الفهرس 
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Abstract
The recent development in nanotechnology has led to manipulate
the size of biomaterials in nanoscale which is known as bionanotechnology. This development enhances the intrinsic properties of
biomaterials resulting due to the increase of their surface area to volume
ratio by decreasing their size below 100 nm.
It has been recently announced that β-glucan with globular small
particles size has higher disease resistant capacity due to its induced
strong immune and antitumor activity. Herein, this work was designed
to extract the nano β-glucan (NGs) by a novel direct modified approach
from mushrooms using acid base procedure. Lentinula edodes (Shiitake)
and Pleurotus ostreatus (oyster) mushrooms were used for production
of NGs-s and NGs-o, respectively, then characterized by different
analytical tools.
Usually, the isolation of nano beta-1,3/1,6-glucans is a multistage
process which includes many steps of preparation of beta-glucan first
then converting it to nano scale by different methods and steps. But our
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current work studied a novel and modified method that can isolate and
extract of beta-glucan nanoparticles in one step only that make of
simplifying and increasing the efficiency of the procedure for isolating
nano-beta glucans
Two mushrooms, namely, Lentinula edodes (Shiitake) and
Pleurotus ostreatus (oyster) mushrooms were used for the production of
NGs-s and NGs-o, respectively, through boiling with 5% NaOH for 24
h followed by neutralization with HCl and washed several times prior
drying. The yield % of the extracted NGs-s was higher than it from
oyster mushroom NGs.
The extracted NGs were then Comprehensive physicochemical
characterized and performed using LC-MS, H1
-NMR, FTIR, UVvisible spectroscopy, particle size and zeta potential, SEM and TEM
Their chemical structure was investigated against standard βglucan. The results emphasized the close and identical structure of both
NGs to the standard β-glucans, as seen from LC mass, H1NMR and
FTIR, with high purity. Furthermore, the UV-visible light absorption
pattern was kinetically monitored in the range of 260-300 nm
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In addition, morphological characteristics of NGs were obtained
using SEM and TEM. SEM revealed the porosity of the surface with
homogeneous distribution, particularly in the case of NGs-s. TEM also
emphasized the formation of NGs of size ranging from 10-25 nm and
40-50nm for NGs-s and NGs-o, respectively, with needle edge shapes
as in NGs-s. However, NGs-o showed loosely aggregated irregular
shapes which may be attributed to their low zeta potential
Incurrentresults, NGs-swas exploited tosynthesizeAuNPswithout
any additional reducing, stabilizing and capping agents, AuNPs were
synthesized by reducing of HAuCl4 with 0.05% (w/v) NGs-ssolution by
usingmicrowave technique in different exposure radiation time.
The optimized conditions were determined by UV–Vis absorption
spectroscopy at λ max =530 nm at 60 secs and 0.4 mM of HAuCl4.
Optimization of AuNPs preparation followed by different
characterizations to indicate the morphological, chemical and crystalline
structure of AuNPs:
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1. Particle size indicated for AuNPs was the main range of 25.2 nm.
AuNPs had a promising zeta potential value of -31.8 mV formed from
NGs solution, the zeta potential value with high negative charge referred
to good quality and stability
2. FTIR analysis showed the peaks of AuNPs and reduction process
of NGs that confirmed the purity of AuNPs preparation.
3. Crystallinity of AuNPs was also cleared with specific peaks The
XRD pattern of the AuNPs exhibited four characteristic peaks at (111),
(200), (220), and (311) planes of gold, respectively. These results
confirmed that natural biopolymer NGs-s had potential to be used as a
reducing and stabilizing agent for AuNPs
4. TEM disclosed that, the formed AuNPs were spherical, uniform
in size and shape with size range of 10-20 nm.
5. AFM of the prepared AuNPs, by virtue of NGs were formed in
well dispersed, homogenous and spherical shape with height 108 nm.
The synthesized AuNPs were tested to show bactericidal and
fungicidal effects against certain microorganisms. Experiments
confirmed that the AuNPs had good antibacterial effects on Gram-
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negative, Gram-positive bacteria and certain fungal isolates. Compared
with both NGs and commercial antibiotics.
AuNPs had highest antibacterial activity with inhibition diameter
36 mm for B. subtilis followed by S. enterica with 35 mm. then B.
cereus, E coli, St. aureus and P. aeruginosa showed zone of inhibition
with diameter equal 28,25,22,18 mm respectively. As well as, showed
high inhibitory effect on the growth of most tested fungi. C. glabrata
was found to be the most susceptible fungi to AuNPs the diameter of
inhibition zone was 21 mm followed by C. albicans and P. expansum
showing 20, 14 mm inhibition zone of fungal growth, respectively.
Cancer is one of the most common causes of deaths worldwide.
Antitumor activity of NGs-s, NGs-o and AuNPs were tested by using
MTT assay against two carcinoma cell lines (Colon HCT116 and Breast
MCF7) which compared to cytotoxicity on normal cells.
NGs and AuNPs showed a significant toxicity against HCT-116
cells, the IC50 was 200 ,125 and 85.3 µg/ml for NGs-o, NGs-s and
AuNPs, respectively. Whereas, in the case of anti-breast cancer (MCF7)
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activity with IC50 at170, 156.6 and 15 µg/ml for NGs-o, NGs-s and
AuNPs, respectively.
In vitro cytotoxicity assays affirmed the potential use of AuNPs as
antitumor materials against carcinoma breast and colon cells at low
doses compared to human normal Vero cell
These findings confirm that despite the safe high dose of NGs to
human normal Vero cells up to 500 µg/L, it showed highly anticarcinogenicity towards breast MCF7 and colon HCT-116 cells at low
and moderate doses as observed fromNGs-s (113 – 125 µg/L) and NGso (126 – 196 µg/L). In addition, NGs-s exhibited higher antitumor
activity than that observed from NGs-o. The treatment with AuNPs and
NGs did not induce cytotoxic effect causing significant damage or death
of the treated normal cells. NGs-s showed a high potential antitumor
activity compared with NGs-o.
NGs-s extracted from shiitake mushroom were used in the preparation
of hydrogels with carrageenan biopolymer, by gelation of NGs by
gamma radiation 10 kg/y (to provide sufficient gel strength) and adding
to carrageenan solution in the presence of CaCl2 as a crosslinker.
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Natural NGs/Cr hydrogels were successfully fabricated using
physical crosslinking approach, without using any toxic solvent during
the preparation method to exploit the resultant hydrogel in different
medical applications.
.
Different concentrations of NGs and CaCl2 (crosslinker) were used
as factors affecting the preparation and optimization of NGs/Cr
hydrogels.
The optimized conditions were detected by % of ESR in dist.H2O and
were summarized by increasing the concentration of NGs up to 2 g with
ratio 1:2 for carrageenan and NGs respectively, the hydrogel reached the
highest ESR of 40% after 24h at room temperature compared with the
18 % of carrageenan. In addition, based on the crosslinker concentration,
the ESR of NGs/Cr hydrogels follows the order: 1.5 % > 2.0% > 1% >
0.5%.
SEM and BET experiments also indicated the optimization by
crosslinker concentration. At concentration of 1.5% CaCl2, NGs/Cr
hydrogel showed homogenous and enjoyed well-proportioned network
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structure with highly connected irregular pores by SEM having the size
ranging from 100 – 190 µ. While BET analysis observed the specific
surface area and total pore volume of the NGs/Cr hydrogel with 1.5 %
CaCl 2 were found 5.674 m 2 /g and 4.984 cc/g, respectively.
Resulted NGs/Cr hydrogel was characterized by FTIR spectroscopy
that showed the peaks of NGs with its skeleton structure and FTIR
spectrum of қ-carrageenan shows the characteristic bands at 1214 cm-1
,1157 cm-1
and 928, corresponding to the sulfate of carrageenan C–O–
SO3, C-o bridge, and sulfate esters O=S=O groups linked to the 3,6-
glucan ring.
The prepared optimized NGs/Cr hydrogel was tested for responsivity
to different pH, the swelling ratio increased by the use of alkaline
medium than acidic. The ESR ratio followed the order respect to pH
11>9>7>5>2, which the ESR % attained 55, 45, 41, 25 and 18 %
respectively.
Furthermore, the prepared pH responsive hydrogel was used in vitro
and in vivo medicinal applications. We evaluated AuNPs as a model of
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in vitro drug release from the NGs/Cr responsive. As well as used this
hydrogel in vivo as dressing in skin wound healing of rats.
AuNPs as a model of drug release from the NGs/Cr pH responsive
hydrogel using a UV-Vis spectrophotometer to draw the standard curve
at 530 nm. NG/Cr hydrogel was dipped into an aqueous AuNPs solution
for 24 h. The drug release was determined by placing it in two different
pH (2 and 10), the drug was released from the NGs/Cr hydrogel,
reaching 80% at approximately 90 min in acidic pH 2 faster than the
basic medium.
AuNPs loaded on the NGs/Cr hydrogel and showed well-organized
three-dimensional network and strong cross-linking structure onto the
pores of NGs/Cr hydrogel when examined under SEM.
On the other hand, Animal experiments were used to evaluate the
efficiency of using NGs/Cr hydrogel and AuNPs loaded on NGs/Cr
dressing in promoting wound healing comparing with negative and
positive control. The initial area diameter of wound was contracted from
1.5 cm to 0.2 ,0.5,0.6,0.9 cm for AuNPs loaded hydrogel, NG/Cr, Iodine
and negative control dressing respectively.
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After 10 days the wound size reduction % were 86, 67, 65.4%, for
groups treated with the AuNPs loaded, NGs/Cr, and iodine solution
dressings, respectively compared with 35.5 % for the control group
(dressing without treatment).In addition, histological analysis was
investigated after 10 days of wounding and the results were recorded by
photomicrographs of skin sections.
Skin in this case of negative dressing control showed area of gapped
wound with scanty granulation tissue composed of excess fibroblasts
and few thick collagen fibers, and covered by ulcerated and necrotic
epidermis. In case of positive control (iodine dressing), skin showed area
of healthy skin and area of ulcerated wound and necrotic crust, with
underlying scar composed of fibroblasts more in deep dermis and excess
thick collagen fibers, small areas of hemorrhage, and markedly
edematous muscles.
In addition, Skin sections which healed with NGs/Cr dressing, the
skin showed area of healthy skin and area of scar tissue composed of
excess fibroblasts and thick collagen fibers, scattered inflammatory
infiltrate and small areas of hemorrhage, and covered by intact
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epithelialized and keratinized epidermis.
On the other hand, skin showed area of healthy skin and area of
completely healed wound with scar composed of excess collagen and
few fibroblasts, covered by intact epithelialized and keratinized
epidermis when treated by AuNPs loaded on NGs/Cr hydrogel dressing.
The hydrogel containing AuNPs promoted best wound healing
activity by stimulating re-epithelialization, cell proliferation and
collagen biosynthesis with complete healing within 10 days.
This work promotes the use of natural polysaccharide for the
biosynthesis of nanomaterials, extending their potential applications in
nanomaterial fields. Finally, the results of the study demonstrated the
NGs/AuNPs hydrogel as a biomaterial for use in the wound dressing
sector whose market is growing and promising.