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

Figures & data

Table 1 The average particle diameter, zeta potential, encapsulation efficiency of CRM in CRM-LIP and in CRM-CL/LIP, and encapsulation efficiency of NGF in NGF-LIP and in NGF-CL/LIP (n=3)

	Particle diameter (nm)	Zeta potential (mV)	Encapsulation efficiency of CRM (%)	Encapsulation efficiency of NGF (%)
CRM-LIP	133.6±2.1	−5.8±1.2	45.2±4.3	–
CRM-CL/LIP	142.9±5.4	−18.3±2.1	56.7±2.6	–
NGF-LIP	122.3±4.2	−5.2±0.8	–	20.5±3.1
NGF-CL/LIP	135.2±6.8	−17.1±1.6	–	36.6±3.8

Abbreviations: 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.

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 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.