Wheat germ agglutinin-conjugated liposomes incorporated with cardiolipin to improve neuronal survival in Alzheimer's disease treatment

Abstract

Curcumin (CRM) and nerve growth factor (NGF) were entrapped in liposomes (LIP) with surface wheat germ agglutinin (WGA) to downregulate the phosphorylation of kinases in Alzheimer's disease (AD) therapy. Cardiolipin (CL)-conjugated LIP carrying CRM (CRM-CL/LIP) and also carrying NGF (NGF-CL/LIP) were used with AD models of SK-N-MC cells and Wistar rats after an insult with β-amyloid peptide (Aβ). We found that CRM-CL/LIP inhibited the expression of phosphorylated p38 (p-p38), phosphorylated c-Jun N-terminal kinase (p-JNK), and p-tau protein at serine 202 and prevented neurodegeneration of SK-N-MC cells. In addition, NGF-CL/LIP could enhance the quantities of p-neurotrophic tyrosine kinase receptor type 1 and p-extracellular signal-regulated kinase 5 for neuronal rescue. Moreover, WGA-grafted CRM-CL/LIP and WGA-grafted NGF-CL/LIP significantly improved the permeation of CRM and NGF across the blood-brain barrier, reduced Aβ plaque deposition and the malondialdehyde level, and increased the percentage of normal neurons and cholinergic activity in the hippocampus of AD rats. Based on the marker expressions and in vivo evidence, current LIP carriers can be promising drug delivery systems to protect nervous tissue against Aβ-induced apoptosis in the brain during the clinical management of AD.

Keywords: Alzheimer’s disease; blood–brain barrier; liposome; neurodegeneration; wheat germ agglutinin; β-amyloid.

Figure 1

Characteristics of particle morphology (A), drug release kinetics (B), physical stability (C) of LIP carriers and BBB integrity (D), transmigration efficacy across the BBB (E), and cytotoxicity after treatment with LIP carriers (F). Notes: (A) (a) SEM image of CRM-LIP, (b) SEM image of WGA-CRM-CL/LIP, (c) TEM image of CRM-LIP, and (d) TEM image of WGA-CRM-CL/LIP; (B) (Δ) CRM-LIP, (◊) CRM-CL/LIP, (○) WGA-CRM-CL/LIP, and (□) WGA-NGF-CL/LIP; (C) (○) WGA-CRM-CL/LIP and (□) CRM-LIP; (D) a: control, b: free CRM, c: free NGF, d: CRM-CL/LIP, e: NGF-CL/LIP, f: WGA-CRM-CL/LIP, and g: WGA-NGF-CL/LIP, empty column for transendothelial electrical resistance and column with skew lines for permeability for propidium iodide; (E) empty column for CRM, column with skew lines for NGF (^#P<0.05); and (F) a: CRM, b: NGF, c: CRM-CL/LIP, d: NGF-CL/LIP, e: WGA-CRM-CL/LIP, and f: WGA-NGF- CL/LIP, empty column for HBMECs and column with skew lines for SK-N-MC cells; n=3. Abbreviations: LIP, liposomes; CRM, curcumin; CL, cardiolipin; WGA, wheat germ agglutinin; TEER, transendothelial electrical resistance; PI, propidium iodide; BBB, blood–brain barrier; SEM, scanning electron microscopic; CL/LIP, CL-conjugated LIP; WGA-CL/LIP, CL-conjugated LIP modified with WGA; CRM-LIP, liposomes loaded with CRM; WGA-CRM-CL/LIP, WGA-grafted and CL-conjugated liposomes loaded with CRM; TEM, transmission electron microscopic; CRM-CL/LIP, CL-conjugated liposomes loaded with CRM; WGA-NGF-CL/LIP, WGA-grafted and CL-conjugated liposomes loaded with CRM; NGF, nerve growth factor; NGF-CL/LIP, CL-conjugated liposomes loaded with NGF; HBMEC, human brain-microvascular endothelial cell.

Figure 1

Characteristics of particle morphology (A), drug release kinetics (B), physical stability (C) of LIP carriers and BBB integrity (D), transmigration efficacy across the BBB (E), and cytotoxicity after treatment with LIP carriers (F). Notes: (A) (a) SEM image of CRM-LIP, (b) SEM image of WGA-CRM-CL/LIP, (c) TEM image of CRM-LIP, and (d) TEM image of WGA-CRM-CL/LIP; (B) (Δ) CRM-LIP, (◊) CRM-CL/LIP, (○) WGA-CRM-CL/LIP, and (□) WGA-NGF-CL/LIP; (C) (○) WGA-CRM-CL/LIP and (□) CRM-LIP; (D) a: control, b: free CRM, c: free NGF, d: CRM-CL/LIP, e: NGF-CL/LIP, f: WGA-CRM-CL/LIP, and g: WGA-NGF-CL/LIP, empty column for transendothelial electrical resistance and column with skew lines for permeability for propidium iodide; (E) empty column for CRM, column with skew lines for NGF (^#P<0.05); and (F) a: CRM, b: NGF, c: CRM-CL/LIP, d: NGF-CL/LIP, e: WGA-CRM-CL/LIP, and f: WGA-NGF- CL/LIP, empty column for HBMECs and column with skew lines for SK-N-MC cells; n=3. Abbreviations: LIP, liposomes; CRM, curcumin; CL, cardiolipin; WGA, wheat germ agglutinin; TEER, transendothelial electrical resistance; PI, propidium iodide; BBB, blood–brain barrier; SEM, scanning electron microscopic; CL/LIP, CL-conjugated LIP; WGA-CL/LIP, CL-conjugated LIP modified with WGA; CRM-LIP, liposomes loaded with CRM; WGA-CRM-CL/LIP, WGA-grafted and CL-conjugated liposomes loaded with CRM; TEM, transmission electron microscopic; CRM-CL/LIP, CL-conjugated liposomes loaded with CRM; WGA-NGF-CL/LIP, WGA-grafted and CL-conjugated liposomes loaded with CRM; NGF, nerve growth factor; NGF-CL/LIP, CL-conjugated liposomes loaded with NGF; HBMEC, human brain-microvascular endothelial cell.

Figure 2

Phosphorylation of p38, JNK, Ser202, TrkA, and ERK5 of SK-N-MC cells after inducing with 10 µM of fibrillar Aβ for 24 h. Notes: (A) Western blot of p-p38 and p-JNK, (B) relative ratio of p-p38/GAPDH and p-JNK/GAPDH, (C) Western blot of p-Ser202, (D) relative ratio of p-Ser202/GAPDH, (E) Western blot of p-TrkA and p-ERK5, and (F) relative ratio of p-TrkA/GAPDH and p-ERK5/GAPDH. (A, C, and E) Symbol (+) indicates the ingredient added to the medium and symbol (−) indicates no such ingredient was added to the medium. (B, D, and F) ^#P<0.05, n=3. Abbreviations: JNK, c-Jun N-terminal kinase; Ser202, serine 202; TrkA, tyrosine kinase receptor type 1; ERK5, extracellular signal-regulated kinase 5; Aβ, β-amyloid peptide; p-p38, phosphorylated p38; p-JNK, phosphorylated JNK; p-Ser202, phosphorylated serine 202; p-TrkA, phosphorylated TrkA; p-ERK5, phosphorylated ERK5; DPBS, Dulbecco’s phosphate-buffered saline; CRM, curcumin; CRM-LIP, liposomes loaded with CRM; CRM-CL/LIP, cardiolipin-conjugated liposomes loaded with CRM; NGF, nerve growth factor; NGF-LIP, liposomes loaded with NGF; NGF-CL/LIP, CL-conjugated liposomes loaded with NGF.

Figure 3

Fluorescent images of staining against p-p38 (A), p-JNK (B), and p-ERK5 (C) in SK-N-MC cells after induction with 10 µM of fibrillar Aβ for 24 h. Notes: (A) (a) Control, (b) Aβ, (c) Aβ + CRM, (d) Aβ + CRM-LIP, and (e) Aβ + CRM-CL/LIP; (B) (a) control, (b) Aβ, (c) Aβ + CRM, (d) Aβ + CRM-LIP, and (e) Aβ + CRM-CL/LIP; and (C) (a) control, (b) Aβ, (c) Aβ + NGF, (d) Aβ + NGF-LIP, and (e) Aβ + NGF-CL/LIP; (1) merged image, (2) blue channel for nuclei, (3) green channel for LIP carriers, and (4) red channel for p-JNK, p-p38, or p-ERK5. Abbreviations: p-p38, phosphorylated p38; JNK, c-Jun N-terminal kinase; p-ERK5, phosphorylated extracellular signal-regulated kinase 5; Aβ, β-amyloid peptide; CRM, curcumin; CRM-LIP, liposomes loaded with CRM; CRM-CL/LIP, cardiolipin-conjugated liposomes loaded with CRM; NGF, nerve growth factor; NGF-LIP, liposomes loaded with NGF; NGF-CL/LIP, cardiolipin-conjugated liposomes loaded with NGF; p-JNK, phosphorylated JNK.

Figure 3

Fluorescent images of staining against p-p38 (A), p-JNK (B), and p-ERK5 (C) in SK-N-MC cells after induction with 10 µM of fibrillar Aβ for 24 h. Notes: (A) (a) Control, (b) Aβ, (c) Aβ + CRM, (d) Aβ + CRM-LIP, and (e) Aβ + CRM-CL/LIP; (B) (a) control, (b) Aβ, (c) Aβ + CRM, (d) Aβ + CRM-LIP, and (e) Aβ + CRM-CL/LIP; and (C) (a) control, (b) Aβ, (c) Aβ + NGF, (d) Aβ + NGF-LIP, and (e) Aβ + NGF-CL/LIP; (1) merged image, (2) blue channel for nuclei, (3) green channel for LIP carriers, and (4) red channel for p-JNK, p-p38, or p-ERK5. Abbreviations: p-p38, phosphorylated p38; JNK, c-Jun N-terminal kinase; p-ERK5, phosphorylated extracellular signal-regulated kinase 5; Aβ, β-amyloid peptide; CRM, curcumin; CRM-LIP, liposomes loaded with CRM; CRM-CL/LIP, cardiolipin-conjugated liposomes loaded with CRM; NGF, nerve growth factor; NGF-LIP, liposomes loaded with NGF; NGF-CL/LIP, cardiolipin-conjugated liposomes loaded with NGF; p-JNK, phosphorylated JNK.

Figure 3

Fluorescent images of staining against p-p38 (A), p-JNK (B), and p-ERK5 (C) in SK-N-MC cells after induction with 10 µM of fibrillar Aβ for 24 h. Notes: (A) (a) Control, (b) Aβ, (c) Aβ + CRM, (d) Aβ + CRM-LIP, and (e) Aβ + CRM-CL/LIP; (B) (a) control, (b) Aβ, (c) Aβ + CRM, (d) Aβ + CRM-LIP, and (e) Aβ + CRM-CL/LIP; and (C) (a) control, (b) Aβ, (c) Aβ + NGF, (d) Aβ + NGF-LIP, and (e) Aβ + NGF-CL/LIP; (1) merged image, (2) blue channel for nuclei, (3) green channel for LIP carriers, and (4) red channel for p-JNK, p-p38, or p-ERK5. Abbreviations: p-p38, phosphorylated p38; JNK, c-Jun N-terminal kinase; p-ERK5, phosphorylated extracellular signal-regulated kinase 5; Aβ, β-amyloid peptide; CRM, curcumin; CRM-LIP, liposomes loaded with CRM; CRM-CL/LIP, cardiolipin-conjugated liposomes loaded with CRM; NGF, nerve growth factor; NGF-LIP, liposomes loaded with NGF; NGF-CL/LIP, cardiolipin-conjugated liposomes loaded with NGF; p-JNK, phosphorylated JNK.

Figure 4

Representative illustration of the therapeutics used to promote the pathway of cell survival and to inhibit the pathway of apoptosis. Abbreviations: Aβ, β-amyloid peptide; CREB, cAMP response element binding protein; JNK, c-Jun N-terminal kinase; ERK5, extracellular signal-regulated kinase 5; MEK5, MAP/ERK kinase 5; NGF, nerve growth factor; TrkA, tyrosine kinase receptor type 1.

Figure 5

Immunohistochemistry for amyloid plaques (A), Nissl staining (B), AChE activity (C), and MDA level (D) in the AD brain of rats after treating with LIP carriers. Notes: (A) Binding of anti-Aβ (brown), (a-1 and a-2) control (sham), (b-1 and b-2) Aβ, (c-1 and c-2) Aβ + CRM, (d-1 and d-2) Aβ + WGA-CRM-LIP, and (e-1 and e-2) Aβ + WGA-CRM-CL/LIP; (B) Nissl body (purple) in the hippocampus, (a) control (sham), (b) Aβ, (c) Aβ + CRM, (d) Aβ + WGA-CRM-LIP, and (e) Aβ + WGA-CRM-CL/LIP, (1) 40×, (2) 100×, (3) 200×, and (4) 400×; (C) AChE activity in brain parenchyma; and (D) MDA expression in brain parenchyma. (C and D) ^#P<0.05, *P>0.05, n=5. Abbreviations: AChE, acetylcholinesterase; MDA, malondialdehyde; AD, Alzheimer’s disease; LIP, liposomes; Aβ, β-amyloid peptide; CRM, curcumin; WGA-CRM-LIP, wheat germ agglutinin-grafted liposomes loaded with CRM; WGA-CRM-CL/LIP, WGA-grafted and cardiolipin-conjugated liposomes loaded with CRM.