Polymeric micelles for potentiated antiulcer and anticancer activities of naringin

Figures & data

Figure 1 Structures of the drug and the copolymer.

Notes: (A) Naringin and (B) PF68.

Abbreviations: EO, ethylene oxide; PF68, pluronic F68; PO, propylene oxide.

Table 1 Drug entrapment and loading efficiencies at different drug-to-polymer ratios

Preparation	Drug-to-polymer ratio (w/w)	EE%	LE%
Naringin–PF68 micelles	1:30	69.99±2.81	2.26±0.09
	1:40	83.53±1.74	2.04±0.04
	1:50	96.14±2.29	1.89±0.05

Note: Data are expressed as mean ± SD (n=3).

Abbreviations: EE%, entrapment efficiency; LE%, loading efficiency; PF68, pluronic F68.

Figure 2 Size distribution curve of naringin–PF68 micelles.

Abbreviation: PF68, pluronic F68.

Figure 3 TEM image of naringin–PF68 micelles.

Abbreviations: PF68, pluronic F68; TEM, transmission electron microscope.

Figure 4 Solid characterization.

Notes: (A) FT-IR spectra, (B) DSC curves, and (C) XRD patterns of (I) naringin, (II) PF68, (III) plain micelles, and (IV) naringin–PF68 micelles.

Abbreviations: PF68, pluronic F68; FT-IR, Fourier transform infrared spectroscopy; DSC, differential scanning colorimetry; XRD, X-ray diffractometry.

Figure 5 In vitro release of naringin from its micelles with PF68 compared with free drug in different dissolution media at 37°C±0.5°C.

Notes: (A) SGF (pH 1.2) (0–2 h) followed by SIF (pH 6.8) and (B) PBS (pH 7.4). Each point represents the mean ± SD (n=3).

Abbreviations: PF68, pluronic F68; SGF, simulated gastric fluid; SIF, simulated intestinal fluid; PBS, phosphate buffered saline.

Table 2 Kinetic modeling of drug release data

Release medium	Correlation coefficient (r^2)			Release order	Korsmeyer–Peppas		Main transport mechanism
	Zero order	First order	Higuchi model		r^2	Diffusional exponent (n)
SGF/SIF Burst release phase	0.990	0.990	0.992	Higuchi	0.998	0.702	Non-Fickian
SGF/SIF Gradual release phase	0.994	0.970	0.999	Higuchi	0.988	0.132	Fickian
PBS (pH 7.4) Burst release phase	0.977	0.994	0.997	Higuchi	0.998	0.647	Non-Fickian
PBS (pH 7.4) Gradual release phase	0.975	0.992	0.999	Higuchi	0.999	0.137	Fickian

Abbreviations: SGF, simulated gastric fluid; SIF, simulated intestinal fluid; PBS, phosphate-buffered saline.

Table 3 Storage stability of lyophilized naringin micelles at room temperature 25°C±1°C

Parameter	0 month	3 months
LE%	2.26±0.09	1.73±0.05
EE%	96.14±2.29	88.41±2.66
Mean particle size, nm	74.80±6.56	80.50±0.70
PDI	0.30±0.11	0.23±0.02
Drug retention (%)	–	91.99±3.24

Note: Data are expressed as mean ± SD (n=3).

Abbreviations: EE%, entrapment efficiency; LE%, loading efficiency; PDI, polydispersity index.

Table 4 Parameters of macroscopical evaluation of ethanol-induced ulcers in rats

Animal group	Number of ulcers Mean ± SE n=6	Percentage incidence of animals	Paul’s index	AA	Severity of inflammation
I (normal control)	–	0	0	–	− (no inflammation)
II (positive control)	26.50±2.32	100	26.50	–	+++ (severe)
III (100 mg/kg free naringin)	18.00±1.81#	83.33	14.99	1.77	++ (moderate)
IV (200 mg/kg free naringin)	7.50±1.04###,$$	66.67	5.00	5.30	+ (mild)
V (100 mg/kg naringin–PF68 micelles)	8.75±0.85###,$	66.67	5.83	4.55	+ (mild)

Notes:

# P<0.05 and

### P<0.001 vs positive control group (II).

$ P<0.05 and

$$ P<0.01 vs 100 mg/kg free naringin pretreated group (III).

Abbreviations: AA, antiulcer activity; SE, standard error; PF68, pluronic F68.

Figure 6 Gross appearance of stomach tissues.

Notes: (A) Normal control with normal gastric mucosa, (B) positive control showing multiple gastric ulcers and intensely hemorrhagic mucosa, (C) rats pretreated with naringin (100 mg/kg) displaying some gastric ulcers and moderately hemorrhagic mucosa, and rats pretreated with either (D) naringin (200 mg/kg) or (E) naringin–PF68 micelles (100 mg/kg) showing minimal gastric ulcers and mildly hemorrhagic mucosa. Arrows indicate gastric ulcers and hemorrhagic mucosa.

Abbreviation: PF68, pluronic F68.

Figure 7 Histological examination (HE, 100×) of rats stomach.

Notes: (A) Normal control showing normal gastric mucosa (arrow) and submucosa (arrow head), (B) positive control with coagulative necrosis of entire mucosal thickness with intense hemorrhage and desquamation of necrotic glandular epithelium (arrow) as well as congestion of blood vessels, severe edema, and neutrophilic infiltrations in the submucosa (arrow head), (C) orally pretreated with naringin (100 mg/kg) showing coagulative necrosis of luminal half of the gastric mucosa with hemorrhage and desquamation of necrotic glandular epithelium (arrow) as well as severe edema (arrow head) and neutrophilic infiltrations in the submucosa, and orally pretreated with either (D) naringin (200 mg/kg) or (E) naringin–PF68 micelles (100 mg/kg) displaying coagulative necrosis of only superficial layer of gastric mucosa (arrow) and mild desquamation of necrotic glandular epithelium, besides mild edema and neutrophilic infiltrations in the submucosa (arrow head).

Abbreviations: HE, hematoxylin and eosin; PF68, pluronic F68.

Figure 8 Microphotographs of gastric tissue of rats using immunohistochemical staining for (A) TNF-α and (B) caspase-3 in comparison with normal and positive control groups (IHC, 100×).

Notes: (I) Normal control displaying minimal brown-stained gastric mucosal epithelium (arrow), (II) positive control showing intense brown staining in gastric mucosal epithelium (arrow), gastric glands, and submucosa (arrow head), (III) rats orally pretreated with naringin (100 mg/kg) showing moderate brown staining of the gastric mucosal epithelium (arrow), gastric glands, and submucosa (arrow head), and orally pretreated with either (IV) naringin (200 mg/kg) or (V) naringin–PF68 micelles (100 mg/kg) displaying mild brown staining of superficial gastric mucosal epithelium (arrow) and gastric glands and submucosa (arrow head).

Abbreviations: IHC, immunohistochemical; PF68, pluronic F68; TNF-α, tumor necrosis factor-alpha.

Figure 9 Effects of naringin oral pretreatment on ethanol-induced increase of gastric expression of TNF-α and caspase-3 in rats in comparison with normal and positive control groups (IHC, 100×).

Notes: (A) TNF-α and (B) caspase-3. Data are mean ± SD, n=6. ***P<0.001 vs normal control group. ^#P<0.05 and ^###P<0.001 vs positive control group. ^$$P<0.01, and ^$$$P<0.001 vs 100 mg/kg naringin pretreated group.

Abbreviations: IHC, immunohistochemical; PF68, pluronic F68; TNF-α, tumor necrosis factor-alpha.

Figure 10 Effects of naringin oral pretreatment on ethanol-induced changes in oxidative stress markers.

Notes: (A) MDA, (B) GSH, and (C) SOD in comparison with normal and positive control groups. Data are mean ± SD, n=6. ***P<0.001 vs normal control group. ^##P<0.01, and ^###P<0.001 vs positive control group. ^$P<0.05 and ^$$P<0.01 vs 100 mg/kg naringin pretreated group.

Abbreviations: GSH, reduced glutathione; MDA, malondialdehyde; PF68, pluronic F68; SOD, superoxide dismutase.

Figure 11 Effects of naringin oral pretreatment on ethanol-induced cytokines expression in comparison with normal and positive control groups using ELISA.

Notes: (A) NF-κB and (B) IL-6. Data are mean ± SD, n=6. **P<0.01, and ***P<0.001 vs normal control group. ^#P<0.05 and ^###P<0.001 vs positive control group. ^$P<0.05 vs 100 mg/kg naringin pretreated group.

Abbreviations: ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin-6; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PF68, pluronic F68.

Table 5 The in vitro cytotoxicity estimated as the IC₅₀ values after 72 h

Treatment	IC50 (μM)
	HepG2	Caco-2	MCF-7
Naringin	25±0.17	7.67±0.14	30.40±1.34
Naringin–PF68 micelles	14.00±0.17@@@,***	0.19±0.01@@@,***	23.00±1.32@@@,*
PF68 plain micelles	98.00±0.08	25.00±0.04	34.00±0.01
Cisplatin	30.75±0.22	9.80±1.13	20.51±0.57

Notes: Data are expressed as mean ± SD (n=3).

@@@ P<0.001 vs free naringin.

* P<0.05, and

*** P<0.001 vs cisplatin.

Abbreviations: Caco-2, colorectal carcinoma; HepG2, human hepatocellular carcinoma; IC₅₀, half maximal inhibitory concentration; MCF-7, human breast cancer; PF68, pluronic F68.

Table 6 Effects of micelled naringin (100 mg/kg) on tumor growth in mice-bearing EAC cells in comparison with cisplatin (1 mg/kg) and free naringin (100 mg/kg)

Treatment group	Tumor size (mm^3)
	Day 0	Day 14	ΔT/ΔC (%)	Percentage of inhibition
EAC-bearing mice	72.40±4.27	722.4±41.91	100	0
Cisplatin	71.33±3.97	206.2±12.70‡	20	80
Naringin	70.00±3.86	346.0±19.26‡,*	42	58
Naringin–PF68 micelles	74.33±4.44	258.0±13.35‡,@	28	72

Notes: Data are expressed as mean ± SE (n=6). ΔT, changes of tumor size in the treatment group; ΔC, changes of tumor size in the EAC-bearing mice; ΔT/ΔC (%), percentage of tumor growth.

‡ P<0.05 vs EAC-bearing mice group,

@ P<0.05 vs free naringin group,

* P<0.05 vs cisplatin group.

Abbreviations: EAC, Ehrlich ascites carcinoma; PF68, pluronic F68.

Figure 12 Survival (%) of mice of different groups at the end of tumor growth study.

Abbreviations: EAC, Ehrlich ascites carcinoma; PF68, pluronic F68.