Neutrophil-mediated and low density lipoprotein receptor-mediated dual-targeting nanoformulation enhances brain accumulation of scutellarin and exerts neuroprotective effects against ischemic stroke

Delivery of poorly permeable drugs across the blood-brain barrier (BBB) is a great challenge in the treatment of ischemic stroke. In order to construct a suitable delivery system for this purpose, we developed a dual-targeting nanoformulation to transfer therapeutic agents targeting the inflammatory sites of the ischemic brain. The matrix of this system is a hydroxyl-terminated polyamidoamine dendrimer with excellent biodegradability. The surface of the matrix is functionalized with two targeting peptides: Angiopep-2 is a low density lipoprotein receptor-mediated peptide with high BBB transcytosis capacity with ligands expressed on brain endothelial cells; N-acetylated proline-glycine-proline (PGP) has high affinity to CXCR2 expressed on infiltrating neutrophils. This system proved to be a high-loading formulation for the neuroprotective compound, scutellarin (STA), and significantly improved its therapeutic efficacy in a rodent model of ischemic stroke. The molecular mechanism underlying the therapeutic efficacy of this formulation is associated with significant down-regulation of the inflammatory cytokines, neutrophils infiltration and intracellular calcium overload and blockade of the inflammatory signaling pathway HMGB1/TLRs/MyD88/TRIF/NF-κB. Our results suggest that this dual-targeting delivery system is a promising drug delivery vehicle for ischemic stroke, and possibly other CNS diseases where neuroinflammation is involved.

The 1 H NMR and 13 C NMR spectra of hydroxyl-terminated G5.0 PAMAM dendrimer (dissolved in D 2 O) were analyzed in Bruker AVANCE 500 MHz NMR spectrometer (Switzerland). The sample of hydroxyl-terminated G5.0 PAMAM was ground, mixed with KBr and pressed into pellets. The infrared spectra were recorded in the spectral range of 400～4000 cm -1 , using a Nicolet 5700 FT-IR spectrometer with a resolution of 2 cm -1 .

Synthesis and Characterization of NHS-PEG-MAL-PAMAM Diblock Copolymers
PEGylated PAMAM copolymers were designed and synthesized by conjugating bifunctional NHS-PEG 3400 -MAL to the amine groups of the synthetic hydroxylterminated G5.0 PAMAM dendrimers. Briefly, the synthesized hydroxyl-terminated The 1 H NMR and 13 C NMR spectra of NHS-PEG-MAL-PAMAM diblock copolymers (dissolved in D 2 O) were analyzed in Bruker AVANCE 500 MHz NMR spectrometer (Switzerland). The infrared spectra were recorded in the spectral range of 400～4000 cm -1 , using a Nicolet 5700 FT-IR spectrometer with a resolution of 2 cm -1 .

Cytotoxicity assay in vitro
The cytotoxicity of dual-ligand modified nanoformulation and single-ligand modified nanocarriers on brain capillary endothelial cells (BCECs) were evaluated as previously described 3

Stability and Drug Release
To evaluate the stability of Angiopep  In tissue distribution study (n = 6 each group), SD rats were sacrificed at predetermined time points, and then the brain tissue and other major organs including heart, liver, spleen, lung, brain and kidney were excised, followed by quick washing with cold saline to remove surface blood and stored at -20℃ until analysis.
Briefly, blood was cleared prior to organ homogenization. The tissues were homogenized in ice saline, and then extracted three times with acetic ether. The organic phase was combined after centrifugation at 14,000 rpm for 10 min, and dried under a stream of nitrogen at 37℃. The dry residues were dissolved in 100 μL methanol, and the concentration of STA was determined by the UPLC-QTOF-MS for analysis. The experiments were performed in triplicates.

Induction of focal cerebral ischemia and experimental design
Briefly, the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) of SD rats were isolated via a ventral midline incision.
Branches of ECA were cauterized. The ECA was ligated and cut off at the distance of 5 mm from crotch of CCA. A nylon monofilament (φ 0.22～0.24 mm) with a rounded tip was inserted into right ICA about 18～20 mm through the broken end of ECA, until laser Doppler flow metry as an abrupt 80%～90% reduction of cerebral blood flow, indicating the occlusion of the origin of middle cerebral artery. Sham-operated control rat received the same procedure except filament insertion. After 2 h of ischemia, the nylon suture was withdrawn to establish reperfusion.
For ICR mice, after 1 h of ischemia, the nylon suture was withdrawn to establish reperfusion.

Assessment of neurological function and infract volume
The neurological function assessment was carried out by an examiner blinded to the experimental groups at 24 h after MCAO, all of the 70 rats from each group (n = 10) were assessed on a modified scoring system that developed from our previous studies 6,7 , as follows: 0, normal spontaneous movements; 1, difficulty in fully extending the contralateral forelimb; 2, unable to extend the contralateral forelimb; 3, mild circling to the contralateral side; 4, severe circling to the contralateral side; 5, an animal was unresponsive to noxious stimulus or unconsciousness.
Infarct volume (mm 3 ) was determined by 2, 3, 5-triphenyltetrazolium chloride (TTC) after the neurological assessment (n = 6 per group). Animals were euthanized and the brains quickly collected. Brain tissue was sliced into 1 mm-thick slice, stained with a 2% solution of TTC at 37℃ for 30 min, followed by fixation with 4% paraformaldehyde 7 . Normal tissue was stained deep red, while the infarct area was stained a pale gray color. TTC-stained sections were photographed and infarct volumes (mm 3 ) were quantified using image analysis software (Image-Pro Plus 5.1, USA) to calculate the infarct ratio (%).

Characterization of G5.0 PAMAM
The successful synthesis of hydroxyl-terminated G5.0 PAMAM polymers was demonstrated by multiple characteristic peaks belonged to the branching units of PAMAM. Detailed spectral analysis is presented in the Figure S2 and Figure S4. In 1 H NMR spectrum ( Figure S2

Characterization of NHS-PEG-MAL-PAMAM Diblock Copolymers
PEGylated PAMAM nanocarriers were designed and synthesized by conjugating  Figure S6). As shown in FT-IR spectrum ( Figure S8), NHS-PEG-MAL-PAMAM showed its unique characteristic peaks in the region from 1400 cm -1 to 1100 cm -1 . Furthermore, stretching peak of primary amines near 1198 cm -1 attributed to hydroxyl-terminated G5.0 PAMAM disappeared, and a new peak was observed at 3429.32 cm -1 , which was just the primary amino peak of hydroxylterminated G5.0 PAMAM present at 3288.68 cm -1 ( Figure S8) and red-shifted to 3439.32 cm -1 in IR spectrum of PEG-PAMAM ( Figure S8). The peak at 2918.13 cm -1 was the stretching vibration peak of -CH 2 belonged to PEG-PAMAM, and the peak at 1101.92 cm -1 is the stretching vibration peak of C-O-C in PEG. So it can be concluded that NHS-PEG 3400 -MAL reacted with the primary amino groups on the surface of hydroxyl-terminated G5.0 PAMAM.