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. 2021 Jan 26;11(1):2216.
doi: 10.1038/s41598-021-81183-x.

Formulation, optimization and characterization of allantoin-loaded chitosan nanoparticles to alleviate ethanol-induced gastric ulcer: in-vitro and in-vivo studies

Reham Mokhtar Aman  1 , Randa A Zaghloul  2 , Marwa S El-Dahhan  3
Affiliations

Formulation, optimization and characterization of allantoin-loaded chitosan nanoparticles to alleviate ethanol-induced gastric ulcer: in-vitro and in-vivo studies

Reham Mokhtar Aman et al. Sci Rep. .

Abstract

Allantoin (ALL) is a phytochemical possessing an impressive array of biological activities. Nonetheless, developing a nanostructured delivery system targeted to augment the gastric antiulcerogenic activity of ALL has not been so far investigated. Consequently, in this survey, ALL-loaded chitosan/sodium tripolyphosphate nanoparticles (ALL-loaded CS/STPP NPs) were prepared by ionotropic gelation technique and thoroughly characterized. A full 24 factorial design was adopted using four independently controlled parameters (ICPs). Comprehensive characterization, in vitro evaluations as well as antiulcerogenic activity study against ethanol-induced gastric ulcer in rats of the optimized NPs formula were conducted. The optimized NPs formula, (CS (1.5% w/v), STPP (0.3% w/v), CS:STPP volume ratio (5:1), ALL amount (13 mg)), was the most convenient one with drug content of 6.26 mg, drug entrapment efficiency % of 48.12%, particle size of 508.3 nm, polydispersity index 0.29 and ζ-potential of + 35.70 mV. It displayed a sustained in vitro release profile and mucoadhesive strength of 45.55%. ALL-loaded CS/STPP NPs (F-9) provoked remarkable antiulcerogenic activity against ethanol-induced gastric ulceration in rats, which was accentuated by histopathological, immunohistochemical (IHC) and biochemical studies. In conclusion, the prepared ALL-loaded CS/STPP NPs could be presented to the phytomedicine field as an auspicious oral delivery system for gastric ulceration management.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Chemical structure of ALL.
Figure 2
Figure 2
Three dimensional surface plots representing the effect of the interaction between the concentrations of both CS (X1) and STPP (X2) on Q and DEE % (A and B) and (C and D) at the maximum and minimum levels of both CS:STPP volume ratio (X3) and ALL amount (X4), respectively, using Design-Expert version 12 (/https://www.statease.com/software/design-expert).
Figure 3
Figure 3
Contour plots representing the effect of the interaction between the concentrations of both CS (X1) and STPP (X2) on Q and DEE % (A and B) and (C and D) at the maximum and minimum levels of both CS:STPP volume ratio (X3) and ALL amount (X4), respectively, using Design-Expert version 12 (/https://www.statease.com/software/design-expert).
Figure 4
Figure 4
Optimization graph. (A) Contour and (B) three dimensional plots with desirability score using Design-Expert version 12 (/https://www.statease.com/software/design-expert).
Figure 5
Figure 5
(A) TEM image of ALL-loaded CS/STPP NPs (F-9). The image was captured using Gatan Microscopy Suite Software, version 2.11.1404.0, (http://www.gatan.com/products/tem-analysis/gatan-microscopy-suite-software). (B) size distribution curve of ALL-loaded CS/STPP NPs (F-9).
Figure 6
Figure 6
Solid characterization. (A) FT-IR spectra, (B) DSC thermograms, and (C) PXRD patterns of pure ALL (i), CS (ii), STPP (iii), physical mixture of ALL, CS and STPP (iv), plain CS/STPP NPs (v) and ALL-loaded CS/STPP NPs (F-9) (vi). DSC thermograms were fitted with a Shimadzu TA-60 series (https://www.ssi.shimadzu.com/products/thermal-analysis/labsolutions-ta-software.html).
Figure 7
Figure 7
The in vitro release pattern of ALL from ALL-loaded CS/STPP NPs (F-9) in comparison with its diffusion from aqueous solution at three different pH values (A) pH 1.2, (B) pH 6.8 and (C) pH 7.4. Each point represents the mean ± SD (n = 3) and the graph was plotted using GraphPad Prism 5.00 (http://www.graphpad.com).
Figure 8
Figure 8
ζ-potential of mucin solution (0.5 mg/mL), CS solution (1.5% (w/v)), mucin solution + CS solution and ALL-loaded CS/STPP NPs (F-9) before and after incubation with mucin at 37 °C for 1 h. Results represents the mean ± SD (n = 3) and the graph was plotted using Microsoft Excel (2010), version 14.0.4734.1000, (www. Microsoft Office 2010.com).
Figure 9
Figure 9
(A) Macroscopically investigation of mucosal lesions in glandular part of the stomachs. (a) N group, (b) ulcer group, (c) ome group, (d) plain group, (e) ALL group, (f) NanoAL group, and (g) NanoAH group. (B) Microscopical examination of glandular part of stomach sections stained by (H&E) (X: 100). (a) N group, (b) ulcer group, (c) ome group, (d) plain group, (e) ALL group, (f) NanoAL group, and (g) NanoAH group. Green arrow points to visual changes than the normal mucosa present in N group, thick black arrow points to mucosal ulceration, thin black arrow points to erosion, yellow arrow points to necrosis, asterisk points to edema, red arrow points to dilated blood vessel, and blue arrow points to leukocytic cells infiltration.
Figure 10
Figure 10
Histochemical staining of stomach sections of rats from the different treated groups with alcian blue stain to detect gastric mucus (X: 100). (a) N group, (b) ulcer group, (c) ome group, (d) plain group, (e) ALL group, (f) NanoAL group, and (g) NanoAH group.
Figure 11
Figure 11
Oxidative status of (A) gastric MDA (nmol/g tissue) and (B) gastric GSH (mg/g tissue) in rats’ gastric tissues. (C) Immunostaining of Nrf-2 in rats’ gastric tissues (X: 100). (a) N group, (b) ulcer group, (c) ome group, (d) plain group, (e) ALL group, (f) NanoAL group, and (g) NanoAH group. X: 100. Data are expressed as mean ± SEM, #p < 0.001 compared to N group, *p < 0.05 compared to ulcer group, ***p < 0.001 compared to ulcer group, @p < 0.01 compared to ome group, $p < 0.001 compared to ALL group, Ap < 0.05 compared to plain group using GraphPad Prism 5.00 (http://www.graphpad.com). Positive stainings are shown by yellow arrow.
Figure 12
Figure 12
Levels of pro-inflammatory mediators; (A) serum level of IL-6 (pg/ml) and (B) gastric level of NO (μM of nitrite/g tissue). (C) Immunostaining of TNF-α in rats’ gastric tissues (X: 100). (a) N group, (b) ulcer group, (c) ome group, (d) plain group, (e) ALL group, (f) NanoAL group, and (g) NanoAH group. X: 100. Data are expressed as mean ± SEM, #p < 0.001 compared to N group, **p < 0.01 compared to ulcer group ***p < 0.001 compared to ulcer group, @p < 0.01 compared to Ome group, $p < 0.001 compared to ALL group, Ap < 0.01 compared to plain group, %p < 0.001 compared to NanoAL group using GraphPad Prism 5.00 (http://www.graphpad.com). Positive stainings are shown by yellow arrow.
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