abinScience Supports Army Medical University Team in Validating the Targeting Mechanism of a Baicalein–Rapamycin Self-Assembled Nanoplatform

Figure 1. Schematic illustration of ABR NPs construction and their therapeutic mechanism against CIRI

01Assembly of Baicalein–Rapamycin Self-Assembled Nanoparticles

The study began with two pharmacologically distinct molecules: baicalein (BLN), a natural flavonoid free-radical scavenger extracted from the Chinese medicinal herb Scutellaria baicalensis, and rapamycin (RAP), a well-established mTOR inhibitor and immunosuppressant. Researchers systematically evaluated self-assembly at various molar ratios and identified 1:1 as the optimal formulation (particle size 134 nm, PDI 0.19 — the best across all tested ratios), designated BR NPs. Molecular dynamics simulations confirmed that BLN and RAP form stable nanoaggregates through hydrogen bonding and van der Waals interactions, while TEM imaging revealed a regular spherical morphology. The nanoparticles were subsequently surface-modified with DSPE-PEG2k-Angiopep-2 to yield ABR NPs (particle size 138.5 nm, zeta potential −40.53 mV, Figure 1C–D) — endowed with the ability to cross the BBB and actively target neurons. Total drug loading reached approximately 77% w/w, substantially higher than that of conventional nanocarriers (typically <20%).

Figure 2. BLN and RAP self-assemble into spherical nanoparticles subsequently surface-modified with Angiopep-2 to yield ABR NPs

02PEGylation Is Essential for Colloidal Stability in Physiological Media

Unmodified BR NPs exhibited significant aggregation within 30 minutes of incubation in PBS, whereas PEG NPs and ABR NPs both maintained stable size distributions under the same conditions — demonstrating that PEGylation is critical for preserving colloidal stability in physiological saline. ABR NPs were further incubated at 37°C for 12 hours in PBS, PBS containing 10% FBS, and PBS containing 50% mouse serum; neither particle size nor PDI showed significant fluctuation across any condition, indicating that the platform can maintain its structural integrity under conditions that more closely approximate the in vivo environment.

Figure 3. PEGylation enables ABR NPs to remain stable across multiple physiological media

03Angiopep-2 Modification Enhances Cellular Uptake Efficiency

Researchers first established a safe concentration window for ABR NPs in three cell lines — PC12 (neuron-like cells), BV2 (microglia), and bEnd.3 (cerebrovascular endothelial cells) — identifying 10 μM as the uniform working concentration for subsequent experiments. At this concentration, confocal laser scanning microscopy (CLSM) and flow cytometry (FCM) revealed that ABR NPs produced markedly stronger fluorescence uptake signals in both PC12 and bEnd.3 cells compared with untargeted PEG NPs, at both 2- and 4-hour time points — indicating that Angiopep-2 modification genuinely enhances nanoparticle internalization by these two cell types. Mechanistic studies showed that uptake was substantially reduced at 4°C, confirming that the internalization process is energy-dependent — consistent with receptor-mediated endocytosis. The specific receptor responsible was ultimately identified using an LRP1 inhibitor supplied by abinScience.

Figure 4. Cellular uptake of ABR nanoparticles

04Dual Validation via In Vitro BBB Model and In Vivo Imaging

Researchers constructed an in vitro blood-brain barrier model using a bEnd.3 monolayer, with PC12 cells placed in the lower chamber to simulate brain parenchyma. At both 12 and 24 hours, the fluorescence intensity transported to the lower chamber and internalized by PC12 cells was significantly higher in the ABR NPs group than in the free FITC or PEG NPs groups. In a more pathophysiologically relevant setting — the middle cerebral artery occlusion (MCAO) mouse model — real-time brain fluorescence distribution was monitored at 3, 6, 9, 12, and 24 hours via an IVIS in vivo imaging system. ABR NPs consistently produced significantly stronger brain signals than PEG NPs throughout the observation period, while free Cy7 was rapidly cleared from systemic circulation. Ex vivo brain slice imaging further demonstrated that ABR NPs predominantly accumulated in the ischemic hemisphere, with minimal signal detected contralaterally; accumulation intensity was also markedly greater than that of PEG NPs. Confocal imaging of cryosectioned brain tissue corroborated this targeted distribution. Taken together, these findings establish that Angiopep-2 modification confers active BBB-crossing capability and ischemic lesion-targeting, rather than relying solely on the passive retention afforded by PEGylation.

Figure 5. Transcytosis capability of ABR nanoparticles across the BBB

05Antioxidant Efficacy Validated in Cell-Free Systems and at the Cellular Level

In cell-free chemical assays, ABR NPs demonstrated dose-dependent scavenging activity against DPPH radicals, ABTS radicals, hydrogen peroxide, and hydroxyl radicals. At the cellular level, researchers employed an oxygen-glucose deprivation/reoxygenation (OGD/R) model to simulate ischemia-reperfusion, using the DCFH-DA fluorescent probe to quantify intracellular ROS levels in PC12 cells. Flow cytometry showed that OGD/R treatment elevated intracellular ROS fluorescence intensity to 18.69-fold that of controls — confirming successful model induction — while ABR NPs treatment reduced this figure to 2.59-fold, outperforming PEG NPs (4.18-fold) and non-targeted BR NPs (6.76-fold). CLSM observations were fully consistent with the flow cytometry data. These results demonstrate that ABR NPs not only possess chemical antioxidant activity but can also efficiently neutralize excess ROS generated by ischemia-reperfusion at the intracellular level. Biosafety evaluation confirmed a hemolysis rate well below the 5% safety threshold, and a 30-day in vivo toxicity study revealed no abnormalities in blood counts, hepatic or renal function, or histology of major organs — indicating that the nanoplatform carries a favorable safety profile.

Figure 6. ROS scavenging activity of ABR nanoparticles

06Therapeutic Efficacy in the MCAO Model

At the cellular level, the late apoptosis rate of OGD/R-challenged PC12 cells dropped from 21.2% in controls to 13.1% following ABR NPs treatment. More strikingly, ABR NPs demonstrated the most potent neuroprotective effect in the MCAO mouse model. At 24 hours after intravenous administration post-reperfusion, TTC staining revealed a cerebral infarct volume of 37.88% in the saline group, which was dramatically reduced to 7.84% in the ABR NPs group — significantly outperforming PEG NPs (16.87%), BR NPs (27.14%), and all other control groups. Zea-Longa neurological deficit scores also showed marked functional improvement in ABR NPs-treated mice. Histopathological analyses provided further corroboration: H&E staining showed largely preserved neuronal architecture in the ABR NPs group; Nissl staining revealed substantially reduced disorganization and morphological disruption; TUNEL staining demonstrated a marked decrease in apoptotic cell numbers; and NeuN staining confirmed a higher density of surviving neurons compared with all other treatment groups.

Figure 7. Therapeutic effects of ABR nanoparticles on CIRI

07Dual-Pathway Mechanism: ROS Scavenging and Microglial M1/M2 Polarization Modulation

To elucidate the anti-inflammatory mechanism, researchers established an LPS-induced BV2 microglial inflammation model and confirmed that RAP-containing formulations exert anti-inflammatory effects by suppressing mTOR phosphorylation. After ABR NPs treatment, pro-inflammatory markers (CD86, TNF-α, IL-6) were downregulated while anti-inflammatory markers (CD206, IL-10, TGF-β) were upregulated; consistent in vivo results confirmed that ABR NPs promote the transition of microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Taken together, ABR NPs achieve synergistic neuroprotection against ischemia-reperfusion injury through a dual-pathway mechanism — baicalein-mediated ROS scavenging combined with rapamycin-driven microglial polarization modulation — delivered to the ischemic lesion via Angiopep-2-mediated BBB transcytosis.

Figure 8. ABR nanoparticles suppress M1 microglial polarization and reshape the anti-inflammatory microenvironment