Liposomal flucytosine capped with gold nanoparticle formulations for improved ocular delivery

Figures & data

Table 1 Composition of both positively and negatively charged liposomal formulations in molar ratio

Liposomal formulation	Soy bean phosphatidylcholine	Cholesterol	Span 60	Stearylamine	Dicetyl phosphate
F1	3	1	0.10	0	0
F2	2	1	0.10	0	0
F3	1	1	0.10	0	0
F4	1	1	0.10	0.05	0
F5	1	1	0.10	0.10	0
F6	1	1	0.10	0.15	0
F7	1	1	0.10	0	0.05
F8	1	1	0.10	0	0.10
F9	1	1	0.10	0	0.15

Abbreviations: F1, PC:Ch:Span 60 at a molar ratio of 3:1:1; F2, PC:Ch:Span 60 at a molar ratio of 2:1:1; F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F4, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.05; F5, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.10; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F7, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.05; F8, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.10; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Table 2 Entrapment efficiency of the prepared neutral and charged liposomal formulations

Formulation	Entrapment efficiency of loaded flucytosine (% w/w)	Entrapment efficiency of loaded gold nanoparticles (% w/w)
F1	70.52±1.86	29.05±1.71
F2	73.84±2.92	31.21±2.48
F3	80.85±2.77	35.19±1.08
F4	79.01±1.43	34.44±1.99
F5	75.72±0.85	36.17±2.27
F6	74.22±1.10	40.57±2.49
F7	81.41±0.99	31.46±1.50
F8	84.37±1.82	29.87±1.97
F9	85.52±1.64	24.73±3.01

Note: Mean ± standard deviation, n=3.

Abbreviations: F1, PC:Ch:Span 60 at a molar ratio of 3:1:1; F2, PC:Ch:Span 60 at a molar ratio of 2:1:1; F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F4, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.05; F5, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.10; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F7, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.05; F8, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.10; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Table 3 Represents the average size, polydisersity index, and zeta potential of the prepared formulations

Formulation	Average particle size (nm)	Polydispersity index	Zeta potential (mV)
F1	88.1±10.6	0.239	−46.4±2.01
F2	107.5±9.1	0.290	−52.8±1.82
F3	120.1±10.1	0.204	−55.4±1.41
F4	122.9±5.5	0.309	23.1±1.54
F5	129.0±4.2	0.240	28.4±1.76
F6	135.1±12.0	0.270	42.5±2.12
F7	150.5±14.1	0.445	−56.9±2.25
F8	158.9±4.9	0.258	−59.1±1.70
F9	174.1±8.9	0.413	−63.0±0.59
Gold nanoparticles	13.3±4.0	0.189	−29.74±2.45

Notes: Average size: mean ± standard error, n=3. Zeta potential: mean ± standard deviation, n=3.

Abbreviations: F1, PC:Ch:Span 60 at a molar ratio of 3:1:1; F2, PC:Ch:Span 60 at a molar ratio of 2:1:1; F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F4, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.05; F5, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.10; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F7, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.05; F8, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.10; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Figure 1 Transmission electron photomicrographs of flucytosine, gold-loaded liposomes, and gold nanoparticles according to the prepared formulations.

Notes: (A) F3; (B) F6; (C) F9; and (D) gold nanoparticles. The arrows indicate gold nanoparticles.
Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Figure 2 The effect of molar ratio of phosphatidylcholine/cholesterol and Span 60 on the cumulative release profile of flucytosine (A) and gold nanoparticles (B) from the prepared liposomes (F1, F2, and F3) in comparison to the free drug.

Note: Mean ± standard deviation, n=3.
Abbreviations: F1, PC:Ch:Span 60 at a molar ratio of 3:1:1; F2, PC:Ch:Span 60 at a molar ratio of 2:1:1; F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; Ch, cholesterol; PC, phosphatidylcholine; SA, stearylamine.

Figure 3 The effect of varying molar ratios of the positive charge inducer (stearylamine) and the negative charge inducer (dicetyl phosphate) on the cumulative release profile of flucytosine (A) and gold nanoparticles (B) from the prepared positive liposomes (F4, F5, and F6) and negative liposomes (F7, F8 and F9) compared with the free drug and the liposomal formulation F3.

Note: Mean ± standard deviation, n=3.
Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F4, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.05; F5, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.10; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F7, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.05; F8, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.10; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Figure 4 Higuchi plot for the cumulative percentage of flucytosine released versus the square root of time.

Notes: Neutral liposomal formulation (F3) (

). Positive liposomal formulation (F6) (■). Negative liposomal formulation (F9) (▲).
Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Figure 5 Zero-order plot for the cumulative percentage of gold nanoparticles released versus time.

Notes: Neutral liposomal formulation (F3) (

). Positive liposomal formulation (F6) (■). Negative liposomal formulation (F9) (▲).
Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Table 4 Entrapment efficiency of gold nanoparticle flucytosine-loaded liposomes (F3, F6, and F9) after the storage period of 3 months kept at 4°C±1°C, 25°C±1°C, and 37°C±1°C, 60%±5% relative humidity

Storage temperature after 3 months	Entrapment efficiency (%) of:
	F3: neutral		F6: positive		F9: negative
	Nanogold	Flucytosine	Nanogold	Flucytosine	Nanogold	Flucytosine
Initial	35.19±1.08	80.85±2.77	40.57±2.49	74.22±1.10	24.73±2.09	85.52±1.64
4°C±1°C	32.64±2.57	77.64±3.52	38.95±3.07	72.54±5.42	22.72±5.31	81.71±3.21
25°C±1°C	22.94±1.89	69.23±4.10	29.57±3.74	65.32±5.32	16.97±3.54	72.05±4.12
37°C±1°C	18.25±1.96	53.98±4.35	21.12±2.85	50.41±3.65	12.97±3.54	59.52±4.32

Note: The values are the mean ± standard deviation from three parallel measurements.

Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Table 5 Particle size of the prepared liposomes (F3, F6, and F9) and the released gold nanoparticles after the storage period of 3 months kept at 4°C±1°C, 25°C±1°C, and 37°C±1°C, 60%±5% relative humidity

Storage temperature after 3 months	Entrapment efficiency (%) of:
	F3: neutral		F6: positive		F9: negative
	Liposomal vesicles	Nanogold	Liposomal vesicles	Nanogold	Liposomal vesicles	Nanogold
Initial	120.10±10.10	13.90±4.00	135.10±12.00	12.50±2.40	174.10±8.90	13.00±3.50
4°C±1°C	131.72±4.65	15.10±3.52	139.16±5.78	13.70±1.41	182.08±5.17	14.20±2.25
25°C±1°C	148.31±4.42	22.30±5.20	166.24±5.25	20.60±4.60	206.45±4.24	21.50±4.54
37°C±1°C	166.47±4.94	31.1±2.85	199.89±8.41	30.2±3.84	245.45±7.23	30.4±3.67

Note: The values are the mean ± standard deviation from three parallel measurements.

Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Table 6 Intraocular penetration distance of colloidal gold nanoparticle solution and neutral, positively, and negatively charged gold nanoliposomes across rabbits’ eyes was measured using computed tomography

Time (hours)	Mean ocular penetration depth (mm)
	Gold nanoparticles	F3, neutral charged liposome	F6, positively charged liposome	F9, negatively charged liposome
0.5	1.71±0.0241	3.12±0.225	4.13±0.149	3.72±0.062
1	1.95±0.0421	4.38±0.262	7.20±0.114	6.95±0.302
2	1.97±0.0711	7.82±0.201	10.12±0.425	9.33±0.310
6	1.96±0.085	9.34±0.160	10.22±0.113	10.08±0.053

Note: Mean ± standard deviation in millimeters, n=3.

Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Table 7 One-way analysis of variance of ocular penetration of neutral (F3), positively (F6), and negatively (F9) charged gold nanoparticle-loaded liposomes across rabbits’ eyes after 6 hours

Source of variance	Degrees of freedom	Sum of squares	Mean square	F	P
Residuals (between columns)	2	1.342	0.671	48.965	0.0001
Treatments (between columns)	6	0.082	0.014
Total	8	1.424

Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Table 8 In vitro susceptibility test of different liposomal formulations and control gold nanoparticle solution by disc diffusion method

Liposomal formulations	Mean diameter of growth inhibition zone	Student’s t-test compared with formulation F3
F3	31.08±1.52
F6	34.66±2.30	0.0320
F9	29.52±1.85	0.102
Gold nanoparticle solution	24.66±0.57	0.0142

Note: Mean ± standard deviation in millimeters, n=3.

Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Figure 6 The growth inhibition zone on using different formulations of flucytosine and gold nanoparticle-loaded liposomes, flucytosine solution (0.2 µL, 0.8% w/v), and colloidal gold nanoparticle solution (0.2 µL, 150 µM).

Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Figure 7 Bio-distribution of gold nanoparticles on instillation of different liposomal formulations (F3, F6, and F9) and Au NPs when computed tomography images of the rabbits’ eyes were captured at different time intervals.

Notes: (A) Zero time, (B) 0.5 hours, (C) 1 hour, (D) 2 hours, and (E) 6 hours using phosphate buffer as a control. The black arrows indicate the accumulation of the nanoparticles (if any) throughout the study. “Cont neg” indicates nothing was applied on the eye.
Abbreviations: Au NPs, gold nanoparticles; Con, control solution (isotonic phosphate buffer); Cont neg, nothing is applied; F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Table 9 Pharmacokinetic parameters of flucytosine in aqueous humor, post-topical instillation of flucytosine solution (0.3% w/v) or flucytosine-loaded liposomal formulations F3, F6, and F9

Formulation	Cmax ± SD (μg/mL)	Tmax (hours)	AUC (μg·h/mL)	Frel (%)
Flucytosine solution	4.90±1.1	1	8.32±3.1	1
Liposomal formulation F3	14.90±2.1	1	60.37±14.2	7.25
Liposomal formulation F6	17.50±2.4	1	97.82±17.3	11.75
Liposomal formulation F9	15.80±2.9	1	83.70±15.2	10.05

Notes: Equivalent to 0.3 mg of flucytosine. n=3, P<0.05.

Abbreviations: AUC, area under the curve maximum; C_max, maximum flucytosine concentration in aqueous humor; F_rel (%), relative bioavailability; T_max, the time to reach C_max; F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Figure 8 Concentration time profiles in aqueous humor of rabbits, following topical instillation of flucytosine as solution eye drops or liposomal formulations (F3, F6, and F9).

Notes: In vivo pharmacokinetic parameters of flucytosine from the studied groups. P<0.05 compared with flucytosine as solution eye drops.
Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Table 10 The percentage and the time of healing of rabbits’ eyes infected with Candida albicans were measured when nanoliposomes formulations F3, F6, and F9 were applied

Group number	Antifungal formulations	Number of examined eyes	Numbers of eyes treated during the								χ^2	P-value
			First week		Second week		Third week		Fourth week
			N*	%**	N*	%**	N*	%**	N*	%**
1	Flucytosine solution	8	0	0	1	12.5	2	25	2	25	2	>0.01 NS
2	F3: neutral	8	3	37.5	3	37.5	5	62.5	7	87.5	7.54	<0.01 S
3	F6: positive	8	5	62.5	5	62.5	7	87.5	8	100	8	<0.01 S
4	F9: negative	8	4	50	5	62.5	6	75	7	87.5	7.54	<0.01 S

Abbreviations: %**, percentage of eye healing; N*, number of the healed eyes; NS, not significant; S, significant; F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.

Figure 9 Photographs show different stages of the healing and the disappearance of Candida keratitis when infected rabbits’ eyes were treated with flucytosine solution, F3, F6, and F9, respectively.

Abbreviations: F3, PC:Ch:Span 60 at a molar ratio of 1:1:1; F6, PC:Ch:Span 60:SA at a molar ratio of 1:1:1:0.15; F9, PC:Ch:Span 60:DCP at a molar ratio of 1:1:1:0.15; Ch, cholesterol; DCP, dicetyl phosphate; PC, phosphatidylcholine; SA, stearylamine.