Zein/Phospholipid Composite Nanoparticles for Successful Delivery of Gallic Acid into aHSCs: Influence of Size, Surface Charge, and Vitamin A Coupling

Figures & data

Table 1 Levels of the Independent Variables in Box-Behnken Statistical Design and the Optimization Criteria of the Dependent Variables

Factors (Independent Variables)	Levels of Variables
	Low	Medium	High
X1: effect of drug loading	5 mg	12.5 mg	20 mg
X2: homogenization time	1 min	1.5 min	2 min
X3: ratio between phospholipid and zein	2:1	3:1	4:1
Responses (dependent variables)	Constraints
Y1: PS	Minimum
Y2: PDI	Minimum
Y3: ZP	Maximum in terms of absolute value
Y4: EE%	Maximum

Table 2 Different Runs’ Composition Corresponding to Box-Behnken Design with Their Resultant Dependent Variables (n=3)

Run	Phospholipid: Zein Ratio	Homogenization Time (min)	Drug Loading (mg)	Mean PS ± SD (nm)	Mean PDI ± SD	Mean ZP ± SD (mV)	Mean EE ± SD (% w/w)
BB1	3:1	1.5	12.5	301.35±3.75	0.25±0.01	−20.15±0.21	49.00±2.82
BB2	4:1	1	12.5	305.55±5.02	0.22±0.03	−26.90±0.10	54.30±2.12
BB3	4:1	1.5	5	281.60±4.52	0.20±0.01	−27.60±1.13	64.50±1.27
BB4	3:1	1.5	12.5	302.70±3.53	0.20±0.01	−23.80±0.70	42.80±2.26
BB5	3:1	1	20	293.90±4.38	0.16±0.01	−21.75±0.21	42.40±7.63
BB6	2:1	1.5	5	290.95±8.13	0.19±0.01	−25.25±0.49	48.10±1.41
BB7	4:1	1.5	20	282.20±10.18	0.17±0.01	−23.40±0.28	49.10±4.66
BB8	2:1	2	12.5	279.95±3.74	0.16±0.01	−16.65±0.49	38.70±2.26
BB9	2:1	1	12.5	303.50±4.24	0.21±0.01	−20.70±1.13	44.00±1.27
BB10	3:1	1	5	294.65±1.48	0.18±0.02	−26.25±0.21	59.70±2.40
BB11	3:1	2	20	269.75±7.71	0.18±0.01	−19.05±0.49	45.50±2.12
BB12	3:1	1.5	12.5	302.00±0.84	0.21±0.01	−23.35±0.91	44.40±0.84
BB13	3:1	2	5	301.05±7.42	0.18±0.02	−27.35±0.91	49.50±2.54
BB14	3:1	1.5	12.5	276.35±3.63	0.17±0.02	−22.25±0.49	50.70±7.35
BB15	3:1	1.5	12.5	313.25±0.49	0.22±0.02	−22.80±0.56	47.00±3.53
BB16	4:1	2	12.5	290.85±1.62	0.21±0.02	−24.25±0.70	48.00±2.82
BB17	2:1	1.5	20	290.45±6.43	0.20±0.02	−15.75±1.34	38.30±2.12

Table 3 Levels of the Independent Variables in D-Optimal Statistical Design and the Optimization Criteria of the Dependent Variables

Factors (Independent Variables)	Levels of Variables
	Low Level	High Level
X1: type of positive charge inducers	SA	DDAB
X2: amount of positive charge inducers	5 mg	30 mg
X3: sonication time	60 s	240 s
Responses (dependent variables)	Constraints
Y1: PS	Minimum
Y2: PDI	Minimum
Y3: ZP	Maximum in terms of absolute value
Y4: EE%	Maximum

Table 4 Different Runs’ Composition Corresponding to D-Optimal Design with Their Resultant Dependent Variables (n=3)

Run	Amount of Positive Charge Inducer (mg)	Type of Positive Charge Inducer	Sonication Time (s)	Mean PS ± SD (nm)	Mean PDI ± SD	Mean ZP ± SD (mV)	Mean EE ± SD (% w/w)
DO1	5	SA	150	212.90±1.69	0.23±0.01	33.60±0.42	42.30±0.56
DO2	15.62	DDAB	60	180.70±1.41	0.22±0.02	40.05±3.04	32.30±2.96
DO3	5	DDAB	163.38	183.70±2.82	0.19±0.02	20.35±0.49	37.30±3.11
DO4	30	SA	240	182.10±1.83	0.30±0.04	30.40±0.28	47.90±2.96
DO5	17.5	SA	60	167.50±1.41	0.22±0.01	50.95±1.62	52.10±2.82
DO6	17.79	DDAB	150	174.15±2.05	0.24±0.01	42.80±2.40	25.00±2.54
DO7	30	SA	60	224.95±0.07	0.27±0.01	42.45±0.49	63.15±0.63
DO8	19.30	DDAB	240	317.90±7.07	0.63±0.12	40.45±1.76	32.00±0.14
DO9	5	SA	240	206.85±4.03	0.20±0.02	34.95±1.48	34.60±1.97
DO10	30	DDAB	217.50	194.65±3.18	0.29±0.04	52.90±0.28	27.40±1.92
DO11	30	SA	240	205.10±2.40	0.24±0.01	44.60±0.14	67.30±2.40
DO12	19.30	DDAB	240	205.05±0.07	0.31±0.10	52.85±1.34	38.00±2.82
DO13	5	DDAB	163.38	196.05±0.49	0.26±0.01	36.55±0.91	32.70±3.25
DO14	5	SA	60	238.65±5.02	0.35±0.08	27.95±0.35	48.60±3.39
DO15	15.62	DDAB	60	177.00±1.27	0.23±0.01	41.25±0.77	36.20±3.11
DO16	17.5	SA	240	163.25±2.33	0.23±0.01	42.35±0.63	27.60±1.97
DO17	30	SA	60	215.70±4.94	0.27±0.01	44.70±0.56	66.50±2.40
DO18	17.50	SA	150	161.15±0.77	0.22±0.01	43.25±0.35	46.40±0.84
DO19	30	DDAB	136.21	183.30±3.39	0.31±0.06	49.90±0.84	32.30±1.83

Table 5 Primers Used for qRT-PCR

Gene	Sequence
Col1A1	Forward: 5ʹ-GATCCTGCCGATGTCGCTA-3ʹ
	Reverse: 5ʹTGTAGGCTACGCTGTTCTTGCA-3ʹ
TGF-β1	Forward: 5ʹ-TGAGTGGCTGTCTTTTGACG-3ʹ
	Reverse: 5ʹ-TGGGACTGATCCCATTGATT-3ʹ
α-SMA	Forward: 5ʹ-CGAAGCGCAGAGCAAGAGA −3ʹ
	Reverse: 5ʹ-CATGTCGTCCCAGTTGGTGAT-3ʹ
Caspase-3	Forward: 5ʹ-GAGACAGACAGTGGAACTGACGATG-3ʹ
	Reverse: 5ʹ-GGCGCAAAGTGACTGGATGA-3ʹ
GAPDH	Forward: 5ʹ-GACAACTTTGGCATCGTGGA-3ʹ
	Reverse: 5ʹ-ATGCAGGGATGATGTTCTGG-3ʹ

Figure 1 Pareto chart showing the standardized effect of independent variables on (A) PS, (B) ZP, (C) EE% of GA-ANCNPs.

Abbreviations: PS, Particle size; ZP, Zeta potential; EE%, Entrapment efficiency percentage; GA, Gallic acid; ANCNP, Anionic zein/phospholipid composite nanoparticles.

Figure 2 Response surface plots for the effects of drug loading and phospholipid: zein ratio on (A) ZP and (B) EE% of GA-ANCNPs.

Abbreviations: ZP, Zeta potential; EE%, Entrapment efficiency percentage; GA, Gallic acid; ANCNP, Anionic zein/phospholipid composite nanoparticles.

Figure 3 Pareto chart showing the standardized effect of independent variables and their interactions on (A) PS, (B) ZP, (C) EE% of GA-CACNPs.

Abbreviations: PS, Particle size; ZP, Zeta potential; EE%, Entrapment efficiency percentage; GA, Gallic acid; CACNP, Cationic zein/phospholipid composite nanoparticles.

Figure 4 (A) Response surface plot for the effects of amount of positive charge inducer and sonication time on ZP, Line plots for (B) the main effect of type of positive charge inducer (SA and DDAB) on EE%, (C) the interaction between type of positive charge inducer and sonication time on PS and (D) the interaction between type and amount of positive charge inducer on EE% of GA-CACNPs.

Abbreviations: ZP, Zeta potential; EE%, Entrapment efficiency percentage; PS, Particle size; GA, Gallic acid; CACNP, Cationic zein/phospholipid composite nanoparticles; SA, Stearylamine; DDAB, Didodecyldimethylammonium bromide.

Figure 5 Transmission electron micrographs of (A) optimized GA-ANCNP, (B) optimized GA-CACNP and (C) GA-CACNP/VA (bar = 100 nm, magnification power= 60000X).

Abbreviations: GA, Gallic acid; ANCNP, Anionic zein/phospholipid composite nanoparticles; CACNP, Cationic zein/phospholipid composite nanoparticles; CACNP/VA, Cationic zein/phospholipid composite nanoparticles coupled with vitamin A.

Figure 6 In vitro release profiles of GA from different systems relative to GA solution in phosphate buffer (pH 6.8). Results are expressed as means ± SD, n=3.

Abbreviations: GA, Gallic acid; ANCNP, anionic zein/phospholipid composite nanoparticles; CACNP, cationic zein/phospholipid composite nanoparticles; CACNP/VA, cationic zein/phospholipid composite nanoparticles coupled with vitamin A.

Figure 7 FTIR spectra of (A) VA, (B) optimized GA-CACNP system and (C) GA-CACNP/VA system.

Abbreviations: VA, Vitamin A; GA, Gallic acid; CACNP, Cationic zein/phospholipid composite nanoparticles; CACNP/VA, Cationic zein/phospholipid composite nanoparticles coupled with vitamin A.

Figure 8 DSC thermograms of GA, zein, phospholipid, SA, VA, sucrose, physical mixture, and GA-CACNP/VA system.

Abbreviations: GA, Gallic acid; SA, Stearylamine; VA, vitamin A; CACNP/VA, Cationic zein/phospholipid composite nanoparticles coupled with vitamin A.

Figure 9 Effect of GA-CNPs on cellular uptake and cytotoxicity activities in aHSCs; (A) microscopic fluorescent image showing aHSCs uptake of Rhodamine-loaded-CNP, (B, C) cytotoxicity/antiproliferative activity of the free GA and GA-CNPs against (B) aHSCs and (C) primary hepatocytes after 48 h exposure. Each experiment is performed in triplicate (n=3).

Abbreviations: GA, Gallic acid; aHSCs, activated hepatic stellate cells; ANCNP, Anionic zein/phospholipid composite nanoparticles; CACNP, Cationic zein/phospholipid composite nanoparticles; CACNP/VA, Cationic zein/phospholipid composite nanoparticles coupled with vitamin A.

Figure 10 Antifibrogenic effects of GA-CNPs on fibrosis markers in aHSCs; (A&B) Representative graphs showing mRNA expression levels of activation [(A) TGF-β1 and (B) α-SMA] in aHSCs; (C) representative images of migration of aHSCs at 0 and 48 h treated with medium, plain CNPs,free GA, and GA-CNPs (x40 magnification); (D) representative graph showing the percentage of wound closure after 48 h; (E&F) representative graphs showing mRNA expression levels of (E) collagen synthesis (COL1A1) and (F) apoptosis (Caspase-3) markers in aHSCs; (G) representative fluorescent photomicrographs showing nuclear morphological changes of apoptotic nuclei (red arrows) in DAPI stained aHSC-T6 treated with medium, plain CNPs, free GA and GA-CNPs. Results are expressed as mean ± SD of three independent experiments, (n=3). ^ap < 0.05 vs control untreated aHSCs; ^bp < 0.05 vs GA-treated aHSCs; ^cp < 0.05 vs GA-ANCNP; ^dp < 0.05 vs GA-CACNP.

Abbreviations: GA, Gallic acid; aHSCs, activated hepatic stellate cells; ANCNP, Anionic zein/phospholipid composite nanoparticles; CACNP, Cationic zein/phospholipid composite nanoparticles; CACNP/VA, Cationic zein/phospholipid composite nanoparticles coupled with vitamin A; TGF-β1, transforming growth factor-β1, α-SMA: α-smooth muscle actin; COL1A1, pro-collagen type I, DAPI: 4ʹ,6-diamidino-2-phenylindole.

Figure 11 In vivo biodistribution of Rhodamine B-labelled-CNPs in vital organs; (A) representative graphs showing mean fluorescence intensity percentages of vital organs after treatment with Rhodamine B loaded-CNPs. Results are expressed as means ± SD, (n=3); (B) photomicrographs showing confocal laser scanning of vital organs treated with Rhodamine B-loaded-CACNP/VA. ^ap < 0.05 denotes statistically significant difference between organs vs liver within the same formula; ^bp < 0.05 vs Rhodamine B-loaded-ANCNP within liver; ^cp < 0.05 vs Rhodamine B-loaded-CACNP within liver.

Abbreviations: ANCNP, Anionic zein/phospholipid composite nanoparticles; CACNP, Cationic zein/phospholipid composite nanoparticles; CACNP/VA, Cationic zein/phospholipid composite nanoparticles coupled with vitamin A.