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. 2015 Apr 1:10:2653-72.
doi: 10.2147/IJN.S79528. eCollection 2015.

Rescuing apoptotic neurons in Alzheimer's disease using wheat germ agglutinin-conjugated and cardiolipin-conjugated liposomes with encapsulated nerve growth factor and curcumin

Yung-Chih Kuo  1 , Ching-Chun Lin  1
Affiliations

Rescuing apoptotic neurons in Alzheimer's disease using wheat germ agglutinin-conjugated and cardiolipin-conjugated liposomes with encapsulated nerve growth factor and curcumin

Yung-Chih Kuo et al. Int J Nanomedicine. .

Abstract

Liposomes with cardiolipin (CL) and wheat germ agglutinin (WGA) were developed to permeate the blood-brain barrier and treat Alzheimer's disease. WGA-conjugated and CL-incorporated liposomes (WGA-CL-liposomes) were used to transport nerve growth factor (NGF) and curcumin (CUR) across a monolayer of human brain-microvascular endothelial cells regulated by human astrocytes and to protect SK-N-MC cells against apoptosis induced by β-amyloid1-42 (Aβ(1-42)) fibrils. An increase in the CL mole percentage in lipids increased the liposomal diameter, absolute zeta potential value, entrapment efficiency of NGF and CUR, release of NGF, biocompatibility, and viability of SK-N-MC cells with Aβ(1-42), but decreased the atomic ratio of nitrogen to phosphorus and release of CUR. In addition, an increase in the WGA concentration for grafting enhanced the liposomal diameter, atomic ratio of nitrogen to phosphorus, and permeability of NGF and CUR across the blood-brain barrier, but reduced the absolute zeta potential value and biocompatibility. WGA-CL-liposomes carrying NGF and CUR could be promising colloidal delivery carriers for future clinical application in targeting the blood-brain barrier and inhibiting neurotoxicity.

Keywords: Alzheimer’s disease; cardiolipin; curcumin; liposome; nerve growth factor; wheat germ agglutinin.

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Figures

Figure 1
Figure 1
Schematic illustration of the structure of a WGA-CL-NGF-CUR-liposome. Abbreviations: DSPE-PEG(2000), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanol-amine-N-[methoxy(polyethylene glycol)-2000]; DSPE-PEG(2000)-CA, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000]; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin.
Figure 2
Figure 2
Effect of CL mole percentage on the average diameter of WGA-CL-NGF-CUR-liposomes. (○) CL-liposome; (•) CL-NGF-liposome; (□) WGA-CL-liposome; (△) CL-CUR-liposome; (⋄) WGA-CL-NGF-CUR-liposome. Note: CWGA =5 mg/mL (n=3). Abbreviations: CWGA, WGA concentration in grafting medium (mg/mL); rCL, CL mole percentage in lipids (%); D, average diameter of WGA-CL-NGF-CUR-liposomes (nm); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin.
Figure 3
Figure 3
Micrographs of WGA-CL-NGF-CUR-liposomes. Notes: (A–H) Transmission electron microscopic images. (I–L) Scanning electron microscopic images. (A, I) rCL =0%; (B, J) rCL =5%; (C, K) rCL =10%; (D, L) rCL =20%; (E) CWGA =2.5 mg/mL and rCL =10%; (F) CWGA =2.5 mg/mL and rCL =20%; (G) CWGA =5 mg/mL and rCL =10%; (H) CWGA =5 mg/mL and rCL =20%. Abbreviations: rCL, CL mole percentage in lipids (%); CWGA, WGA concentration in grafting medium (mg/mL); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin.
Figure 3
Figure 3
Micrographs of WGA-CL-NGF-CUR-liposomes. Notes: (A–H) Transmission electron microscopic images. (I–L) Scanning electron microscopic images. (A, I) rCL =0%; (B, J) rCL =5%; (C, K) rCL =10%; (D, L) rCL =20%; (E) CWGA =2.5 mg/mL and rCL =10%; (F) CWGA =2.5 mg/mL and rCL =20%; (G) CWGA =5 mg/mL and rCL =10%; (H) CWGA =5 mg/mL and rCL =20%. Abbreviations: rCL, CL mole percentage in lipids (%); CWGA, WGA concentration in grafting medium (mg/mL); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin.
Figure 4
Figure 4
X-ray photoelectron spectra of WGA-CL-NGF-CUR-liposomes. The inlet graphs on each spectrum are the magnified nitrogen signals. Notes: (A) CWGA =2.5 mg/mL and rCL =10%; (B) CWGA =2.5 mg/mL and rCL =20%; (C) CWGA =5 mg/mL and rCL =10%; (D) CWGA =5 mg/mL and rCL =20%. Abbreviations: rCL, CL mole percentage in lipids (%); CWGA, WGA concentration in grafting medium (mg/mL); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin.
Figure 5
Figure 5
Effect of CL mole percentage on NGF entrapment efficiency (A) and CUR entrapment efficiency (B) in CL-NGF-CUR-liposomes. (A): (○) CL-NGF-liposome; (□) CL-NGF-CUR-liposome. (B): (○) CL-CUR-liposome; (□) CL-NGF-CUR-liposome (n=3). Abbreviations: rCL, CL mole percentage in lipids (%); ECUR, CUR entrapment efficiency in CL-NGF-CUR-liposomes (%); ENGF, NGF entrapment efficiency in CL-NGF-CUR-liposomes (%); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor.
Figure 6
Figure 6
Dissolution profile for NGF (A) and CUR (B) from WGA-CL-NGF-CUR-liposomes. (⋄) rCL =0%; (□) rCL =5%; (○) rCL =10%; (△) rCL =20%. CWGA =5 mg/mL (n=3). Abbreviations: CWGA, WGA concentration in grafting medium (mg/mL); rCL, CL mole percentage in lipids (%); PCUR, accumulated percentage of CUR released from WGA-CL-NGF-CUR-liposomes (%); PNGF, accumulated percentage of NGF released from WGA-CL-NGF-CUR-liposomes (%); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin.
Figure 7
Figure 7
Viability of HBMECs, HAs, HBVPs, and SK-N-MC cells after treatment with WGA-CL-NGF-CUR-liposomes. CWGA =5 mg/mL and rCL =20% (n=4). Abbreviations: CWGA, WGA concentration in grafting medium (mg/mL); rCL, CL mole percentage in lipids (%); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin; HBMECs, human brain-microvascular endothelial cells; HAs, human astrocytes; HBVPs, human brain-vascular pericytes; PCV, percentage of cell viability (%).
Figure 8
Figure 8
Permeability of NGF (A) and CUR (B) across HBMEC/HA using WGA-CL-NGF-CUR-liposomes. (b–e) Black bar, CL-NGF-CUR-liposomes; gray bar, WGA-CL-NGF-CUR-liposomes, CWGA =5 mg/mL. (b) rCL =0%; (c) rCL =5%; (d) rCL =10%; (e) rCL =20%. (A): (a) free NGF; (b)-(e) CL-NGF-liposome; (B): (a) free CUR; (b–e) CL-CUR-liposome (n=4). Abbreviations: CWGA, WGA concentration in grafting medium (mg/mL); rCL, CL mole percentage in lipids (%); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin; HBMEC, human brain-microvascular endothelial cell; HA, human astrocyte.
Figure 9
Figure 9
Temporal variation in viability of Aβ1–42-incubated SK-N-MC cells after rescue with WGA-CL-NGF-CUR-liposomes. Notes: (A) Aβ1–42 at 5 μM; (B) Aβ1–42 at 10 μM. CWGA =5 mg/mL and rCL =20% (n=4). P<0.05 between CL-CUR-liposome group and free CUR group and between WGA-CL-NGF-CUR-liposome group and free CUR group after 24 hours. Abbreviations: PCV,SK-N-MC, viability of SK-N-MC cells after an insult with Aβ1–42 fibrils and treatment with WGA-CL-NGF-CUR-liposomes (%); CWGA, WGA concentration in grafting medium (mg/mL); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin.
Figure 10
Figure 10
(A) Immunochemical staining images of WGA-CL-NGF-CUR-liposomes interacting with HBMECs. (a–d) CL-NGF-CUR-liposomes; (e–h) WGA-CL-NGF-CUR-liposomes; (a) rCL =0%; (b) rCL =5%; (c) rCL =10%; (d) rCL =20%; (e) CWGA =2.5 mg/mL and rCL =10%; (f) CWGA =2.5 mg/mL and rCL =20%; (g) CWGA =5 mg/mL and rCL =10%; (h) CWGA =5 mg/mL and rCL =20%. Green WGA-CL-NGF-CUR-liposomes are adjacent to red O-linked N-acetylglucosamine near blue HBMEC nuclei. (B) Fluorescent images of WGA-CL-NGF-CUR-liposomes interacting with SK-N-MC cells with an insult of Aβ1–42. (a) Control; (b–e) CL-NGF-CUR-liposomes; (b) rCL =0%; (c) rCL =5%; (d) rCL =10%; (e) rCL =20%; (f) CWGA =5 mg/mL, rCL =10%; (g) CWGA =5 mg/mL, rCL =20%. The Aβ1–42 concentration is 10 μM. Green WGA-CL-NGF-CUR-liposomes attached on red Aβ1–42, which is deposited near blue SK-N-MC cell nuclei. Abbreviations: CWGA, WGA concentration in grafting medium (mg/mL); rCL, CL mole percentage in lipids (%); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin; HBMECs, human brain-microvascular endothelial cells; Aβ1–42, β-amyloid1–42.
Figure 10
Figure 10
(A) Immunochemical staining images of WGA-CL-NGF-CUR-liposomes interacting with HBMECs. (a–d) CL-NGF-CUR-liposomes; (e–h) WGA-CL-NGF-CUR-liposomes; (a) rCL =0%; (b) rCL =5%; (c) rCL =10%; (d) rCL =20%; (e) CWGA =2.5 mg/mL and rCL =10%; (f) CWGA =2.5 mg/mL and rCL =20%; (g) CWGA =5 mg/mL and rCL =10%; (h) CWGA =5 mg/mL and rCL =20%. Green WGA-CL-NGF-CUR-liposomes are adjacent to red O-linked N-acetylglucosamine near blue HBMEC nuclei. (B) Fluorescent images of WGA-CL-NGF-CUR-liposomes interacting with SK-N-MC cells with an insult of Aβ1–42. (a) Control; (b–e) CL-NGF-CUR-liposomes; (b) rCL =0%; (c) rCL =5%; (d) rCL =10%; (e) rCL =20%; (f) CWGA =5 mg/mL, rCL =10%; (g) CWGA =5 mg/mL, rCL =20%. The Aβ1–42 concentration is 10 μM. Green WGA-CL-NGF-CUR-liposomes attached on red Aβ1–42, which is deposited near blue SK-N-MC cell nuclei. Abbreviations: CWGA, WGA concentration in grafting medium (mg/mL); rCL, CL mole percentage in lipids (%); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin; HBMECs, human brain-microvascular endothelial cells; Aβ1–42, β-amyloid1–42.
Figure 10
Figure 10
(A) Immunochemical staining images of WGA-CL-NGF-CUR-liposomes interacting with HBMECs. (a–d) CL-NGF-CUR-liposomes; (e–h) WGA-CL-NGF-CUR-liposomes; (a) rCL =0%; (b) rCL =5%; (c) rCL =10%; (d) rCL =20%; (e) CWGA =2.5 mg/mL and rCL =10%; (f) CWGA =2.5 mg/mL and rCL =20%; (g) CWGA =5 mg/mL and rCL =10%; (h) CWGA =5 mg/mL and rCL =20%. Green WGA-CL-NGF-CUR-liposomes are adjacent to red O-linked N-acetylglucosamine near blue HBMEC nuclei. (B) Fluorescent images of WGA-CL-NGF-CUR-liposomes interacting with SK-N-MC cells with an insult of Aβ1–42. (a) Control; (b–e) CL-NGF-CUR-liposomes; (b) rCL =0%; (c) rCL =5%; (d) rCL =10%; (e) rCL =20%; (f) CWGA =5 mg/mL, rCL =10%; (g) CWGA =5 mg/mL, rCL =20%. The Aβ1–42 concentration is 10 μM. Green WGA-CL-NGF-CUR-liposomes attached on red Aβ1–42, which is deposited near blue SK-N-MC cell nuclei. Abbreviations: CWGA, WGA concentration in grafting medium (mg/mL); rCL, CL mole percentage in lipids (%); CL, cardiolipin; CUR, curcumin; NGF, nerve growth factor; WGA, wheat germ agglutinin; HBMECs, human brain-microvascular endothelial cells; Aβ1–42, β-amyloid1–42.
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