Targeting brain cells with glutathione-modulated nanoliposomes: in vitro and in vivo study

Figures & data

Table 1 Flucytosine nanoliposomes expressed as molar ratios of lipid components, whereas glutathione–malemide–PEG₂₀₀₀–distearoyl phosphatidyle ethanolamine is expressed as percentage molar substitution

Liposomal formulae	Soya bean phosphatidylcholine (PC)	Cholesterol	Span 65	Glutathione–maleimide–PEG2000–distearoyl phosphatidyl ethanolamine (expressed as percent molar substitution)
	Molar ratio (M)
F1	1	1	1	0
F2	1	1	1	0.25
F3	1	1	1	0.50
F4	1	1	1	0.75

Note: Glutathione was coupled to the liposomal formulae (F1–F4) in escalating mole percent ranging from 0 mol% to 0.75 mol%.

Table 2 Entrapment efficiency of flucytosine within targeted liposomes when different ratios of glutathione moiety were used (means ± SD [n=3])

Liposomal formulae	Entrapment efficiency (% w/w)	± SD
F1	76.85	±2.49
F2	76.06	±3.05
F3	77.52	±1.71
F4	75.65	±1.58

Table 3 Zeta potential values of glutathione-targeted nanoliposomes (measurements ± SD [n=3])

Liposomal formulae	Zeta potential (−mV)	± SD
F1	−36.4	±1.41
F2	−43.7	±2.70
F3	−49.3	±1.77
F4	−64.3	±1.49

Table 4 Particle size measurements and polydispersity index of targeted liposomes (measured ± SD [n=3])

Liposomal formulae	Particle size (nm) ± SD	Polydispersity index
F1	105.7±2.4	0.59±0.02
F2	99.0±3.2	0.27±0.01
F3	96.1±3.1	0.29±0.01
F4	90.0±1.7	0.29±0.005

Figure 1 Transmission electron micrographs of flucytosine-loaded nanoliposomes: (A) represents F1 liposomes without targeting moiety, (B) represents F2, (C) represents F3, and (D) represents F4.

Figure 2 Percent drug released against time (h) from both glutathione-free and glutathione-modulated liposome formulae (F1–F4).

Note: Glutathione was coupled to the liposomes in escalating mole percent ranging from 0 to 0.75 mol%.

Table 5 The mean particle size measurements (nm) of the prepared vesicles during storage at 4°C±1°C, 25°C±1°C, and 37°C±1°C for three months (n=3, the values were the means ± SD)

Liposomal formulae	Initial	After 3 months
		4°C±1°C	25°C±1°C	37°C±1°C
F1	105.7±2.4	110.72±2.44	129.21±2.41	151.24±2.74
F2	99.0±3.2	102.16±2.78	121.24±1.45	154.21±2.42
F3	96.1±3.1	100.01±1.47	116.45±1.24	155.42±3.25
F4	90.0±1.7	93.01±1.47	108.25±2.12	142.21±4.21

Note: The values were the means ± standard deviation from three parallel measurements.

Table 6 The entrapment efficiency measurements (%) of the prepared vesicles during storage at 4°C±1°C, 25°C±1°C, and 37°C±1°C for 3 months (n=3, the values were the means ± SD)

Liposomal formulae	Initial	After 3 months
		4°C	25°C	37°C
F1	76.85±2.49	74.92±2.75	67.19±4.1	57.21±3.12
F2	76.06±3.05	74.54±3.84	69.57±2.31	55.37±2.41
F3	77.52±1.71	75.87±2.13	68.89±3.02	56.78±3.25
F4	75.65±1.58	74.87±2.87	68.05±3.45	56.74±2.54

Note: The values were the means ± standard deviation from three parallel measurements.

Figure 3 Intracellular uptake of liposomes functionalized with 0.75 mol% of glutathione moiety at different time intervals: (A) 0 hour and (B) at 6 hours, and intracellular uptake of primary cell culture pretreated with excess free glutathione and then incubated with liposomes functionalized with 0.75 mol% of glutathione moiety at different times intervals (C) 0 hour and (D) 6 hours.

Note: The blue fluorescence represents cell nuclei stained with 4′,6-diamidino-2-phenylindole, while the green fluorescence represents the uptake of calcein-loaded liposomes.

Figure 4 A dot-plot representation of flow cytometry data of green fluorescent cells LR (lower right quarter) and nonfluorescent cells LL (lower left quarter) within cultured primary rat brain cells are represented.

Notes: The cells were incubated with glutathione-modulated liposomes (F1, F2, F3, and F4), functionalized with 0 mol%, 0.25 mol%, 0.5 mol%, and 0.75 mol% of glutathione moiety. The measurements were taken at different time intervals: (a) 1 hour, (b) 2.5 hours, (c) 5 hours, (d) 6 hours, and (e) 24 hours. The results are average of three measurements ± standard error.

Figure 5 The uptake of glutathione-labeled liposomes (F1–F4) when added into the primary brain cells of rats and also the competition assay of formula (F4) incubated with the cells (pretreated with excess glutathione) during a 24-hour incubation period.

Note: The results are expressed as a percent of total green fluorescent cell versus time in comparison to the cellular uptake of free calcein solution (control).

Figure 6 Flow cytometric measurements of glutathione-modulated liposomes when incubated with cultured primary rat brain cells for 24 h.

Note: The results are expressed as percentage of total green fluorescent cells within the medium.

Figure 7 Flucytosine concentration (μg/g) in the plasma and brain 30 minutes postintravenous injection of flucytosine solution (10 mg/kg) or flucytosine-loaded liposomes functionalized with 0%, 0.25%, 0.50%, or 0.75% mole/mole glutathione moiety (F1–F4).

Notes: Each formula was equivalent to 10 mg of flucytosine. Each point represents the mean ± standard deviation of three experiments. P<0.05 versus flucytosine.

Figure 8 Comparison of (C_brain/C_plasma)flucytosine ratios of flucytosine or flucytosine-loaded liposomes (F1–F4). C_brain, C_plasma: flucytosine concentration (μg/g) in the brain or plasma 30 minutes postintravenous injection of flucytosine solution (10 mg/kg) or flucytosine-loaded liposomes functionalized with 0%, 0.25%, 0.50%, or 0.75% mole/mole glutathione moiety (F1–F4).

Notes: Each formula was equivalent to 10 mg of flucytosine. Each point represents the mean ± standard deviation of three experiments (P<0.05 versus flucytosine).

Figure 9 Flucytosine concentration in the plasma and brain (μg/g) versus time postintravenous injection of flucytosine-loaded liposomes functionalized with 0% and 0.75% mole/mole glutathione moiety (F1 and F4, respectively).

Notes: Each injected formula was equivalent to 10 mg of flucytosine. Each point represents the mean ± SD of three experiments. P<0.05 versus flucytosine.

Figure 10 (C_brain/C_plasma)_flucytosine ratios of flucytosine-loaded conventional liposomes (F1) and flucytosine-loaded liposomes (F4) versus time (h). (C_brain/C_plasma)_flucytosine concentration (μg/g) in the brain or plasma postintravenous injection of flucytosine-loaded conventional liposomes (F1) (equivalent to 10 mg/kg) or flucytosine-loaded liposomes functionalized with 0.75% mole/mole glutathione moiety (F4).

Notes: Each formula was equivalent to 10 mg of flucytosine. Each point represents the mean ± standard deviation of three experiments. P<0.05 versus flucytosine.

Table 7 Pharmacokinetic parameters of flucytosine in brain, plasma, and certain tissues of rats, postintravenous injection of flucytosine-loaded 0% mole/mole glutathione-modulated liposome (F1) or flucytosine-loaded 0.75% mole/mole glutathione-modulated liposome, F4 (equivalent to 10 mg/kg of flucytosine) (n=3, P<0.05)

Tissue	Flucytosine 0.75% mole/mole glutathione-modulated liposome, F4			Flucytosine-loaded 0% mole/mole glutathione-modulated liposome, F1
	AUC0–12 (μg/g·h)	Cmax (μg/g)	MRT0–t (h)	AUC0–12 (μg/g·h)	Cmax (μg/g)	MRT0–t (h)
Plasma	126.27±14.89	29.47±4.57	1.83±0.089	176.14±21.14	51.118±6.75	1.62±0.041
Brain	65.82±12.24	21.64±4.81	1.62±0.078	25.011±5.74	13.61±2.75	1.13±0.076
Heart	21.85±3.78	6.01±1.02	0.98±0.089	20.72±4.74	5.07±1.05	1.51±0.078
Lung	22.21±3.65	8.76±2.54	0.90±0.014	15.45±4.35	6.08±2.35	1.12±0.069
Liver	41.81±4.89	12.12±2.84	1.01±0.092	30.25±3.74	8.78±2.78	1.34±0.157
Kidney	29.54±4.52	7.59±3.65	0.89±0.823	15.87±4.95	5.87±1.91	1.71±0.126
Spleen	12.75±2.55	3.97±0.95	0.95±0.0628	14.51±2.81	4.02±0.98	1.48±0.045

Table 8 Targeting efficiency of flucytosine-loaded 0.75% mole/mole glutathione-modulated liposome (F4) compared with flucytosine-loaded 0% mole/mole glutathione-modulated liposome (F1) postintravenous injection of flucytosine-loaded liposomal formulae (equivalent to 10 mg/kg) in male rats (n=3)

	Brain	Heart	Lung	Liver	Kidney	Spleen
REa	2.632±0.089	1.054±0.011	1.437±0.017	1.382±0.019	1.861±0.064	0.878±0.052
CEb	1.590±0.049	1.185±0.071	1.440±0.037	1.380±0.045	1.293±0.085	0.988±0.074
DTIc	3.670±0.824	1.470±0.074	2.005±0.045	1.927±0.039	2.596±0.098	1.225±0.074

Notes:

a RE = (AUC)_{liposomal formulation, F4}/(AUC)_{liposomal formulation, F1}.

b CE = (C_max)_{liposomal formulation, F4}/(C_max)_{liposomal formulation, F1}.

c DTI = (AUC_tissue/AUC_plasma)_{liposomal formulation, F4}/(AUC_tissue/AUC_plasma) _{liposomal formulation, F1}.

Abbreviations: AUC, area under the curve; CE, concentration efficiency; DTI, drug-targeting index; RE, relative efficiency.

Table S1 Molar concentration of lipids and surfactants in the prepared liposomal formulations

Formulation	Soya bean phosphatidyl choline (PC), molar ratio	Cholesterol, molar ratio	Span 65, molar ratio	Span 80,molar ratio
S1	3	1
S2	2	1
S3	1	1
S4	3	1	1
S5	2	1	1
S6	1	1	1
S7	3	1		1
S8	2	1		1
S9	1	1		1

Table S2 Entrapment efficiency values of flucytosine in flucytosine-loaded liposomes formulae (means ± SD [n=3])

Formula no	Entrapment efficiency EE (% w/w) ± SD	Particle size (nm) ± SD	Polydispersity index
S1	32.41±2.37	333±4.5	0.322
S2	35.04±1.52	396±1.1	0.232
S3	40.7±3.11	458±15.5	0.822
S4	59.32±2.95	120.7±1.6	0.408
S5	66.84±1.98	109.7±2.9	0.425
S6	76.85±3.05	105.7±2.4	0.595
S7	45.52±2.83	149.29±5.6	0.427
S8	49.52±1.74	163.77±6.8	0.458
S9	57.56±3.67	181.77±3.6	0.200

Table S3 Zeta potential values of flucytosine-loaded liposomes (measured ± SD [n=3])

Formula no	Zeta potential (mV)
S1	−25.7±1.02
S2	−27.3±2.02
S3	−33.0±2.21
S4	−43.3±1.91
S5	−40.3±1.05
S6	−36.4±1.41
S7	−48.0±1.08
S8	−53.8±2.32
S9	−59.4±2.13

Figure S1 Percent drug released against time (h) from both free drug and flucytosine-loaded liposome formulae (S1–S9).