Rescuing cholinergic neurons from apoptotic degeneration by targeting of serotonin modulator-and apolipoprotein E-conjugated liposomes to the hippocampus

Abstract

β-Amyloid (Aβ)-targeting liposomes (LIP) with surface serotonin modulator (SM) and apolipoprotein E (ApoE) were utilized to facilitate the delivery of nerve growth factor (NGF) across the blood-brain barrier (BBB) for neuroprotection in the hippocampus. The therapeutic efficacy of SM- and ApoE-grafted LIP carrying NGF (NGF-SM-ApoE-LIP) was assessed by an in vitro Alzheimer's disease (AD) model of degenerated SK-N-MC cells and an in vivo AD model of Aβ-insulted Wistar rats. The experimental evidences revealed that the modified SM and ApoE on the surface of LIP increased the permeation of NGF across the BBB without serious damage to structural integrity of tight junction. When compared with free NGF, NGF-SM-ApoE-LIP upregulated the expression of phosphorylated neurotrophic tyrosine kinase receptor type 1 on cholinergic neurons and significantly improved their survival. In addition, NGF-SM-ApoE-LIP could reduce the secretion of acetylcholinesterase and malondialdehyde and rescue hippocampal neurons from apoptosis in rat brains. The synergistic effect of SM and ApoE is promising in the induction of NGF to inhibit the neurotoxicity of Aβ and NGF-SM-ApoE-LIP can be a potent antiapoptotic pharmacotherapy for clinical care of patients with AD.

Keywords: Alzheimer’s disease; apolipoprotein E; blood–brain barrier; liposome; nerve growth factor; serotonin modulator.

Figure 1

Physicochemical characterization of NGF-SM-ApoE-LIP. Notes: (A) Average particle diameter and zeta potential; C_SM =40 μg/mL; C_ApoE =40 μg/mL; (○) average particle diameter; n=3; (□) zeta potential; n=3; inset: corresponding SEM images at r_CL =10%, r_CL =20%, r_CL =30%, (B) TEM images; r_CL =30%; (a) NGF-ApoE-LIP; C_ApoE =40 μg/mL; (b) NGF-SM-LIP; C_SM =40 μg/mL; (c) NGF-SM-ApoE-LIP; C_SM =40 μg/mL; C_ApoE =40 μg/mL, (C) grafting efficiency; (○) grafting efficiency of ApoE on NGF-LIP; C_SM =0 μg/mL; n=3; (□) grafting efficiency of SM on NGF-ApoE-LIP; C_ApoE =40 μg/mL; n=3, (D) XPS spectra; (a) NGF-LIP; r_CL =0%; (b) NGF-LIP; r_CL =30%; (c) NGF-ApoE-LIP; r_CL =30%; C_ApoE =40 μg/mL; (d) NGF-SM-ApoE-LIP; r_CL =30%; C_SM =40 μg/mL; C_ApoE =40 μg/mL. Abbreviations: C_SM, concentration of serotonin modulator; C_ApoE, concentration of apolipoprotein E; r_CL, weight percentage of cardiolipin in bilayer (%).

Figure 2

Effect of NGF-SM-ApoE-LIP on the BBB permeation. Notes: r_CL =30%. (A) Viability of HBMECs and HAs; C_SM =40 μg/mL; C_ApoE =40 μg/mL; n=3, (B) TEER and permeability coefficient of PI across HBMEC/HA; (○) TEER; NGF-ApoE-LIP; (X₁ and Y₁ axes); (●) TEER; NGF-SM-ApoE-LIP; C_ApoE =40 μg/mL; X₂ and Y₁ axes; (- - -): intact TEER; Y₁ axis; n=3, (□) permeability coefficient of PI; NGF-ApoE-LIP; X₁ and Y₂ axes; (■) permeability coefficient of PI; NGF-SM-ApoE-LIP; C_ApoE =40 μg/mL; X₂ and Y₂ axes; (– – –): intact permeability coefficient of PI; Y₂ axis; n=3, (C) permeability coefficient of NGF across HBMEC/HA; (○) NGF-ApoE-LIP; (●) NGF-SM-ApoE-LIP; C_ApoE =40 μg/mL; n=3, (D) fluorescent NGF-SM-ApoE-LIP internalized by HBMECs; C_ApoE =40 μg/mL; (a) NGF-LIP; (b) NGF-ApoE-LIP; (c) NGF-SM-ApoE-LIP; C_SM =20 μg/mL; (d) NGF-SM-ApoE-LIP; C_SM =40 μg/mL; (a1, b1, c1, d1), (a2, b2, c2, d2), (a3, b3, c3, d3), and (a4, b4, c4, d4) are red (against LDLR antibody in [a] and [b] and against both LDLR and SR antibody in [c] and [d]), green (LIP carrier), blue (DAPI for nucleus), and merged channels, respectively. a1, a2, and a3 are obtained from excitation at 555 nm, 490 nm, and 350 nm, respectively. a4 is a merged image from a1–a3. The difference between a1–a4 is the same for b, c, and d. Abbreviations: r_CL, weight percentage of cardiolipin in bilayer (%); C_SM, concentration of serotonin modulator; C_ApoE, concentration of apolipoprotein E; HBMEC, human brain-microvascular endothe lial cell; HA, human astrocyte; BBB, blood–brain barrier; TEER, transendothelial electrical resistance; LIP, liposome; SM, serotonin modulator; NGF, nerve growth factor.

Figure 2

Effect of NGF-SM-ApoE-LIP on the BBB permeation. Notes: r_CL =30%. (A) Viability of HBMECs and HAs; C_SM =40 μg/mL; C_ApoE =40 μg/mL; n=3, (B) TEER and permeability coefficient of PI across HBMEC/HA; (○) TEER; NGF-ApoE-LIP; (X₁ and Y₁ axes); (●) TEER; NGF-SM-ApoE-LIP; C_ApoE =40 μg/mL; X₂ and Y₁ axes; (- - -): intact TEER; Y₁ axis; n=3, (□) permeability coefficient of PI; NGF-ApoE-LIP; X₁ and Y₂ axes; (■) permeability coefficient of PI; NGF-SM-ApoE-LIP; C_ApoE =40 μg/mL; X₂ and Y₂ axes; (– – –): intact permeability coefficient of PI; Y₂ axis; n=3, (C) permeability coefficient of NGF across HBMEC/HA; (○) NGF-ApoE-LIP; (●) NGF-SM-ApoE-LIP; C_ApoE =40 μg/mL; n=3, (D) fluorescent NGF-SM-ApoE-LIP internalized by HBMECs; C_ApoE =40 μg/mL; (a) NGF-LIP; (b) NGF-ApoE-LIP; (c) NGF-SM-ApoE-LIP; C_SM =20 μg/mL; (d) NGF-SM-ApoE-LIP; C_SM =40 μg/mL; (a1, b1, c1, d1), (a2, b2, c2, d2), (a3, b3, c3, d3), and (a4, b4, c4, d4) are red (against LDLR antibody in [a] and [b] and against both LDLR and SR antibody in [c] and [d]), green (LIP carrier), blue (DAPI for nucleus), and merged channels, respectively. a1, a2, and a3 are obtained from excitation at 555 nm, 490 nm, and 350 nm, respectively. a4 is a merged image from a1–a3. The difference between a1–a4 is the same for b, c, and d. Abbreviations: r_CL, weight percentage of cardiolipin in bilayer (%); C_SM, concentration of serotonin modulator; C_ApoE, concentration of apolipoprotein E; HBMEC, human brain-microvascular endothe lial cell; HA, human astrocyte; BBB, blood–brain barrier; TEER, transendothelial electrical resistance; LIP, liposome; SM, serotonin modulator; NGF, nerve growth factor.

Figure 3

Effect of NGF-SM-ApoE-LIP on the survival of neurons treated with Aβ_1–42. C_SM =40 μg/mL; C_ApoE =40 μg/mL. Notes: (A) Viability of SK-N-MC cells; r_CL =30%; *P<0.05; **P<0.01; n=3, (B) p-TrkA level of SK-N-MC cells; r_CL =30%; (a): Western blot; (b): R_p, *P<0.05; n=3, (C) immunochemical staining images of NGF-SM-ApoE-LIP interacting with SK-N-MC cells; (a) NGF-LIP; r_CL =0%; (b) NGF-LIP; r_CL =30%; (c) NGF-SM-LIP; r_CL =30%; (d) NGF-SM-ApoE-LIP; r_CL =30%; (a1, b1, c1, d1), (a2, b2, c2, d2), (a3, b3, c3, d3), and (a4, b4, c4, d4) are red (against Aβ_1–42 antibody), green (LIP carrier), blue (DAPI for nucleus), and merged channels, respectively. a1, a2, and a3 are obtained from excitation at 555 nm, 490 nm, and 350 nm, respectively. a4 is a merged image from a1–a3. The difference between a1–a4 is the same for b, c, and d. Abbreviations: C_SM, concentration of serotonin modulator; C_ApoE, concentration of apolipoprotein E; r_CL, weight percentage of cardiolipin in bilayer (%); R_p, protein ratio of p-TrkA to GAPDH; LIP, liposome; SM, serotonin modulator; NGF, nerve growth factor.

Figure 3

Effect of NGF-SM-ApoE-LIP on the survival of neurons treated with Aβ_1–42. C_SM =40 μg/mL; C_ApoE =40 μg/mL. Notes: (A) Viability of SK-N-MC cells; r_CL =30%; *P<0.05; **P<0.01; n=3, (B) p-TrkA level of SK-N-MC cells; r_CL =30%; (a): Western blot; (b): R_p, *P<0.05; n=3, (C) immunochemical staining images of NGF-SM-ApoE-LIP interacting with SK-N-MC cells; (a) NGF-LIP; r_CL =0%; (b) NGF-LIP; r_CL =30%; (c) NGF-SM-LIP; r_CL =30%; (d) NGF-SM-ApoE-LIP; r_CL =30%; (a1, b1, c1, d1), (a2, b2, c2, d2), (a3, b3, c3, d3), and (a4, b4, c4, d4) are red (against Aβ_1–42 antibody), green (LIP carrier), blue (DAPI for nucleus), and merged channels, respectively. a1, a2, and a3 are obtained from excitation at 555 nm, 490 nm, and 350 nm, respectively. a4 is a merged image from a1–a3. The difference between a1–a4 is the same for b, c, and d. Abbreviations: C_SM, concentration of serotonin modulator; C_ApoE, concentration of apolipoprotein E; r_CL, weight percentage of cardiolipin in bilayer (%); R_p, protein ratio of p-TrkA to GAPDH; LIP, liposome; SM, serotonin modulator; NGF, nerve growth factor.

Figure 4

Biochemical analysis and histochemical staining of the brain of AD rats treated with NGF-SM-ApoE-LIP. r_CL =30%; C_SM =40 μg/mL; C_ApoE =40 μg/mL. Notes: (A) MDA level of the brain CA1 tissue after treating with LIP nanocarriers; *P<0.05; ^#P>0.05; **P<0.01; n=6, (B) AChE activity of the brain CA1 tissue after treating with LIP nanocarriers; *P<0.05; **P<0.01; n=6, (C) Nissl staining of the brain CA1 tissue after treating with LIP nanocarriers; (a) control; (b) Aβ_1–42; (c) Aβ_1–42 + NGF; (d) Aβ_1–42 + NGF-LIP; (e) Aβ_1–42 + NGF-SM-ApoE-LIP; (a1, b1, c1, d1, e1) 40×; (a2, b2, c2, d2, e2) 100×; (a3, b3, c3, d3, e3) 200×; (a4, b4, c4, d4, e4) 400×. a1, a2, a3, and a4 were obtained from magnification at 40×, 100×, 200×, and 400×, respectively. The difference between a1–a4 is the same for b, c, d, and e. Abbreviations: r_CL, weight percentage of cardiolipin in bilayer (%); C_SM, concentration of serotonin modulator; C_ApoE, concentration of apolipoprotein E; LIP, liposome; SM, serotonin modulator; NGF, nerve growth factor; MDA, malondialdehyde.