Protein Nanoparticles Modified with PDGF-B as a Novel Therapy After Acute Cerebral Infarction

Characterization of PDGF-B HSPNP. A, The size of small HSP 16.5 NPs (HSPNPs) and PDGF-B modified HSPNPs (PDGF-B HSPNPs), determined using dynamic light scattering. B, Immunoblot analyses for Akt, phospho-Akt, and β-actin in cultured human brain vascular pericyte cells treated with PBS, HSPNPs, PDGF-BB, and PDGF-B HSPNPs. β-Actin was used as the loading control. d: diameter.

Dose and time effects of PDGF-B HSPNP accumulation in the infarct area. A, Dose effects of PDGF-B HSPNP accumulation in the infarct area 6 h after administration (2.5, 5, and 10 nmol; n = 3–10). B, Temporal profile of PDGF-B HSPNP accumulation (3, 7, 15 d after administration of PDGF-B HSPNPs; n = 3–10). C, Frozen sections 3 d after PDGF-B HSPNP administration. Left panel, MAP-2 staining to confirm the infarct area. Right panel, Fluorescence microscopy. Scale bars: 200 μm. NPs are widely distributed in the infarct area on day 3.

Therapeutic effects of administration of PDGF-B HSPNPs. Evaluation of infarct volume with MRI. The infarct volume was calculated using the following formula: infarct volume ratio = (ipsilateral T2 high intensity area)/(contralateral hemisphere × 2). Because of individual differences in cerebral infarct volume, we evaluated the infarct volume before injection of NPs (24 h after MCAO) as a control. A, The infarct volume decreased in the PDGF-B HSPNP group (1.04 μmol/l) compared with the PBS or PDGF-BB protein group. B, MRI on days 3 and 7 after administration of PBS, PDGF-BB, and PDGF-B HSPNPs. C, The infarct volume decreased in the PDGF-B HSPNP group (1.04 μmol/l) compared with that in the HSPNP group but not in the group administered a lower concentration of PDGF-B HSPNPs (0.104 μmol/l). Shown is mean ± SD (n = 5–8; *p < 0.01; n.s., not significant). D, MRI on days 3 and 7 after administration of HSPNPs, PDGF-B HSPNPs (low), and PDGF-B HSPNPs. E, MAP-2 staining demonstrated that the infarct volume decreased in the PDGF-B HSPNP group compared with that in the HSPNP group on day 7. MAP-2-negative ratio = (ipsilateral MAP-2-negative area)/(contralateral hemispheric area × 2). Values are mean ± SD (n = 5; *p < 0.05; n.s., not significant). F, MAP-2 staining on days 3 and 7 after administration of HSPNPs and PDGF-B HSPNPs. Scale bars: 100 μm. Dotted areas indicate infarct areas. G, H, Motor functional evaluation using the cylinder test before MCAO, before administration of NPs (day 0), 3 d after administration (day 3), and 7 d after administration (day 7). D, Motor function improved significantly on day 7 in the PDGF-B HSPNP group (1.04 μmol/l) compared with the PBS or PDGF-BB group. E, Motor function improved significantly on day 7 in the PDGF-B HSPNP group (1.04 μmol/l) compared with that in the groups administered HSPNP or lower concentration of PDGF-B HSPNP (0.104 μmol/l). Values are mean ± SD (n = 5–8; †p < 0.01, PBS and PDGF-BB vs PDGF-B HSPNP), *p < 0.05, HSPNP versus PDGF-B HSPNP.

Akt phosphorylation by PDGF-B HSPNPs. A, Temporal profile of phospho-Akt (Ser 473) expression using immunohistochemistry on days 3 and 7 after administration of HSPNPs and PDGF-B HSPNPs. Scale bars: 100 μm. B, Phospho-Akt-positive cells were counted by hybrid cell count in the peri-infarct area and ischemic core. Data are presented as mean ± SD (n = 5; *p < 0.05; n.s., not significant). C, Immunoblot analyses for Akt and phospho-Akt (Ser 473) 3 d after administration of HSPNPs and PDGF-B HSPNPs. D, Densitometry for phospho-Akt/total Akt. Data are presented as mean ±SD (n = 9; *p < 0.05). E, Immunofluorescent double labeling of phospho-Akt (p-Akt; red) and PDGFRβ (green) in the ischemic core on day 3 after PDGF-B HSPNP administration. Scale bar: 50 μm.

Effects of PDGF-B HSPNPs on the expression of NTs and astrogliosis in peri-infarct area in a murine transient middle cerebral artery occlusion model of stroke. NT expression 3 d after administration of PDGF-B HSPNPs was investigated with ELISA using a multi-NT rapid screening ELISA kit. A, Neurotrophin-3 (NT-3) (n = 10; *p < 0.05). B, Nerve growth factor (NGF) (n = 8; n.s., not significant.). C, Brain-derived neurotrophic factor (BDNF) (n = 8). D, Neurotrophin-4/5 (NT-4/5) (n = 8). E, Immunoblot analyses for GFAP 7 d after administration of HSPNPs and PDGF-B HSPNPs. GFAP–positive cells were counted by hybrid cell count in the peri-infarct area. Data are presented as mean ± SD (*p < 0.05).