the CP@tPA group were reduced by 41.08%
and 33.58% compared to the saline group and tPA group, respectively (Fig. 3d). This dual therapeutic
effect (ROS scavenging by CP polymersomes and enhanced tPA delivery) likely underlies the observed
thrombolytic enhancement. Laser speckle imaging confirmed superior perfusion recovery in CP@tPA-
treated subjects. Lower extremity arterial thrombi showed 79.98 ± 5.56% flow restoration versus 28.51 ±
5.19% (saline) and 63.51 ± 2.17% (tPA) (Supplementary Fig. 14a,b). H&E staining of vascular sections
at the thrombus site revealed thrombus areas in CP@tPA group were reduced to 26.53% of saline group
(Supplementary Fig. 14c,d). Comparable efficacy advantages were observed in carotid artery thrombus
model (Supplementary Fig. 15a-c).

We further evaluated the thrombolytic efficacy of CP@tPA using the MCAO model, a well-
established preclinical model for studying ischemic stroke. Thrombolytic therapy was initiated 2 h
poststroke, and regional cerebral blood flow (rCBF) was continuously monitored. Thrombus injection
into the middle cerebral artery led to a precipitous drop in rCBF, with flow persistently restricted to less
than 30%, mimicking the severe ischemic conditions observed in stroke patients (Fig. 3e,f). 2 h after
administration, free tPA restored partial reperfusion, with rCBF recovering to approximately 50%.
In contrast, CP@tPA treatment achieved significantly superior recovery, with rCBF reaching 71.86 ± 8.57%.
To quantify thrombus dissolution, we measured serum levels of fibrin degradation products (FDP), a
biomarker of fibrinolysis34. As shown in Fig. 3g, FDP level in the CP@tPA group surged compared to
the MCAO and tPA groups. The superior thrombolytic efficacy of CP@tPA can be attributed to its unique
properties, including prolonged blood half-life, thrombus targeting ability, and ROS-responsive drug
release characteristics. These features collectively enhance the bioavailability and therapeutic effect of
tPA at the site of thrombosis.

CD177+ neutrophils induce hemorrhagic transformation
Despite the superior thrombolytic efficacy demonstrated by CP@tPA, it has not effectively alleviated the
HT side effect after thrombolysis (Fig. 3h and Supplementary Fig. 16). To elucidate the mechanisms
underlying tPA-induced HT, we collected peripheral blood samples from ischemic stroke patients who
developed HT following thrombolysis (Fig. 4a). RNA sequencing (RNA-seq) analysis revealed
significant changes in the expression of 485 genes (320 upregulated and 165 downregulated) in HT
patients (Fig. 4b). Pathway enrichment analyses using KEGG and Reactome indicated that tPA
significantly impacts pathways related to the immune system, innate immune system and inflammatory
signaling, including MAPK, NF-κB, Toll-like receptors signaling pathways, and Th17 cell differentiation
(Fig. 4c and Supplementary Fig. 17). Notably, the KEGG analysis detected an enrichment of genes
associated with NET formation, suggesting a potential association between tPA-induced HT and
neutrophils. By analyzing the GO enrichment of differentially expressed genes within the innate immune
system, these genes are involved in neutrophil activation, particularly through the upregulation of CXCR2
and CD177 (Fig. 4d), as well as their participation in various immune response pathways (Supplementary
Fig. 18). Notably, CD177 expression in the HT group was 16.6 times higher than that in the stroke group
(Supplementary Fig. 19). Previous studies have proved that CD177 can bind with CD31 to promote
neutrophil transmigration18-21.

To further elucidate the potential relationship between CD177⁺ neutrophils and tPA-induced HT, we
performed flow cytometry and immunofluorescence staining on peripheral blood and brain tissues from
tPA-treated MCAO mice that developed HT (Fig. 4e). The number of CD177+ neutrophils in the HT
mouse blood was 1.8-fold higher compared to that in the MCAO group (Fig. 4f,g and Supplementary Fig.
20). Moreover, a marked increase in CD177+ neutrophil infiltration was observed in the brains of HT
mice, reaching levels approximately 3.6 times higher than those in the MCAO group (Fig. 4h,i). The
expression of CD177 can also be upregulated when treating the isolated mouse neutrophils with tPA
(Supplementary Fig. 21). These findings, combined with clinical data, suggest that tPA-induced HT
involves the upregulation of CD177+ neutrophils and their infiltration into brain tissue. The process of
neutrophil migration may contribute to BBB disruption, which could serve as a critical prerequisite for
HT development. To further investigate the role of CD177+ neutrophils in BBB permeability, we created
a cranial window above the ischemic brain to monitor the real-time dynamics of CD177+ neutrophil
transmigration and vascular leakage (Fig. 4j). Following tPA administration, CD177+ neutrophils were
initially adhering to the luminal side of the vascular wall, and subsequently infiltrated into the ischemic
brain in a time-dependent manner (Fig. 4k). Concurrently, we observed that as CD177+ neutrophils
migrated from the vasculature into brain tissue, they induced BBB disruption, leading to FITC-dextran
leakage. The extravasation was significantly enhanced in regions where CD177+ neutrophil infiltration
occurred (Supplementary Fig. 22).

The increased BBB permeability mediated by CD177+ neutrophils is likely due to their enhanced
ability to generate NETs, which play a pivotal role in tPA-induced HT. Specifically, histones and
proteases within NETs directly degrade tight junction proteins and the basal lamina, leading to increased
vascular permeability35,36. Additionally, NETs enhance thrombo-inflammation by activating platelets and
amplifying the release of pro-inflammatory cytokines, further compromising BBB integrity37. To prove
this, we evaluated NET formation after 90 min of tPA stimulation. Neutrophils triggered by tPA displayed
markedly intensified SYTOX signals compared to the MCAO group, confirming enhanced NET
generation (Supplementary Fig. 23b). Quantitative analysis using enzyme-linked immunosorbent assay
(ELISA) further supported these observations, with NET and MPO levels in the tPA group elevated by
2.2-fold and 1.9-fold, respectively, relative to the MCAO group (Supplementary Fig. 23b,c). These
findings collectively uncover a critical pathway underlying HT: tPA drives CD177+ neutrophil
recruitment to the brain, where excessive NET production compromises BBB integrity, culminating in
hemorrhagic complications.

CP@rCD177 polymersomes improved tPA-induced HT
Intervention against CD177+ neutrophil infiltration represents a promising therapeutic strategy to
mitigate HT. Given that CD177+ neutrophils interact with CD31 through surface receptors, an interaction
blockable by recombinant CD177 (rCD177)38, we evaluated rCD177's efficacy in suppressing neutrophil
migration using transwell assays (Fig. 5a). By counting the cell number in the lower chamber, the result
revealed that rCD177 reduced neutrophil migration rates by 34.8% (from 78.4% to 51.1%, Fig. 5b). Flow
cytometry further demonstrated a 42.5% decrease in tPA-induced CD177+ neutrophil transmigration with
rCD177 treatment (Fig. 5c and Supplementary Fig. 24). Notably, NET production in the lower chamber
was dramatically reduced to 23.4% of tPA-treated group levels (Supplementary Fig. 25). These findings
position rCD177 as a potential therapeutic agent that attenuates CD177+ neutrophil-mediated BBB
disruption, thereby ameliorating HT progression.

To enhance therapeutic precision, we engineered CP@rCD177 by encapsulating rCD177 into CP
polymersomes. CP@rCD177 possessed the particle size of 124.3 nm and zeta potential of -3.85 ± 0.75
mV (Supplementary Table 3). The drug loading content and encapsulation efficiency of CP@rCD177 are
6.62% and 72.82%, respectively. Similarly, CP@rCD177 also exhibit excellent colloidal stability and
long-term particle stability (Supplementary Fig. 26a,b), comparable to that of CP@tPA (Supplementary
Fig. 6a,b). This nanoplatform achieves ROS-triggered payload release (Supplementary Fig. 26c,d) and
prolonged systemic circulation (3.88-fold half-life extension vs free rCD177; Supplementary Fig. 26e and
Supplementary Table 4), ensuring sustained bioactivity in vivo. We next assessed CP@rCD177's capacity
to inhibit brain infiltration of CD177+ neutrophils (Fig. 5d). Immunofluorescence at 22 h post-tPA
administration revealed perivascular CD177+ neutrophil accumulation in mouse brains (Fig. 5e). When
mice were pre-treated with CP@rCD177 before thrombolysis, there was a marked overlap between CD31
and rCD177 at the vascular interfaces, along with a substantial reduction in the number of CD177+
neutrophils in the brain tissue (Fig. 5f). Flow cytometry analysis demonstrated that either tPA or CP@tPA
treatment upregulated the neutrophil populations (Fig. 5g). In contrast, interventions with rCD177 and
CP@rCD177 significantly reduced this occurrence. Notably, CP@rCD177 therapy decreased cerebral
neutrophils by 82.5% and CD177+ neutrophils by 87.4% compared to the tPA group (Supplementary Fig.
27a-c). Complementary immunofluorescence further validated CD177+ neutrophil suppression with
CP@rCD177 intervention (Supplementary Fig. 28). These findings establish that CP@rCD177-mediated
CD31 blockade disrupts neutrophil-endothelial interactions, effectively preventing CD177+ neutrophil
transmigration into ischemic brain.

To further investigate the potential of CP@rCD177 in preventing hemorrhagic complications, we
evaluated cerebral hemorrhage 22 h after therapy. Photoacoustic imaging (PA) was first employed for
real-time monitoring of intracerebral bleeding (Fig. 5h). Capitalizing on the strong optical absorption
characteristic of hemoglobin at 532 nm39, we utilized PA signals at this specific wavelength for
reconstruction of cerebral vasculature. PA observation revealed distinct spatial distribution patterns of
hemoglobin signals across groups (Fig. 5i). The sham and MCAO groups exhibited signals predominantly
confined within the vascular architecture, whereas thrombolytic treatment groups (tPA and CP@tPA)
demonstrated significant extravasation of hemoglobin signals into the parenchymal tissue, indicative of
HT. Notably, therapeutic intervention with rCD177 formulations substantially attenuated hemoglobin
signal dispersion, with CP@rCD177 therapy showing the most pronounced protective effect against HT.
Quantitative assessment of hemorrhagic volume using a hemoglobin detection kit demonstrated that
CP@rCD177 pretreatment significantly reduced bleeding by 61.6% and 48.7% compared to tPA and
CP@tPA groups, respectively (Fig. 5j). Examination of brain sections corroborated these findings,
revealing differential HT severity across treatment groups (Fig. 5k). We then investigated the hemorrhage
severity based on petechiae quantification in the ischemic hemisphere40. While the overall hemorrhage
rates were 91.7% and 83.3% in tPA and CP@tPA groups, respectively, CP@rCD177 pretreatment
reduced this incidence to 75% (Fig. 5l). More specifically, the intervention demonstrated particular
efficacy in mitigating severe hemorrhage, with HI-II and HI-III classifications both showing a 33.3%
reduction. Concurrently, we observed a 50% increase in NH (no hemorrhage) cases and a 200% elevation
in HI-I (mild hemorrhage) instances.

Neutrophil infiltration is closely associated with endothelial dysfunction and increased BBB
permeability; blocking this infiltration has been shown to effectively attenuate post-stroke BBB injury5.
To determine whether CP@rCD177 could confer similar protective effects, we investigated its capacity
to ameliorate neutrophil-mediated BBB damage. First, Evans Blue (EB) extravasation assay was
performed. The brain sections showed that cerebral ischemia induced substantial EB leakage
(Supplementary Fig. 29a), confirming BBB structural compromise. Notably, MCAO mice subjected to
thrombolytic therapy exhibited a marked elevation in BBB permeability, with spatial colocalization
between dye extravasation zones and hemorrhagic regions. While free rCD177 administration showed
moderate attenuation of BBB disruption, residual EB leakage remained significant compared to sham
group, a phenomenon potentially attributable to the short blood half-life (Supplementary Fig. 26e and
Supplementary Table 4). Notably, pretreatment with CP@rCD177 nanoparticles before CP@tPA
thrombolysis achieved superior BBB protection, reducing EB extravasation to 66.9% of thrombolysis-
only control (Supplementary Fig. 29b). To further assess BBB integrity in the ischemic hemisphere, we
employed intravital microscopy and TEM. Intravital imaging confirmed pronounced vascular leakage in
both thrombolysis-treated and MCAO groups relative to sham controls (Supplementary Fig. 30). While
pre-treatment with CP@rCD177 significantly reduced this extravasation. TEM analysis further
demonstrated disruption of the BBB ultrastructure following HT and ischemia, characterized by loss of
tight junctions and formation of inter-endothelial gaps (Supplementary Fig. 31). Importantly,
administration of CP@rCD177 substantially reversed these pathological changes, reducing the number of
inter-endothelial gaps to approximately 49% of those in the CP@tPA group. This protective effect may
play a critical role in suppressing hemorrhagic complications and maintaining neurovascular function.
Triphenyltetrazolium chloride (TTC) staining was then performed to evaluate the effect of nanodrugs
on ischemic damage. MCAO mice developed substantial cerebral infarction, exhibiting an infarct volume
of 35.68% at 24 h poststroke (Supplementary Fig. 32). Administration of free tPA and CP@tPA reduced
the infarct volume to 28.07% and 19.91%, respectively. Notably, intervention with CP@rCD177 resulted
in a further 39.21% reduction in infarct volume compared to CP@tPA group, culminating in a final infarct
volume of only 12.1%. This outcome underscores its superior efficacy in mitigating ischemic brain
damage by inhibiting CD177+ neutrophils infiltration. The therapeutic efficacy of CP@rCD177/tPA
combination therapy was further substantiated by a significant improvement in survival outcomes in
MCAO mice. Through its ability to stabilize the BBB and mitigate brain infarct, CP@rCD177
intervention enhanced the 24-h survival rate to 66.67% (Supplementary Fig. 33). These comprehensive
findings establish that CP@rCD177 pretreatment serves as an effective neuroprotective strategy,
simultaneously reducing the HT incidence and brain damage while significantly attenuating thrombolysis-
associated mortality risk.

CP@rCD177 polymersomes alleviated neuroinflammation and improved long-term neurological
effects
To assess post-treatment neuroinflammation, key pro-inflammatory cytokines (IL-1β, TNF-α) and anti-
inflammatory cytokines (IL-10, TGF-β) in the ischemic hemispheres were measured on day 3 after
treatment. As shown in Supplementary Fig. 34, both ischemia (MCAO group) and HT (tPA and CP@tPA
groups) resulted in significantly elevated pro-inflammatory cytokines and reduced anti-inflammatory
mediators. This inflammatory response is likely triggered by ischemia-reperfusion injury, tPA-induced
neutrophil chemotaxis, and microglial activation41-44. Importantly, CP@rCD177 therapy significantly
attenuated this response, reducing IL-1β and TNF-α levels by 12.1% and 48.5%, respectively, while
increasing IL-10 and TGF-β by 28.6% and 39.6%, respectively, compared to the MCAO group. These
results suggest that targeting CD177⁺ neutrophils can effectively alleviate thrombolysis-related
neuroinflammation.

We next assessed the long-term neurobehavioral recovery in mice through a comprehensive
behavioral test, which incorporated multiple sensorimotor assessments including the mNSS, corner turn,
rotarod, and adhesive removal tests, as well as the Barnes maze test to evaluate cognitive function. Our
results revealed that both MCAO surgery and post-thrombolysis led to severe motor deficits, accompanied
by significantly elevated mNSS scores (Supplementary Fig. 35). The functional impairments associated
with tPA thrombolysis were closely correlated with the hemorrhagic complications it induced14,45,46. In
contrast, pretreatment with CP@rCD177 prior to thrombolysis markedly improved functional outcomes:
reduced mNSS scores, more symmetrical turning behavior, shorter removal time in the adhesive test, and
longer latency to fall on the rotarod (Supplementary Fig. 36a-g). Spatial memory was assessed using the
Barnes maze on day 28 post-treatment (Supplementary Fig. 36h). Mice in the MCAO, tPA, and CP@tPA
groups required significantly more time, traveled longer distances, and made more errors before locating
the escape box compared to sham mice (Supplementary Fig. 36i-k). CP@rCD177 treatment effectively
attenuated these deficits, indicating preserved cognitive function. Together, these results demonstrate that
the combine use of CP@rCD177 and CP@tPA promotes significant recovery of sensorimotor and
cognitive functions, highlighting its potential as a promising therapeutic strategy for ischemic stroke.

CP@rCD177 inhibited HT by reducing CD177+ neutrophils-associated NET formation
To elucidate the mechanistic basis of CP@rCD177-mediated HT inhibition, we performed transcriptome
profiling of infarct hemispheres 24 h post-MCAO. Ischemia induced substantial transcriptomic changes
with 830 differentially expressed genes (DEGs: 221 upregulated, 609 downregulated) compared with
sham control (Supplementary Fig. 37). Only slight alterations were detected between tPA-mediated HT
group and MCAO group, with 65 DEGs identified (14 upregulated and 51 downregulated), suggesting
shared molecular injury mechanisms between ischemic and hemorrhagic pathologies. The combination
therapy of CP@rCD177/tPA elicited a distinct genomic signature, reversing the HT-induced expression
pattern with 138 DEGs (127 upregulated, 11 downregulated). Heatmap revealed that CP@rCD177/tPA
significantly suppressed neutrophil activation pathways, including chemotaxis, migration, while also
markedly downregulating NET formation genes (Fig. 6a,b). KEGG enrichment analysis further
demonstrated the multimodal action of CP@rCD177/tPA through modulating apoptosis regulation (p53),
inflammatory cascades (JAK-STAT, IL-17) (Fig. 6c). Subsequently, DEGs associated with BBB
disruption were selected for heatmap analysis (Fig. 6d). The results revealed a marked upregulation of
key inflammatory and apoptotic regulators Jun/Fos in the HT group47,48, and genes linked to apoptosis
(Gadd45), oxidative stress (Tsp1), and inflammation (Cox2) were elevated as well49-51. Notably,
chemokines (CCL7, CCL9) and the interferon-stimulated gene Isg15 were also upregulated, suggesting
enhanced neutrophil recruitment to the injury site52-54. Furthermore, the increased expression of Angpt2
likely contributes to heightened vascular permeability55. These transcriptional changes reflect dynamic
BBB damage-repair mechanisms that critically influence neuroinflammatory states and vascular stability.
CP@rCD177 intervention counteracted these effects, suppressing the expression of AP-1 and its
downstream targets, with concordant protein-level validation via Western blot (Fig. 6e). Furthermore,
CP@rCD177 attenuated HT-induced inflammatory cytokines IFN-β and IL-6, underscoring its anti-
inflammatory efficacy (Supplementary Fig. 38).

Integrating these findings with NET dynamics, we identified a mechanistic link between HT and
NET formation. By intravital microscopy monitoring, sham-operated mice exhibited intact vasculature
with minimal NETs, while excessive NETs were generated in HT group — predominantly localized at
sites of vascular leakage and surrounded by abundant neutrophils (Fig. 6f). This observation suggests that
neutrophil-derived NETs actively contribute to BBB destabilization. Strikingly, CP@rCD177/tPA
reduced cerebral NET content by 51.8% compared to tPA monotherapy (Supplementary Fig. 39), likely
due to pre-thrombolytic CP@rCD177 administration limiting CD177+ neutrophil recruitment.
Immunofluorescence mapping revealed NETs in tPA-induced HT brains predominantly co-localized with
microglia (Iba1+), with minimal association with astrocytes (GFAP+) or neurons (NeuN+) (Supplementary
Fig. 40). This spatial specificity implies CD177+ neutrophil-derived NETs exacerbate neuroinflammation
via microglial activation44. Collectively, tPA-induced HT involves a sequential pathological cascade
initiated by the infiltration of CD177+ neutrophils, which drives excessive NET production, subsequently
triggering microglia-mediated inflammation, and ultimately leading to BBB disruption and blood effusion
(Fig. 6g). Preemptive CP@rCD177 administration disrupts this pathogenic axis, preserving BBB integrity
and mitigating HT.

Methods
Materials
Trizol was purchased from TakaRa (Japan). N, N-Carbonyldiimidazole, Mal-Poly(Ethylene Glycol)-OH
(Mal-PEG-OH), D,L-Lactide (D,L-LA), 4-Carboxyphenylboronic acid (PBA), 1,1,1-
Tris(hydroxymethyl)ethane (TME), methacide and tetrahydrofuran (THF) were purchased from Adamas
(China). Dichloromethane (DCM) was purchased from Heowns (China). Sn(Oct)2, DMAP, DCC, Evans
blue, ICG, and FITC-dextran were purchased from Sigma-Aldrich (China). Mal-PEG-OH was purchased
from Ponsure (China). CREKA peptide was purchased from the Chinese Peptide Company (China).
CaAM/PI, CCK-8, and Hemoglobin test kits were purchased from Beyotime Biotechnology (China).
Mouse peripheral blood neutrophil isolating fluid kits were purchased from Servicebio (China). TTC and
Mouse TNF-α, TGF-β ELISA kit were purchased from Solarbio (China). tPA were purchased from
Boehringer Ingelheim (Germany). Hydrogen peroxide assay kit, BCA protein assay kit, and India ink
were purchased from Bioss (China). ELISA kits including sCD40L, ROS, and MPO were purchased from
Saipei (China). ELISA kits including IL-6, IFN-β, and FDP were purchased from Shanghai Enzyme-
linked (China). Mouse NET and IL-1β ELISA kits were purchased from Jiangsu Meimian (China). Mouse
IL-10 ELISA kit was purchased from Multi Sciences (Lianke) Biotech (China). SYTOX Green was
purchased from Invitrogen (USA).

Cells and animals
Bend.3 cell lines (iCell Bioscience Inc, Shanghai, China) were separately cultivated in DMEM medium
(Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. Neutrophils were extracted from the blood
of male C57BL/6J mice.

C57BL/6J mice (8-12 weeks, male) were purchased from Beijing Huafukang Biotechnology Co.,
Ltd. (Beijing, China). All animals were maintained under 12 h of light-dark cycle at 25 ± 1 °C and 50%
~ 60% of humidity with access to food and water ad libitum. All animal tests were approved by the
Institutional Animal Care and Use Committee of Southwest Jiaotong University (SWJTU-2203-
NSFC(007)).

Mouse embolic MCAO model68
The blood clot was first prepared in vitro. Mouse blood was drawn into a PE-50 tube, coagulated at room
temperature for 2 h, and then left at 4 °C for 22 h. The tube containing thrombus was then cut into 5 cm
in length, and one side was connected to PE-10 tube attached with another PE50 tube. Saline was
repeatedly injected into the syringe for rinse. Then 5% isoflurane was applied to C57BL/6J mice by gas
anesthesia. After deep anesthesia, the carotid artery (CCA), external carotid artery (ECA), and internal
carotid artery (ICA) were isolated. The modified PE50 (terminal outer diameter < 0.2 mm) tube was
inserted into the ICA through the ECA. Thrombosis (1.5 cm) was injected with saline to form a central
cerebral artery embolism. The cerebral blood flow (CBF) was monitored by RFLSI III Laser Speckle
Blood Flow Imaging System (RFLSI III., RWD).

Mouse carotid artery and lower extremity arterial thrombus models69
The carotid artery thrombus and lower extremity arterial thrombus were induced by ferric chloride (FeCl3).
For carotid artery thrombus model, mice were anesthetized to expose the CCA blood vessels. Then the
filter paper (4 × 1 mm) soaked with 10% FeCl3 solution was applied to the CCA surface and removed 3
min later. After 10 min, thrombosis was observed under the digital microscope (DOM-1001, RWD). For
lower extremity arterial thrombus model, mice were anesthetized to expose the femoral artery. Then 10%
FeCl3-soaked filter paper (1 × 1 mm) was applied to the vessel surface for 1 min before removal.

Characterization of thrombus from patients and mice
The thrombus of patients with ischemic stroke and patients with carotid artery embolism was removed by
mechanical thrombectomy, and fixed with electron microscopic fixative (2.5% glutaraldehyde). After
dehydration, drying, and gold spraying, the thrombus was observed by scanning electron microscope
(SEM, JSM-IT700HR, Japan). A similar method was used to observe the thrombus structure in mouse
thrombus models.

Synthesis and characterization of CREKA-PEPLA
Synthesis of Mal-PEG-PBA. Mal-PEG-OH (2 g, 1 mmol), PBA (0.17 g, 1 mmol), DMAP (0.37 g, 3 mmol)
and DCC (0.62 g, 3 mmol) were dissolved in a mixture of DCM and THF (v/v, 50:50) under nitrogen
atmosphere for 24 h of reaction in dark. DCM and THF were then removed by rotary evaporation. The
concentrate was precipitated through petroleum ether and dried under vacuum to obtain Mal-PEG-PBA.
Synthesis of Mal-PEG-PBE. Mal-PEG-PBA（0.22 g, 0. 1 mmol) and TME (0.06 g, 0.5 mmol) were
dissolved in anhydrous toluene, and heated at 120 ℃ reflux for 10 h in the Dear Stark dewatering unit.
The mixture was concentrated by rotary evaporation, precipitated with ice ether, and dried under vacuum
to obtain Mal-PEG-PBE.
Synthesis of Mal-PEPLA and Mal-PELA. Mal-PEPLA was synthesized through ring-opening
polymerization (ROP) of D,L-LA initiated by Mal-PEG-PBE catalyzing using Sn(Oct)2. Briefly, Mal-
PEG-PBE (1 g, 0.44 mmol), D,L-LA (4.5 g, 31.25 mmol) and Sn(Oct)2 (0.1 wt%) were quickly added to
round-bottom flask with stopcock. Then the flask was degassed under vacuum for 6 h with continuous
stirring. The ring-opening polymerization reaction was carried out at 150 ℃ for 4 h. After the reaction
finished, the product was dissolved with a small amount of dichloromethane and precipitated by excess
cold ethanol. Mal-PEPLA was obtained by centrifugation and vacuum drying. Mal-PELA was
synthesized as above except using Mal-PEG-OH as initiator.
Synthesis of CREKA-PEPLA (CPEPLA). CREKA-peptide (0.05 mmol, 30.2 mg) and Mal-PEPLA
(0.05 mmol, 600 mg) were dissolved in dichloromethane at 25 ℃ under nitrogen atmosphere. After 12 h
of reaction, the mixture was precipitated with cold ethanol and dried by lyophilization. Then the freeze-
dried powder was dissolved in deionized water and dialyzed (MWCO: 3500) for 24 h, and lyophilized to
obtain CPEPLA.

1H NMR. The chemical structures of polymers were characterized by 1H NMR (AM 300, Bruker,
Germany). CDCl3 was used as solvent, and Tetra-methylsilane (TMS) was used as the internal reference.
Chemical shifts are expressed in parts permillion, ppm.

Preparation of polymersomes
Polymersomes were fabricated by solvent evaporation method. Briefly, 10 mg of CPEPLA was dissolved
in THF (5 mL), and added dropwise into deionized water (5 mL) under stirring. The CPEPLA
polymersomes were obtained with the evaporation of THF. Similarly, PELA and PEPLA polymersomes
were prepared as above. tPA-loaded polymersomes were prepared by electroporation. 400 µg of
polymersomes, 40 μg of rCD177 or tPA, and 360 µL of PBS buffer were added to the electroporation
dish (Biorad, USA). Then electroporation was performed at 250 V, 350 μF, and 50 Ω (Biorad, Gene Pulser
Xcell, USA). The resulting solution was incubated at 37 ℃ for 30 min to recover the structure of
polymersomes, and centrifuged (20,000 × g, 30 min) to get tPA-loaded polymersomes.

Characterization of polymersomes
The particle size, polydispersity index (PDI), and zeta potential of the polymersomes were determined by
DLS (Zeta-Sizer, Malvern Nano-ZS90, Malvern, Ltd., UK). The vesicle morphology was examined by
TEM (TECNAI G2F20FEI, USA). The content of unencapsulated tPA after electroporation was detected
by BCA protein assay kit. The drug loading content (DL%) and encapsulation efficiency (EE%) were
calculated by the following formula:
DL% = (Weight of drug added-Unencapsulated drug content) / Weight of polymersomes × 100% (1)
EE% = (Weight of drug added-Unencapsulated drug content) / Weight of drug added × 100% (2)

Colloidal stability and long-term stability3,70
To assess the colloidal stability of the polymersomes, CP@tPA and CP@rCD177 (2 mg mL-1) were
incubated in FBS at 37 °C for 8 h. The size distribution of the polymersomes was measured by DLS at 0,
2, 4, 6 and 8 h. To evaluate the long-term stability of the polymersomes, CP@tPA and CP@rCD177 (2
mg mL-1) were stored in PBS at 4 °C in the dark. The size change was determined by DLS at day 0, 7, 15
and 30.

ROS responsiveness detection
To verify the ROS responsiveness, the polymersomes (100 μg mL-1) were treated with H2O2 (100 μM) for
10 min. The morphology and particle size change of polymersomes was detected by TEM and DLS, and
the ROS elimination capacity of polymersomes was determined by hydrogen peroxide kit.

In vitro drug release
To detect the in vitro release kinetics, tPA-loaded polymersomes were transferred to a dialysis bag
(MWCO: 300 kDa), which was then dipped into PBS buffer containing H2O2 (100 μM) or not, and shake
well in a 37 °C bath. The cumulative release of tPA was then detected at the scheduled point of time (0,
0.5, 1, 2, 3, 6, 12 and 24 h) by BCA protein test kit.

Pharmacokinetics
FITC-labeled tPA and rCD177 were separately encapsulated into CP polymersomes and intravenously
injected into mice (doses equivalent to 1 mg kg⁻1 tPA or 1 mg kg⁻1 rCD177). Blood samples were collected
at 1, 5, 10, 20, 30, 60, 90, and 120 min post-injection. Plasma was isolated by centrifugation, and the
fluorescence intensity of the plasma at each time point was measured using a fluorescence
spectrophotometer (Hitachi F-7000, Japan). Drug concentrations in the blood were quantified based on
the fluorescence measurements, and pharmacokinetic parameters were calculated using DAS 2.0 software.

In vitro cytotoxicity study71
Cytotoxicity was first evaluated using CCK-8 assay and live/dead staining. For CCK-8 assay, bEnd.3
cells were seeded in 48-well plates at a density of 5 × 104 cells per well and allowed to adhere. The cells
were then treated with various concentrations of CP@tPA (0, 25, 50, 100, and 200 μg mL⁻1) for 24 h.
After treatment, the medium was replaced with 200 µL of CCK-8 working solution (10% CCK-8 reagent
in fresh medium, v/v). The plates were incubated for 0.5 h at 37 °C, after which 100 µL of the resulting
solution from each well was transferred to a 96-well plate for absorbance measurement at 450 nm using
a microplate reader. Cell viability was calculated by the following formula:
Cell vivability (%) = (OD450(Sample)-OD450(Blank)) / (OD450(Control)-OD450(Blank)) × 100% (3)
For live/dead staining, bEnd.3 cells were seeded in 24-well plates at a density of 1 × 105 cells per
well and allowed to adhere. The cells were then treated with CP@tPA polymersomes at the specified
concentrations. Following 24 h of incubation, the cells were washed with PBS, stained with calcein
acetoxymethyl ester (Calcein-AM) (2 µM) for 10 min to label live cells, and propidium iodide (PI) (4
µM) for 10 min to label dead cells. After staining, the cells were washed again to remove excess dye. The
stained cells were imaged using a fluorescence microscope (Axio Observer Z1, Zeiss, Germany). In the
acquired images, viable cells exhibited green fluorescence, whereas dead cells exhibited red fluorescence.

Blood compatibility study
Hemolysis test. The hemocompatibility of CP@tPA was first evaluated via hemolysis assay using mouse
red blood cells (RBCs). Purified RBCs were diluted with saline to a 2% (v/v) concentration. Then, 0.5
mL of the RBC suspension was mixed with 0.5 mL of CP@tPA at various concentrations (0, 25, 50, 100,
and 200 μg mL⁻1) and incubated at 37 °C for 1 h. Saline and 0.1% Triton X-100 were used as the negative
(0% hemolysis) and positive (100% hemolysis) controls, respectively. After incubation, the samples were
centrifuged at 300 × g for 10 min. The absorbance of the supernatant was measured at 540 nm using a
microplate reader (Elx800, BioTek, USA). The hemolysis rate was calculated using the following
formula:
Hemolysis ratio (%) = (As-An) / (Ap-An) × 100% (4)
As, An and Ap represent the absorption at 540 nm of samples, negative control (saline) and positive control
(Triton X-100), respectively.
Coagulation test. 50 μL of recalcified whole blood was introduced into CP@tPA polymersomes with
different concentrations (0, 25, 50, 100, and 200 μg mL⁻1) to initiate coagulation. Deionized water and
saline served as the positive and negative controls, respectively. After 1 min, 10 mL of deionized water
was added to each sample to lyse any unclotted erythrocytes. The mixtures were then collected and
centrifuged at 300 × g for 10 min to isolate the supernatant, which contained hemoglobin released from
lysed red blood cells. The absorbance of the supernatant was measured at 540 nm. The blood clotting
index (BCI) was calculated using the following formula:
BCI (%) = As / Ap × 100% (5)
As is the absorbance of the sample and Ap is the positive control absorbance.

In vitro thrombosis targeting
100 μL of mouse blood was incubated with thrombin (50 U) at 37 ℃ for 30 min to obtain thrombus.
The resulted thrombus was then treated with ICG-loaded PELA (Null@ICG), PEPLA (P@ICG), CPEPLA
(CP@ICG) polymersomes, and observed at 15 min, 30 min, 60 min, 90 min, and 120 min using in vivo
imaging system (AniView Phoenix full-spectrum animal imaging system, China). Besides, the adhesion
of nanoparticles on the thrombus surface was observed by SEM.

In vivo safety study
To evaluate the in vivo safety of the polymersomes, mice were intravenously administered with CP@tPA
at a dose equivalent to 1 mg kg⁻1 of tPA. After 24 h, the mice were euthanized, and major organs (heart,
liver, spleen, lungs, and kidneys) were harvested for histological analysis via hematoxylin and eosin (H&E)
staining. Additionally, blood samples were collected and centrifuged at 700 × g for 5 min to obtain serum.
The serum was subsequently used for blood biochemistry tests to assess liver and kidney function.

In vivo thrombosis targeting72
MCAO mice were intravenously injected with Null@ICG, P@ICG, and CP@ICG polymersomes (400
μg polymersomes/mouse) at 2 h poststroke, and observed at different points by in vivo imaging systems.
2 h post injection, mice were executed to separate brain, heart, liver, spleen, lung and kidney for ex vivo
imaging. The average radiation efficiency was analyzed using Phoenix software. Immunofluorescence
staining was applied to analyze the thrombosis targeting of polymersomes. The brain tissue was cut into
20 μm sections and incubated with primary antibodies including rat anti-CD31 antibody (1:100, Abcam,
ab256569) and rabbit anti-fibrinogen antibody (1:1000, Abcam, ab92572), and secondary antibodies
including Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:1000, Abcam, ab150076) and Alexa
Fluor 488-conjugated goat anti-rat IgG (1:1000, Abcam, ab150157), respectively. The nucleus was
stained with DAPI. Images were obtained by confocal microscope (A1R+, Nikon, Japan).

In vivo thrombolysis
The thrombolysis effect of CP@tPA in carotid artery thrombus model, lower extremity arterial thrombus
model, and MCAO model was investigated. For both the carotid artery thrombus model and lower
extremity arterial thrombus model, saline, tPA, or CP@tPA (150 μL per mouse, equivalent 1 mg kg-1 of
tPA) was administered via tail-vein injection 10 min after model establishment to initiate thrombolytic
therapy. For the MCAO model, 2 h after stroke onset the mice were intravenously given saline, tPA, or
CP@tPA (150 μL per mouse, equivalent 1 mg kg-1 of tPA). Blood perfusion in the ischemic area was
monitored by laser speckle at different time points (0, 30, 60, 90, and 120 min). For the lower extremity
arterial thrombus model, mice were euthanized 2 h after thrombolysis to separate the blood vessels
containing thrombosis for H&E staining. The thrombosis rate was calculated by ImageJ. Besides,
thrombolysis was measured by observing the activated platelet in thrombus. Mice were injected with 100
μL of FITC-dextran (2 mg mL-1, 2000 KDa) and 10 μL of APC-CD62P (Biolegend ,148303), and
observed by two-photon microscopy (A1RMP＋, Nikon, Japan). The thrombus markers FDP, sCD40L in
mouse blood were determined by ELISA.

Cerebral hemorrhage evaluation
At 22 h after treatment with tPA and CP@tPA (1 mg kg-1 of tPA), MCAO mice were sacrificed to collect
brains, which were sliced and photographed. The severity of hemorrhage was classified into four levels:
no hemorrhage (NH); hemorrhage infarction type 1 (HI-I), a single petechia in the ischemic area;
hemorrhage infarction type 2 (HI-II), 2~3 petechiae in the ischemic area; and hemorrhage infarction type
3 (HI-III), more than 3 petechiae in the ischemic area. The tissues were then homogenized and centrifuged,
and the hemoglobin content was determined by measuring the absorbance at 410 nm using a hemoglobin
assay kit. A standard curve was constructed with freshly collected homologous blood to calculate the total
blood volume.

Transcriptome Analysis
Blood was collected from ischemic stroke patients with hemorrhagic transformation (HT) before and after
tPA thrombolysis. The patients ranged in age from 67 to 91 years, including 2 males and 7 females.
Clinical samples were obtained from the Third People Hospital of Chengdu (China). The collected blood
was treated with Trizol to extract RNA. The transcriptome analysis was performed by constructing the
libraries and sequencing by Shanghai Majorbio Bio-Pharm Biotechnology Co., Ltd. (China). The study
was approved by the Ethics Review Committee of the Third People Hospital of Chengdu (2024-S-154),
and informed consent signed by patients was obtained. The transcriptome analysis for the brain tissues
was performed as the similar procedure except isolating the brain to be homogenized for RNA extraction.
To identify differential expression genes (DEGs) between groups, the expression level of each
transcript was calculated according to the transcripts per million (TPM) method. Gene abundance
estimation was performed with RSEM. Differential expression analysis was performed using DESeq2
(v1.24.0). DEGs with |log2FC ≥ 1 and FDR ≤ 0.05(DESeg2) were considered to be significantly different
expressed genes. Functional-enrichment analysis including Gene Ontology (GO), Reactome and KEGG
were performed to identify biological functions and pathways significantly associated with the DEGs. A
Benjamini-Hochberg corrected P-value ≤ 0.05 was used as the threshold for significance against the
whole-transcriptome background. GO enrichment was conducted using Goatools, while KOBAS was
employed for both Reactome and KEGG pathway analyses.

Effect of tPA on activating CD177+ neutrophils
The effect of tPA on the activation of CD177+ neutrophils was explored both in vitro and in vivo. For in
vitro study, neutrophils were isolated from peripheral blood of MCAO mice and treated with tPA (100 μg
mL-1) for 90 min. Simply put, the mouse blood was mixed with PBS and red blood cell sedimentation
solution at a volume ratio of 1:1:1. The mixture was left at room temperature for 30 min and centrifuged
through density gradient to collect the supernatant and obtain the neutrophils. For in vivo study, tPA (1
mg kg-1) was intravenously injected into MCAO mice at 2 h poststroke, and neutrophils were extracted
from peripheral blood of mice with HT (petechia in the ischemic area) 22 h later. These cells were labeled
with FITC-Ly6G (1:100, Thermo Fisher scientific, 11-9668-80) and APC-CD177 (1:50, BD, 566599) for
flow cytometry (CytoFLEX, Beckman Coulter, USA) analysis. The percentage of Ly6G+CD177+ cells
was calculated. Besides, brains were collected from MCAO mice with HT for immunofluorescence
staining. Brain was cut into sections and incubated with primary antibodies including rat anti-Ly6G
antibody (1:100, Abcam, ab25377) and rabbit anti-CD177 antibody (1:1000, Servicebio, GB11316-100),
followed by treating with secondary antibodies including Alexa Fluor 488-conjugated goat anti-rat IgG
(1:1000, Abcam, ab150157), and Alexa Fluor 594-conjugated donkey anti-rabbit IgG (1:1000, Abcam,
ab150076). The nuclei were stained with DAPI. Images were obtained by confocal microscope.

Effect of tPA on NET generation
Neutrophils were isolated from peripheral blood of MCAO mice and treated with tPA (100 μg mL-1) for
90 min. Then the cells were rinsed, fixed with 4% paraformaldehyde at 4 °C, stained with SYTOX Green
(1:500), and observed with fluorescence microscope. The concentration of NETs and MPO were
measured by ELISA.

CD177+ neutrophils promote BBB leakage
After 10 h of tPA thrombolysis, a cranial window was established to observe the BBB penetration of
CD177+ neutrophils. Briefly, mice were anesthetized to fix the head on the brain locator. A circular area
was drilled in the infarct brain with a micro handheld cranial drill and subsequently covered with a
coverslip. Then mice were intravenously injected with FITC-dextran (10 mg kg-1, 40 KDa) and APC-
CD177 (0.1 mg kg-1, BD, 566599), respectively. Images were acquired using two-photon microscopy at
different time points.

Effect of rCD177 on inhibiting CD177+ neutrophils migration
To explore the effect of rCD177 on inhibiting the transendothelial ability of CD177+ neutrophils,
transmigration assay was performed38. bEnd.3 were seeded on the upper chamber of transwell and
cultured to form cell monolayer, which could be used as the BBB model once the value of trans endothelial
electrical resistance (TEER) was higher than 200 Ω cm2. The culture medium was then replaced with
glucose-free and serum-free DMEM medium (Gibco), and the cells were cultured in 95% N2 and 5% CO2
environment to simulate low oxygen condition73. After 3 h of oxygen-glucose deprivation (OGD)
processing, the medium was replaced with normal medium and the cells were transferred to normal
incubator to simulate reperfusion injury. rCD177 (100 μg mL-1) was added to the upper chamber for 10
min, subsequently neutrophils (1×106) and tPA (100 μg mL-1) were added for further incubation. 90 min
later, cells were collected from the lower chamber, and labeled with FITC-Ly6G (1:100, Thermo Fisher
scientific, 11-9668-80) and APC-CD177 (1:50, BD, 566599) for flow cytometry analysis. Besides, the
number of neutrophils in the lower chamber was counted to calculate cell mobility. Then the migrated
cells were stained with SYTOX Green (1:500) and observed with fluorescence microscope. The
concentration of NETs was measured by ELISA kit.

Inhibition of CD177+neutrophils migrating into the brain
rCD177 was fluorescently labeled with FITC and encapsulated into CP polymersomes. The resulting
CP@rCD177 was administered to MCAO mice 2 h poststroke and 10 min prior to thrombolysis. At 22 h
post-treatment, brain tissues were cut into 20 μm sections and subjected to immunostaining using primary
antibodies including rat anti-CD31 antibody (1:100, Abcam, ab256569) and rabbit anti-CD177 antibody
(1:1000, Servicebio, GB11316-100), followed by incubation with secondary antibodies including Alexa
Fluor 594-conjugated donkey anti-rabbit IgG (1:1000, Abcam, ab150076) and Alexa Fluor 647-
conjugated goat anti-rat IgG (1:1000, Abcam, ab150159). Nuclei were stained with DAPI. Images were
obtained by confocal microscope.
For further investigation, mouse brains were collected from saline, tPA, rCD177+tPA, CP@tPA, and
CP@rCD177/tPA groups after treatment. The tissues were homogenized and labeled with FITC-Ly6G
(1:100, Thermo Fisher scientific, 11-9668-80) and APC-CD177 (1:50, BD, 566599) for flow cytometry
analysis. For immunofluorescence staining, brains were cut into sections and incubated with primary
antibodies including rat anti-Ly6G antibody (1:100, Abcam, ab25377) and rabbit anti-CD177 antibody
(1:1000, Servicebio, GB11316-100), followed by treating with secondary antibodies including Alexa
Fluor 488-conjugated goat anti-rat IgG (1:1000, Abcam, ab150157) and Alexa Fluor 594-conjugated
donkey anti-rabbit IgG (1:1000, Abcam, ab150076). Images were obtained by confocal microscope.

Effect of CP@rCD177 on improving tPA-induced HT
MCAO mice were intravenously injected with saline, tPA, rCD177/tPA, CP@tPA, and CP@rCD177/tPA
at 2 h poststroke (1 mg kg-1 of tPA and 1 mg kg-1 rCD177). For rCD177/tPA and CP@rCD177/tPA groups,
rCD177 and CP@rCD177 were injected 10 min prior to tPA and CP@tPA injections, respectively. 22 h
after therapy, the blood leakage in the mouse brains was detected using the photoacoustic multimode
small animal in vivo imaging system (GAni-Plus, Guangzhou G-Cell Technology Co., Ltd.). Mice were
anesthetized and the skin tissue of the mice's head was cut open to expose the skull, which was infiltrated
with deionized water to couple the photoacoustic signals. The hemoglobin fluorescence was detected at
532 nm to reconstruct the hemoglobin signal. Then the mouse brains were collected, sliced and
photographed. The severity of hemorrhage was evaluated by four levels. Additionally, the brain tissue
was homogenized and centrifuged; the hemoglobin content was then quantified by measuring the
absorbance at 410 nm using a hemoglobin assay kit. A standard curve was generated with freshly collected
homologous blood to calculate the total hemorrhage volume.

Evans blue staining
Mice were intravenously injected with 4% Evans blue (4 mL kg−1) 22 h after treatment. 2 h later, mice
were executed and brains were cut into 1 mm slices for photographing by camera. Then the tissues were
homogenized and centrifuged to collect the supernatant. The EB content was determined by measuring
the absorbance at 620 nm, expressed as A620/tissue (g).

Assessment of BBB integrity
BBB integrity was first assessed in vivo using intravital microscopy. At 22 h post-treatment, a cranial
window was implanted over the infarcted hemisphere of MCAO mice, followed by intravenous injection
of 100 μL Evans blue (1 mg mL-1). After 30 min, intravital imaging was performed, revealing
extravasation of the dye from compromised cerebral vessels. In parallel, TEM was employed to examine
the ultrastructure of the BBB at 22 h post-treatment. Brain tissue was collected, fixed in 2.5%
glutaraldehyde, dehydrated, embedded, and sectioned into ultra-thin slices. The sections were stained with
uranyl acetate and lead citrate, and the microarchitecture of the BBB was visualized and imaged under
TEM.

TTC staining
22 h after treatment, the mouse infarcted brains were harvested, and sectioned coronally into five 1 mm-
thick slices. The slices were incubated in a 2% TTC solution at 37 °C for 30 min, fixed in 4%
paraformaldehyde, rinsed with PBS, and imaged using a digital microscope. Viable brain tissue was
stained rose-red, whereas the infarcted areas remained unstained (white). The total area and the infarct
area of each slice were quantified using ImageJ software.
Infarct area (%) = (Total cerebral infarction area / Total brain area) × 100% (6)

Inflammatory cytokine assay
To evaluate neuroinflammatory responses after treatment, mice were euthanized on day 3 post-treatment.
The ischemic brain hemispheres were collected, and the concentrations of key pro-inflammatory
cytokines (IL-1β and TNF-α) as well as anti-inflammatory cytokines (IL-10 and TGF-β) were quantified
using ELISA kits74.

Behavioral test
Modified neurological severity score (mNSS). Mice underwent comprehensive behavioral assessments to
evaluate their motor, sensory, reflexive, and equilibrium functions for the mNSS calculation. The mNSS
scale ranges from 0 to 18 points, where 0 indicates no observable neurological deficits and 18 represents
the most severe neurological impairment.
Corner turning test. The corner turning test was employed to assess behavioral bias in mice. Two
plastic plates were arranged at a 30° angle with a 5 mm gap at the vertex to create a corner that attracted
the mice. Each mouse was allowed to enter the corner and then spontaneously turn either left or right. The
test was repeated ten times per mouse, and the number of left and right turns was recorded. The turning
bias was calculated using the following formula:
Corner turn score (%) = L / (L+R) × 100% (7)
L: left turn, R: right turn.
Rotarod test. The motor coordination of mice was assessed using a rotarod test by Rota Rod System
(LE8200, RWD). Mice were placed on a rotating rod that accelerated gradually from 5 to 40 rpm over a
4-min period. Before formal testing, all mice received a 5-day training regimen consisting of 3 trials per
day. Mice that failed to remain on the rod for 270 s during training were excluded from subsequent
experiments. The latency to fall was recorded and analyzed as the mean residence time.
Adhesive test. Adhesive test was used to evaluate cutaneous sensitivity and sensorimotor integration.
Specifically, a piece of adhesive paper (3 × 3 mm2) was applied to the ipsilateral forepaw of each mouse,
and the time taken for the mouse to contact and remove the paper was recorded. Similarly, mice were
trained before surgery to exclude those that could not remove the paper after 60 s.
Barnes maze test75. Barnes maze test was used to test spatial cognitive function of mice after stroke.
Briefly, mice were placed in the center of the maze, which was covered with an impervious box to limit
their activity to 5 s. Then, the box was removed to expose the mice to strong light above the platform,
forcing them to search for the correct hole connected to the dark box. Before MCAO surgery, the mice
were trained for five consecutive days (three trials per day) to acquire the spatial memory required to
locate the target escape hole, and any mouse that failed to escape within 5 min was excluded. The whole
process of testing was videotaped camera until the mice entered the escape hole. The movement route
was analyzed using the SMART 3.0 behavioral recording system (Panlab, Spain).

Western blot
Protein expression levels were examined through western blot analysis. Brain tissues were collected and
lysed in RIPA buffer. The lysate was then detected using BCA protein analysis kit to quantify the protein
content. Equal amount of protein was isolated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), and transferred to the nitrocellulose membrane. At room temperature, the
membrane was closed by 5% skim milk powder for 2 h, washed with TBST, and incubated with rabbit
anti-GADD45 (1:1000, Thermo Fisher scientific, PA5-43160), rabbit anti-TSP1 (1:1000, Solarbio,
K007665P), rabbit anti-ANGPT2 (1:1000, Solarbio, K001733P), rabbit anti-COX-2 (1:500, Solarbio,
K009752P), and anti-β-actin (1:1000, Cell Signaling Technology, 4967S) overnight. Next, the sample
was washed with TBST, followed by incubating with horseradish peroxidase-conjugated secondary
antibody. Protein strips were imaged with ECL development, the light density of protein strips was
quantified. β-actin was served as loading control.

In vivo NET decomposition
To observe the in vivo NET decomposition, mice after treatment with different formulations were
intravenously injected with SYTOX Green (10 μM), PE-Ly6G (10 μL, BioLegend, 127607) and EB (1
mg mL-1, 100 μL). 30 min later, mice were sacrificed to separate the brains and observed by IntraVital
Microscopy (IVM-CMS-3, Shanghai Aifei Electronic Technology Co., Ltd.). Besides, the brains were
homogenized and centrifuged to obtain the supernatant. The concentrations of NETs and inflammatory
factors (IL-6, IFN-β) were measured by ELISA.

Brain distribution of NETs
HT mice were executed to isolate the brains 24 h poststroke. The brains were cut into sections and
incubated with primary antibodies including rat anti-Iba1 antibody (1:100, Abcam, ab283346), rat anti-
GFAP antibody (1:200, Abcam, ab279291), or rat anti-NeuN antibody (1:100, Abcam, ab279297), and
rabbit anti-H3Cit (1:1000, Abcam, ab219407). Then the brain sections were treated with secondary
antibodies including Alexa Fluor 488-conjugated goat anti-rat IgG (1:1000, Abcam, ab150157) and Alexa
Fluor 594-conjugated donkey anti-rabbit IgG (1:1000, Abcam, ab150076), and visualized using confocal
microscope.

Statistical analysis
Statistical analysis was performed using GraphPad Prism (9.5.0) software. For comparison of means of
samples with normal distribution and homogeneous variance, paired t-test was used for two groups. For
samples with more than two groups, one-way or two-way ANOVA with Bonferroni’s or Tukey’s post-
hoc test was used for multiple comparisons. All data are presented as means ± SDs. *P < 0.05, ** P <
0.01, *** P < 0.001, **** P < 0.0001, n.s, not significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors have developed a thrombus-targeting and ROS-triggered polymersomes to separately load tPA and recombinant CD177 protein (rCD177) for thrombolysis while concurrently inhibiting hemorrhagic transformation. Overall, this manuscript is well organized. There are some issues may be further improved before publication:\n1. The characterization of the CP@rCD177 nanoparticles is incomplete, it is recommended to add experiments to prove the successful loading of the drug.\n2. In the drug release experiment (Figure 1d), less than 25% of the drug was released within 2 h. Why is the thrombolytic effect at 2 h stronger than tPA (Figure 2b)? It is recommended to add an explanation of why thrombolysis can be achieved without drug release.\n3. The colloidal stability (FBS) and long-term particle stability should be provided for in vivo applications and recent reference, such as Nat. Commun. 14, 255 (2023), Angew. Chem. Int. Ed. 61, e202109068 (2022) would be helpful for explanation and discussion.\n4. It is recommended to supplement the determination method of the dosing interval and explain how sequential administration ensures the drug's duration of action.\n5. The bleeding risk was determined in the MCAO model. It is recommended to further verify the bleeding risk in other models to enhance the generalizability of the findings. Additionally, it is suggested to conduct safety evaluations of blood cells and vascular endothelial cells after treatment.\n6. Unable detect the process of CP@rCD177 nanoparticles in vivo. The pharmacokinetics studies should be provided.\n7. The administration method is unreasonable. Clinically, it is impossible to administer CP@rCD177 before the onset of the disease. The authors should attempt to administer the drug simultaneously with model establishment.\n8. Reperfusion injury caused by thrombolysis can easily trigger various cerebral inflammations. It is recommended to measure other inflammatory factors in the brain after treatment. Otherwise, it will be impossible to determine the inflammatory level in the brain and the treatment effect after treatment.\n9. The overall standardization of the manuscript is subpar.\na) The content of sub-figure e-f in Fig. 1 does not match the figure title.\nb) Fig. 4f is a flow cytometry image, which is inconsistent with the description in the article stating that \"Photoacoustic imaging (PA) was used for real - time monitoring of intracerebral hemorrhage.\nc) The content of sub -figures b-c in Fig. 2 is chaotic.", "reviewer_2": "The study by Wang et al. presents a new therapeutic platform to deliver thrombolysis after ischemic stroke in mice with diminished risk of hemorrhagic transformation (HT). The intervention is based on synthetic particles that are targeted to the thrombus by a fibrin-binding peptide and contain encapsulated rtPA. Furthermore, the authors loaded the particles with recombinant CD177 to block neutrophil-endothelial cell interaction which resulted in reduced BBB breakdown and neutrophil migration to the stroked brain. Combinatorial nanoparticles incorporating tPA and rCD177 proved more potent in preventing HT. This is a well-designed study that shows the efficacy of a novel ischemic stroke therapy with potential for clinical translation. However, the quantitative aspects of the study are limited, data are not well presented, and the benefits for stroke outcome have not been investigated. Specific concerns are:\n1. There is no indication that CP@tPA reduced infarct volume and improved neurobehavioral outcome after stroke. These are essential – and the only clinically relevant – parameters for any therapeutic intervention.\n2. It is presumed that 100 µM H2O2 was used for all in vitro experiments. The concentrations of extracellular H2O2 released by neutrophils is thought to be in the low µM range (PMID: 6323407). In addition, the presence of erythrocytes in the clot would act as a H2O2 sponge further reducing available H2O2 for polymer breakdown.\n3. Ferric chloride clots are relative resistant to tPA thrombolysis because of low fibrin incorporation into the clot (PMID: 21326267). What was the recanalization rate in tPA and CP@tPA treated FeCl3 MCAO mice? Why would CP@tPA be superior in dissolving a clot that has minimal fibrin deposition?\n4. Fig 2h. The HT in the CP@tPA coronal sections looks different between tPA and CP@tPA treated animals. While the number of macroscopic petechial bleeds might be higher in tPA treated mice, there is indication of a more diffuse blood extravasation in CP@tPA mice. At least this is my impression from the presented images. In addition, petechial bleeds are also observed in the contralateral hemisphere of CP@tPA mice. Contralateral and ipsilateral hemispheres of all treatment groups should be used to quantitatively determine the hemoglobin content by chemical analysis.\n5. Fig S9 shows extensive accumulation of polymersomes in lung, kidney, and liver. How and when are polymersomes cleared from those organs and what is their effect on lung, kidney, and liver tissue integrity and function?\n6. Many experiments are missing time points of drug treatment and endpoints. Generally, time points should be indicated in the main text and/or figure legends, and not in the methods section.\n7. The SYTOX indicator should be used in conjunction with a neutrophil marker such as Ly6G.\nMinor\n1. In what volume were the polymersomes and tPA delivered?\n2. There are no clinical data for stroke patients and controls.\n3. Methods for RNA-seq and DGE, including statistical analysis, are missing.\n4. Line 504. Please specify “lower extremity artery”.\n5. Fig 1e-f. Reversed order in legend.\n6. Fig S7a. Size markers missing."}, "author_response": "Point-by-point responses to Reviewers’ Comments\nWe would like to express our appreciation for the thoughtful critiques provided by Reviewer and we have used as the basis for an extensive revision of the manuscript. The reviewers' comments are provided in italics. Our response to each comment is provided in plain text.\nWith Regard to the Comments of Reviewer #1: [Full text of rebuttal for Round 1 omitted for brevity, but included in full in the provided source text]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors well address my concerns.", "reviewer_2": "The authors have added relevant clarifications and provided new data and experiments to support their findings, thereby improving the overall quality of the study. One concern remains, that needs to be addressed. While there is an overall decrease in petechiae and larger bleedings in CP@tPA treated animals, the total amount of brain hemoglobin was not reduced (Fig. R4). Furthermore, the authors refer to Fig. 4J as quantitative hemoglobin measurements (“hemoglobin detection kit”) while the graph seems to depict hemoglobin volume obtained by morphometric analysis. This discrepancy needs to be addressed.\nIn addition, the presence of diffuse hemoglobin signals in CP@tPA might reflect microvascular problems, as acknowledged by the authors in their rebuttal, and might be an important factor for the clinical translation of this therapeutic approach. It needs, therefore, further investigation (high resolution histology?), and Fig R4 should be presented as supplemental data. The authors are also encouraged to include these findings into the discussion."}, "author_response": "With Regard to the Comments of Reviewer #2: The authors have added relevant clarifications and provided new data and experiments to support their findings, thereby improving the overall quality of the study. One concern remains, that needs to be addressed. While there is an overall decrease in petechiae and larger bleedings in CP@tPA treated animals, the total amount of brain hemoglobin was not reduced (Fig. R4). Furthermore, the authors refer to Fig. 4J as quantitative hemoglobin measurements (“hemoglobin detection kit”) while the graph seems to depict hemoglobin volume obtained by morphometric analysis. This discrepancy needs to be addressed.\nIn addition, the presence of diffuse hemoglobin signals in CP@tPA might reflect microvascular problems, as acknowledged by the authors in their rebuttal, and might be an important factor for the clinical translation of this therapeutic approach. It needs, therefore, further investigation (high resolution histology?), and Fig R4 should be presented as supplemental data. The authors are also encouraged to include these findings into the discussion.\nRe: Thank you for your insightful comments and for acknowledging the improvements made to our manuscript. We appreciate the opportunity to address the remaining concern regarding the relationship between macroscopic bleeding reduction and total brain hemoglobin content, as well as the clarification on our quantification method. We agree that this point is critical for the interpretation of our findings and its clinical implications. Regarding the methodological clarification for Fig. 4J, we apologize for any confusion caused by the initial wording. The quantitative data presented were obtained not through morphometric analysis, but by homogenizing whole-brain tissue and using a hemoglobin assay kit. The values were calibrated against a standard curve generated from the blood of the same mouse strain and are expressed as hemorrhage volume.2 We have revised the Methods section to describe this procedure with greater precision and clarity.\nWe fully acknowledge the central point of your critique: the observation that while CP@tPA treatment reduces visible petechiae and larger hemorrhages, it does not decrease the total brain hemoglobin content (now presented in Supplementary Figure 16). We believe this apparent discrepancy is highly informative. It aligns with our data in Fig. 2h, confirming that the enhanced thrombolytic efficiency of CP@tPA is indeed accompanied by a more widespread hemorrhagic transformation. This suggests a shift in the bleeding pattern, where focal bleeding may be mitigated but is supplanted by a more diffuse, distributed extravasation of blood throughout the parenchyma. This phenomenon directly supports your hypothesis of underlying \"microvascular problems,\" indicating that the therapeutic intervention, while effective in lysing the primary clot, may inadvertently exacerbate capillary fragility and leakage. This critical limitation underscores why bleeding complications remain a severe constraint for clinical translation1 and powerfully validate the necessity of our follow-up strategy involving rCD177 as an adjunctive therapy designed precisely to protect vascular integrity."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have resolved the remaining concerns by adding further experiments and analyses, and the revised discussion now more clearly reflects the complex aspects of thrombolytic therapies for ischemic stroke."}, "author_response": ""}]

Decision letter