Novel chitosan oligosaccharide-based nanoparticles for gastric mucosal administration of the phytochemical “apocynin”

Figures & data

Figure 1 Chemical structures of the polymer and the drug.

Note: (A) COS and (B) APO.

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide.

Table 1 Composition and characterization of APO-loaded COS-NP_s formulations

Formulation code	COS concentration (% w/v)	TPP concentration (% w/v)	Particle size (nm)	PDI	ZP (mV)	DEE (%)	Mucin-binding efficiency (%)
F1	0.3	0.2	904.20±23.01	0.388±0.22	+6.82±0.67	19.65±1.07	1.30±0.62
F2	0.5	0.2	814.93±70.70	0.347±0.11	+10.67±0.40	24.10±2.10	9.56±2.63
F3	0.3	0.1	833.10±77.99	0.848±0.16	+24.27±0.50	17.15±1.11	36.70±4.58
F4	0.5	0.1	436.20±24.45	0.390±0.07	+38.20±1.47	35.06±1.89	51.22±2.99
F5	0.4	0.15	718.90±74.99	0.653±0.16	+28.63±1.14	22.59±0.98	39.35±3.09

Notes: Each value represents the mean ± SD (n=3). Each formulation contains 8 mg APO.

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; DEE, drug entrapment efficiency; NP_s, nanoparticles; PDI, polydispersity index; TPP, tripolyphosphate; ZP, zeta potential.

Figure 2 FT-IR spectra (A), DSC thermograms (B), and XRD patterns (C).

Notes: (I) APO, (II) COS, (III) TPP, (IV) physical mixture, (V) plain COS-NPs, and (VI) APO-loaded COS-NP_s (F4).

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; DSC, differential scanning calorimetry; FT-IR, Fourier-transform infrared; NP_s, nanoparticles; TPP, tripolyphosphate; XRD, X-ray diffractometry.

Figure 3 TEM images, size and ZP distribution curves of APO-loaded COS-NPs (F4).

Notes: (A) TEM images with different magnifications, (B) size distribution curve, and (C) ZP distribution curve.

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; NP_s, nanoparticles; TEM, transmission electron microscopy; ZP, zeta potential.

Figure 4 The in vitro release pattern of APO from COS-NP_s (F4) in comparison with its diffusion from aqueous solution.

Note: Each point represents the mean ± SD (n=3).

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; NP_s, nanoparticles.

Table 2 Kinetic analysis of the release data of APO from COS-NP_s (F4)

Release phase	Zero order	First order	Higuchi model	Release mechanism	Korsmeyer-Peppas		Drug release mechanism
				Coefficient of determination (R^2)		(R^2)	Diffusional exponent (n)
Burst release phase	0.8326	0.8735	0.9851	Diffusion	0.9817	0.4256	Fickian
Sustained release phase	0.8859	0.8993	0.9073	Diffusion	0.9048	0.1011	Fickian

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; NP_s, nanoparticles.

Table 3 Stability study data of APO-loaded COS-NP_s (F4) after storage at refrigerated (4±1°C) and room (25±2°C/60±5% RH) temperatures

Storage time	Evaluation parameters
	Refrigerated temperature (4±1°C)				Room temperature (25±2°C/60±5% RH)
	Particle size (nm)	PDI	ZP (mV)	Drug retention (%)	Particle size (nm)	PDI	ZP (mV)	Drug retention (%)
Initial	436.20±24.45	0.390±0.07	+38.20±1.47	100.00±0.00	436.20±24.45	0.390±0.07	+38.20±1.47	100.00±0.00
1 month	438.57±4.73	0.354±0.04	+37.80±0.85	99.30±0.50	450.08±31.69	0.438±0.10	+36.10±1.63	99.10±0.79
2 months	440.50±9.30	0.254±0.02	+37.14±1.97	98.08±1.69	461.90±25.64	0.483±0.04	+35.30±1.70	97.23±2.66
3 months	449.73±36.11	0.317±0.14	+36.30±2.04	97.10±2.04	501.40±21.81	0.481±0.06	+33.10±1.13*	96.30±2.48
4 months	477.07±35.29	0.410±0.17	+35.10±1.56	96.90±2.44	511.60±26.61*	0.505±0.08	+31.14±0.41*	96.10±3.12
5 months	478.87±24.48	0.429±0.06	+34.70±1.42	95.60±3.18	541.03±24.77*	0.555±0.11	+28.58±1.22*	95.20±3.81
6 months	489.33±27.74#	0.487±0.05	+33.90±1.26#	94.80±3.69	567.08±28.49*	0.521±0.10	+27.30±0.85*	92.11±4.57

Notes: Each value represents the mean ± SD (n=3). *Significant at p<0.05 monthly vs initial. ^#Significant at p<0.05 refrigerated temperature vs room temperature after 6 months.

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; NP_s, nanoparticles; PDI, polydispersity index; RH, relative humidity; ZP, zeta potential.

Table 4 Macroscopic evaluation parameters of concurrent induction and treatment regimen and post-treatment regimen aganist KP-induced gastric ulcer in rats

Animal group	Evaluation parameters
	Concurrent induction and treatment regimen				Post-treatment regimen
	Number of ulcers Mean ± SEM (n=6)	Percentage incidence of rats with ulcers	PI	AA	Number of ulcers Mean ± SEM (n=6)	Percentage incidence of rats with ulcers	PI	AA
I (normal control)	0	0	0	_	0	0	0	_
II (positive control)	36.83±1.30	100	36.83	_	27.17±1.54	100	27.17	_
III (free APO)	24.50±1.18^###	100	24.50	1.50	15.50±0.76^###	100	15.50	1.75
IV (APO-loaded COS-NPs)	6.83±0.60^###$$$	66.66	4.55	8.09	2.83±0.31^###$$$	50	1.42	19.13

Notes: Oral dose of APO in group III and IV was 14 mg/kg. ^###p < 0.001 vs positive control group (II); ^$$$p < 0.001 vs free APO group (III) relative to the corresponding treatment regimen.

Abbreviations: AA, antiulcerogenic activity; APO, apocynin; COS, chitosan oligosaccharide; KP, ketoprofen; NP_s, nanoparticles; PI, Paul's index; SEM, standard error of the mean.

Figure 5 Macroscopic gross appearance of gastric mucosal tissues following (A) concurrent induction and treatment regimen and (B) post-treatment regimen.

Notes: (I) Normal control group, (II) positive control group, (III) free APO treated group, and (IV) APO-loaded COS-NP_s (F4) treated group. Oral dose of free or loaded APO was 14 mg/kg.

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; NP_s, nanoparticles.

Figure 6 Histopathological examination of glandular stomach of rats following (A) concurrent induction and treatment regimen and (B) post-treatment regimen.

Notes: (I) Normal control group, (II) positive control group, (III) free APO treated group, and (IV) APO-loaded COS-NP_s (F4) treated group. Oral dose of free or loaded APO was 14 mg/kg. Thick black arrow points to extensive areas of ulceration. Thick green arrow points to decreased thickness of mucosa due to loss of superficial epithelial layers. Thick red arrows point to focal erosion. Thin black arrows point to congested blood vessels. Thin red arrows point to leukocytic cells' infiltration. Asterisk points to edema. H&E, 100×.

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; M, mucosa; H & E, hematoxylin and eosin; NP_s, nanoparticles; SM, submucosa.

Figure 7 Histochemical staining of glandular stomach of rats with alcian blue stain following (A) concurrent induction and treatment regimen and (B) post-treatment regimen.

Notes: (I) Normal control group, (II) positive control group, (III) free APO treated group, and (IV) APO-loaded COS-NP_s (F4) treated group. Oral dose of free or loaded APO was 14 mg/kg. Thick black arrows point to loss of glycoproteins' secretion in mucosa near ulcer. Thick green arrow points to loss of glycoproteins' secretion in mucosa near erosion. Thick red arrow points to decreased glycoproteins' deposition in mucosa near erosion. Thin red arrows point to continued surface glycoproteins. Alcian blue stain, 100×.

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; M, mucosa; NP_s, nanoparticles; SM, submucosa.

Figure 8 Histochemical staining of glandular stomach of rats with toluidine blue stain following (A) concurrent induction and treatment regimen and (B) post-treatment regimen.

Notes: (I) Normal control group, (II) positive control group, (III) free APO treated group, and (IV) APO-loaded COS-NP_s (F4) treated group. Oral dose of free or loaded APO was 14 mg/kg. Thick black arrows point to mucosal damage. Thin black arrows in insert point to mast cells. Thin red arrows point to leukocytic cells' infiltration. Toluidine blue stain, 100× and insert 200×.

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; M, mucosa; NP_s, nanoparticles; SM, submucosa.

Figure 9 Positive signal for IHC staining of COX-2 in rats’ gastric tissues following (A) concurrent induction and treatment regimen and (B) post-treatment regimen. Statistical analysis of intensity of COX-2 positive signal in rats’ gastric tissues (C) following (a) concurrent induction and treatment regimen and (b) post-treatment regimen.

Notes: (I) Normal control group, (II) positive control group, (III) free APO treated group, and (IV) APO-loaded COS-NP_s (F4) treated group. Oral dose of free or loaded APO was 14 mg/kg. Thick black arrows point to strong COX-2 expression in areas of mucosal necrosis. Yellow arrows in insert point to positive signal. IHC counterstained with Mayer’s hematoxylin, 100× and insert 200×. Kruskal–Wallis test (non-parametric test) was applied followed by Dunn's multiple comparison test. *p<0.05 and **p<0.01 vs normal control group. ^#p<0.05 vs positive control group. ^$p<0.05 APO-loaded COS-NP_s (F4) treated group vs free APO treated group.

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; COX-2, cyclooxygenase-2; IHC, immunohistochemical; M, mucosa; NP_s, nanoparticles; SM, submucosa.

Figure 10 Levels of oxidative stress markers in rats’ gastric tissues following (A) concurrent induction and treatment regimen and (B) post-treatment regimen.

Notes: (a) MDA, (b) SOD, and (c) GSH. Data are mean ± SEM (n=6). * p<0.05, **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 and ^$$p<0.01 APO-loaded COS-NP_s (F4) treated group vs free APO treated group.

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; GSH, reduced glutathione; MDA, malondialdehyde; NP_s, nanoparticles; SEM, standard error of the mean; SOD, superoxide dismutase.

Figure 11 Levels of NO in rats’ gastric tissues following (A) concurrent induction and treatment regimen and (B) post-treatment regimen.

Notes: Data are mean ± SEM (n=6). *p<0.05 and ***p<0.001 vs normal control group. ^###p<0.001 vs positive control group. ^$p<0.05 APO-loaded COS-NP_s (F4) treated group vs free APO treated group.

Abbreviations: APO, apocynin; COS, chitosan oligosaccharide; NO, nitric oxide; NP_s, nanoparticles; SEM, standard error of the mean.