International Journal of Nanomedicine Dovepress...Correspondence: Elham Abdelmonem Mohamed Department of Pharmaceutics, Faculty of Pharmacy, Mansoura University, Gomhoreyah St., Mansoura
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OR I G I N A L R E S E A R C H
In vitro–in vivo evaluation of chitosan-PLGA
nanoparticles for potentiated gastric retention and
anti-ulcer activity of diosminThis article was published in the following Dove Press journal:
International Journal of Nanomedicine
Walaa Ebrahim Abd El Hady
Elham Abdelmonem Mohamed
Osama Abd El-Aazeem Soliman
Hassan Mohamed EL-Sabbagh
Department of Pharmaceutics, Faculty of
Pharmacy, Mansoura University,
Mansoura 35516, Egypt
Background: Diosmin showed poor water solubility and low bioavailability. Poly(d,l-
lactide-co-glycolide) (PLGA) nanoparticles were successfully used to improve the drugs
solubility and bioavailability. Coating of PLGA nanoparticles with chitosan can ameliorate
their gastric retention and cellular uptake.
Methodology: PLGA nanoparticles of diosmin were prepared using different drug and poly-
mer amounts. Nanoparticles were selected based on entrapment efficiency% (EE%) and particle
size measurements to be coated with chitosan. The selected nanoparticles either uncoated or
coated were evaluated regarding morphology, ζ-potential, solid-state characterization, in vitro
release, storage stability, and mucoadhesion. The anti-ulcer activity (AA) against ethanol-
induced ulcer in rats was assessed through macroscopical evaluation, histopathological exam-
ination, immunohistochemical localization of nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-κB) and transmission electron microscopic examination of gastric tissues
compared to free diosmin (100 mg/kg) and positive control.
Results: Based on EE% and particle size measurements, the selected nanoparticles, either
uncoated or coated with 0.1% w/v chitosan, were based on 1:15 drug-PLGAweight ratio and 20
mg diosmin employing methylene chloride as an organic phase. Examination by scanning electron
microscopy (SEM) and transmission electron microscopy (TEM) revealed nanoscopic spherical
particles. Drug encapsulationwithin the selected nanoparticleswas suggested by Fourier transform-
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PLGA (F14) using methylene chloride was further examined
compared to the corresponding uncoated PLGA nanoparticles
(F5) and the free drug.
Particle size analysis
Average diameter of ≤594.4±13.40 nm and PDI values
≤0.53±0.01, suggesting narrow size distribution, were
recorded. The effects of drug:polymer weight ratio and
the organic phase nature on the particle size of the
uncoated nanoparticles were investigated (Table 1).
A significant (P<0.05) lowering in the average size was
observed on the increase in the polymer concentration. Higher
polymer concentration may have resulted in increased viscos-
ity of the organic phase which might have counteracted the
diffusion and Ostwald ripening, so smaller particles were
obtained.42 Ostwald ripening does not depend on particles
coalescence but on their diffusive transport through the disper-
sion medium.42
Ethyl acetate is partially water-miscible, while methylene
chloride is immiscible withwater.43 Some authors claimed that
smaller particles are obtained with less water-miscible organic
solvents such as methylene chloride that was at the top of the
list of such solvents.25 This may explain the smaller average
Table 1 Characterization of the uncoated PLGA nanoparticles
Formula code Drug:polymer (mg) Size (nm) PDI EE%
F1 10:100 594.40±13.40 0.21±0.02 31.40±3.60
F2 10:150 552.90±18.40 0.16±0.08 50.90±4.80
F3 10:200 165.80±3.30 0.37±0.10 48.50±3.90
F4 20:200 303.10±49.80 0.19±0.03 70.90±5.30
F5 20:300 155.90±3.10 0.20±0.15 75.30±2.60
F6 20:400 133.50±0.20 0.28±0.10 58.60±1.90
F7 30:300 259.10±5.20 0.53±0.01 66.95±3.11
F8 10:100 482.50±2.50 0.33±0.00 48.40±4.30
F9 10:150 432.00±8.71 0.33±0.01 49.60±3.20
F10 10:200 404.30±7.50 0.22±0.00 37.00±2.16
F11 20:200 545.30±15.60 0.20±0.05 49.40±1.40
F12 20:300 450.40±5.30 0.31±0.29 55.60±0.30
F13 20:400 340.20±17.20 0.17±0.14 40.10±2.20
Notes: Data are expressed as mean±SD (n=3); F1–F7, uncoated PLGA nanoparticles prepared using methylene chloride; F8–F13, uncoated PLGA nanoparticles prepared
Notes: Data are expressed as mean±SD (n=3). F14, F15, and F16 were the selected PLGA nanoparticles (F5) coated with 0.10%, 0.15%, and 0.30% w/v chitosan, respectively.
Notes: Data are expressed as mean±SD (n=3). *P<0.05 vs initially determined size at the beginning of the storage period or drug retention % at the first month, ***P<0.001vs initially determined size at the beginning of the storage period.
pretreatment groups (III, IV, and V) showed significantly
(P<0.05) lower mean ulcers number when compared to posi-
tive control rats (II). Hence, highly diminished ulcer inci-
dence and Paul’s index values were estimated for these
groups relative to positive control. In comparison with rats
orally pretreated with free diosmin, a greater AA was
obtained on oral pretreatment with uncoated PLGA nanopar-
ticles. Coating of PLGA nanoparticles with chitosan resulted
in highly potentiated AA of diosmin.
More prolonged residence time of nanoparticles in
ulcerative tissues can be expected due to the greater accu-
mulation and the facilitated uptake by immune cells, such
as macrophages.55
The greater AA of the coated nanoparticles than the
uncoated ones can be attributed to the adherence of the
positively charged chitosan-coated nanoparticles to the
negatively charged cellular membranes56 as well as their
escape from the acidic solution of endosomes–
lysosomes,57 thus inducing their intracellular uptake into
the cytoplasm. The interaction with the cellular membrane
results in a structural reorganization of junction proteins
that is reversed when the contact with chitosan is
terminated.58 Moreover, mucoadhesion enhanced through
the electrical interaction of positively charged chitosan-
coated nanoparticles with the negatively charged mucin
can still account for the potentiated AA.54 In spite of
these biological interactions due to the positively charged
surface of chitosan-coated nanoparticles, chitosan cyto-
toxicity was negated as clarified by cell viability close to
100% after contacting with chitosan or systems based on
it.59 In addition, no significant toxicity was reported fol-
lowing repeated oral administration of microparticles or
nanoparticles incorporating chitosan at a dose range of
100–125 mg/kg as revealed by the absence of abnormal-
ities in hepatic and renal functions or pathological changes
in liver, kidney, and intestinal segments.60,61
Figure 7 Scanning electron microscopy of stomach and duodenum of the different groups orally treated with diosmin (100 mg/kg) or an equivalent dose of either uncoated
or chitosan-coated nanoparticles.
Notes: (A) Two-hour post-dosing and (B) eight-hour post-dosing. (I) and (II) stomach and duodenum of rats treated with diosmin, respectively, (III) and (IV) stomach and
duodenum of rats that administered uncoated PLGA nanoparticles, respectively, (V) and (VI) stomach and duodenum of rats given chitosan-coated nanoparticles,
respectively. Arrows point to the attached nanoparticles.
Figure 9 displays microphotographs of histopathological
examination (HE, 100×) of non-glandular (A) and gland-
ular (B) gastric tissues of the different groups. Normal
control (I) displayed an intact architecture of non-gland-
ular and glandular gastric wall (I). Administration of
ethanol to rats of positive control (II) triggered a severe
gastric injury with high scores of microscopic damage
including extensive congestion and edema in non-
glandular portion, mucosal ulceration and submucosal
inflammation in glandular portion. Submucosal conges-
tion and edema in non-glandular and glandular portions
besides submucosal inflammation in glandular portion
were moderate in rats pretreated with free diosmin (III),
while mild in rats that administered uncoated nanoparti-
cles (IV). Non-glandular and glandular gastric walls
retained their normal histological pictures in rats that
received chitosan-coated nanoparticles (V).
Figure 8 Gross appearance of freshly excised stomachs.
Notes: (A) Normal control with normal gastric mucosa. (B) Positive control showing glandular mucosal ulcerations (red arrowheads) as well as extensive glandular and non-
glandular mucosal congestion and edema. (C) Rats pretreated with diosmin (100 mg/kg) displaying moderate glandular mucosal ulcerations (red arrowheads), glandular and non-
glandular mucosal congestion and edema were moderate. (D) Rats pretreated with an equivalent dose of uncoated PLGA nanoparticles exhibiting mild congestion and edema
appeared in glandular and non-glandular gastric mucosa. (E) Apparently normal gastric mucosa of rats pretreated with an equivalent dose of chitosan-coated nanoparticles.
Figure 9 Histological examination (HE, 100x) of gastric tissues.
Notes: (A) Non-glandular and (B) glandular gastric mucosa. (I) Normal control displaying an intact architecture of non-glandular and glandular gastric wall. (II) Positive
control showing extensive congestion (red arrows) and edema (black asterisk) in non-glandular portion, mucosal ulceration (thick black arrow), and submucosal
inflammation (thin black arrow) in glandular portion. (III)Rrats pretreated with diosmin (100 mg/kg) displaying moderate submucosal congestion (red arrows) and edema
(black asterisk) in non-glandular and glandular portions besides submucosal inflammation (thin black arrow) in glandular portion. (IV) Rats pretreated with uncoated PLGA
nanoparticles exhibiting mild submucosal congestion (red arrows) and edema (black asterisk) in non-glandular and glandular portions besides very mild submucosal
inflammatory cells infiltration in glandular portion (thin black arrow). (V) Non-glandular and glandular gastric walls retained their normal histological pictures in rats
edema, congestion, and inflammation in glandular por-
tions relative to positive control. In comparison with
free diosmin, there was a significant (P<0.05) reduction
in these pathological changes only following oral
administration of coated nanoparticles.
Significantly (P<0.05) elevated edema in non-glandu-
lar portions of gastric tissues was observed in positive
control and rats orally pretreated with either free diosmin
or uncoated nanoparticles than normal rats (Figure 10E).
Rats administered coated nanoparticles encountered a sig-
nificantly (P<0.05) lower edema in non-glandular portions
than positive control, free diosmin, and uncoated nanopar-
ticles. Regarding congestion in non-glandular gastric tis-
sues, there was a significant (P<0.05) increase in positive
control and rats pretreated with free diosmin relative to
normal control (Figure 10F). Significantly (P<0.05) dimin-
ished congestion was obtained in the three pretreatment
25
A B C
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rticles
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les
* *
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*# *
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@
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Figure 10 Statistical analysis of microscopic histopathological scores in gastric mucosa.
Notes: (A–D) Glandular and (E and F) non-glandular mucosa. Data are mean±SD, n=6; statistical differences at P<0.05 considered significant; *vs normal control group; #vs
positive control group; $vs diosmin (100 mg/kg) pretreated group; @vs group pretreated with an equivalent dose of uncoated PLGA nanoparticles.
groups when compared to positive control. In contrast to
uncoated nanoparticles, oral administration of coated
nanoparticles resulted in a significantly (P<0.05) lower
congestion in non-glandular gastric tissue than rats given
free diosmin. There was insignificant difference between
rats orally pretreated with uncoated and coated nanoparti-
cles regarding congestion scores in non-glandular portion.
There was insignificant difference between normal
control and rats received coated nanoparticles regarding
severity of all pathological changes describing ulceration
in both glandular and non-glandular gastric tissues. Thus,
it can be said that coating of PLGA nanoparticles with
chitosan significantly potentiated the cytoprotective activ-
ity of diosmin against ethanol-induced ulcer in rats. These
results may be due to the increased interaction with the
negatively charged cellular membranes and the improved
intracellular uptake into the cytoplasm,56 as well as the
enhanced escape from the acidic solution of endosomes–
lysosomes into the cytoplasm.57 Moreover, the increased
mucoadhesion of positively charged chitosan-coated nano-
particles with the negatively charged mucin and the result-
ing gastric retention (Figure 7) can still explain the
superiority of coated nanoparticles.54
Immunohistochemical loclization of NF-κBFigure 11 illustrates immunohistochemical evaluation of
NF-κB expression in non-glandular (A) and glandular (B)
gastric tissues. Mild positive expression was recognized in
both tissues in normal control group (I). Strong immunor-
eactivity was observed in both mucosae particularly
around area of mucosal damage and stained inflammatory
cells infiltrating submucosa of glandular portion in posi-
tive control (II). Oral pretreatment with free diosmin sus-
pension in 1%w/v CMC (100 mg/kg, III) or an equivalent
Figure 11 Microscopic pictures of rats stomach immunostained against NF-κB.Notes: (A) Non-glandular and (B) glandular gastric mucosa. (I) Normal control showing mild positive expression in glandular and non-glandular gastric mucosa. (II) Positive control
rats displaying strong positive expression in glandular and non-glandular gastric mucosa particularly around area of mucosal damage (thick black arrow) and stained inflammatory
cells infiltrating submucosa of glandular portion. (III) Rats pretreated with diosmin (100 mg/kg) or (IV) an equivalent dose of uncoated PLGA nanoparticles exhibiting mild positive
expression in non-glandular mucosa, as well as moderate positive expression in gastric mucosa and the inflammatory cells infiltrating submucosa of glandular portion. (V) Rats that
received chitosan-coated nanoparticles showing mild positive expression in glandular and non-glandular gastric mucosa. Thin black arrows point to positive signal in mucosa and thin
yellow arrows point to positively stained leukocytes infiltrating submucosa. IHC counterstained with Mayer’s hematoxylin (100×), insert (S, 200×).
Abbreviations: NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PLGA, poly (d,l-lactide-co-glycolide); IHC, immunohistochemistry.
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DovePressInternational Journal of Nanomedicine 2019:147208
Following oral treatment with free diosmin suspension,
there was a slight amelioration in the mucosal cells, yet
wide junctions between cells were observed and some
cytoplasmic organelles were still deleterious, including
swollen mitochondria, pyknotic nucleus, and irregular
microvilli (Figure 13C). Regarding rats that received
uncoated PLGA nanoparticles, a slightly ameliorated
Normal
contr
olPo
sitive
contr
olFr
ee di
osmin
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nopa
rticles
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nano
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les
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olPo
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osmin
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***### ##
###$$$
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2
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)
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ositi
ve s
tain
ing
(0–3
)
A B
Figure 12 Statistical analysis of IHC scores of NF-κB in gastric mucosa.
Notes: (A) Glandular and (B) non-glandular mucosa. Data are mean±SD, n=6;
*P<0.05 and ***P<0.001 vs normal control; ##P<0.01 and ###P<0.001 vs positive control; $$$P<0.001 vs diosmin (100 mg/kg) pretreated group; @P<0.05 and @@@P<0.001 vs
group pretreated with an equivalent dose of uncoated PLGA nanoparticles.
Abbreviations: IHC, immunohistochemistry; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PLGA, poly(d,l-lactide-co-glycolide).
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observed in the mucosal surface (Figure 13D). Oral pre-
treatment with chitosan-coated nanoparticles of diosmin
resulted in obvious amelioration in the mucosal cells,
disappearance of vacuoles, intact cell membrane with
tight junctions and regularly arranged microvilli with
high density as well as normal mitochondria, endoplas-
mic reticulum, and nucleus (Figure 13E).
ConclusionThe selected uncoated nanoparticles consisted of 1:15
drug-PLGA weight ratio using 20 mg diosmin and methy-
lene chloride as an organic phase because they showed the
highest EE% (75.30±2.60%) and particle size <200 nm
(155.90±3.10 nm). Coating of these nanoparticles with
chitosan (0.10%, 0.15%, and 0.30% w/v) significantly
enlarged the size on the increase of chitosan concentration
but EE% did not significantly differ; thus, those coated
Figure 13 TEM examination of the gastric mucosa ultrastructure.
Notes: (A) Normal control rats showing well-arranged microvilli in neat rows with no loss, normal nucleus, high density of mitochondria, regular pattern of rough endoplasmic
reticulum, and dispersed gastric secretion. (B) Positive control rats displaying swollen mitochondria, deleted rough endoplasmic reticulum, abnormal nucleus, cytoplasmic vacuoles,
cells with dilated reticulum, complete loss of microvilli, and several non-homogenated cytoplasmic inclusions. (C) Rats pretreated with free diosmin (100 mg/kg) exhibited a slight
amelioration in the mucosal cells, wide junctions between cells and some deleterious cytoplasmic organelles including swollen mitochondria, pyknotic nucleus, and irregular
microvilli. (D) Rats that received uncoated PLGA nanoparticles showing a slightly ameliorated mitochondria, normal rough endoplasmic reticulum, intact cell membrane, and
regular microvilli. (E) Rats pretreated with chitosan-coated nanoparticles of diosmin showing obvious amelioration in the mucosal cells, disappearance of vacuoles, intact cell
membrane with tight junctions and regularly arranged microvilli with high density as well as normal mitochondria, endoplasmic reticulum, and nucleus.
Abbreviation: TEM, transmission electron microscopy.
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