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Research Article Acetylated Hyaluronic Acid: Enhanced Bioavailability and Biological Studies


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Hyaluronic acid (HA), a macropolysaccharidic component of the extracellular matrix, is common to most species and it is found in many sites of the human body, including skin and soft tissue. Not only does HA play a variety of roles in physiologic and in pathologic events, but it also has been extensively employed in cosmetic and skin-care products as drug delivery agent or for several biomedical applications. The most important limitations of HA are due to its short half-life and quick degradation in vivo and its consequently poor bioavailability. In the aim to overcome these difficulties, HA is generally subjected to several chemical changes. In this paper we obtained an acetylated form of HA with increased bioavailability with respect to the HA free form. Furthermore, an improved radical scavenging and anti-inflammatory activity has been evidenced, respectively, on ABTS radical cation and murine monocyte/macrophage cell lines (J774.A1).
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Research Article
Acetylated Hyaluronic Acid: Enhanced Bioavailability and
Biological Studies
Carmela Saturnino,1Maria Stefania Sinicropi,2Ortensia Ilaria Parisi,2,3
Domenico Iacopetta,2Ada Popolo,1Stefania Marzocco,1Giuseppina Autore,1
Anna Caruso,2,3 Anna Rita Cappello,2Pasquale Longo,4and Francesco Puoci2
2Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende, Italy
3Department of Computer Engineering, Modeling, Electronics and Systems, University of Calabria, 87036 Rende, Italy
4Department of Sciences, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Italy
Correspondence should be addressed to Carmela Saturnino; and Maria Stefania Sinicropi;
Received  February ; Accepted  June ; Published  July 
Academic Editor: Michela Ori
Copyright ©  Carmela Saturnino et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Hyaluronic acid (HA), a macropolysaccharidic component of the extracellular matrix, is common to most species and it is found
in many sites of the human body, including skin and so tissue. Not only does HA play a variety of roles in physiologic and in
pathologic events, but it also has been extensively employed in cosmetic and skin-care products as drug delivery agent or for several
biomedical applications. e most important limitations of HA are due to its short half-life and quick degradation in vivo and its
consequently poor bioavailability. In the aim to overcome these diculties, HA is generally subjected to several chemical changes.
In this paper we obtained an acetylated form of HA with increased bioavailability with respect to the HA free form. Furthermore, an
improved radical scavenging and anti-inammatory activity has been evidenced, respectively, on ABTS radical cation and murine
monocyte/macrophage cell lines (J.A).
1. Introduction
Hyaluronic acid (HA), the main component of the gly-
cosaminoglycans, is a linear biodegradable polymer with
high molecular weight consisting of disaccharide units of N-
acetylglucosamine and D-glucuronic acid, connected alter-
nately by - and - glycosidic b onds. HA is naturally
present in almost all body uids and tissues such as the
synovial uid, eye vitreous humor, connective, epithelial,
and neural tissues and plays important biological functions
in wound healing regulating cell adhesion, motility, dif-
ferentiation, and proliferation. HA assists the early phases
of the inammatory process, improving cell inltration
and, aerwards, the free radical scavenging and antioxidant
characteristics of HA allow suppressing the inammatory
response during the healing process []. is dual role played
during the inammation phases depends on HA molecular
mass indeed; in its native state, it generally exists as a
high-molecular-mass polymer whereas, under inammation,
HA is more polydisperse, with a preponderance of lower-
molecular-mass forms []. Besides, several studies shed light
on other key roles played by HA in inuencing cellular pro-
cesses, for instance, morphogenesis, cancer progression, and
metastasis []. Indeed, many HA fragments have been found
in a wide range of carcinomas, lymphomas, melanocytic,
and neuronal tumors; these fragments exhibit properties, not
normally found in the native HA polymer, whose eects
depend on the molecular size as, for instance, angiogenics
or growth suppressing. e altered HA metabolism and the
amount of itself in the tumor stroma or in the neoplastic cell
compartment are strictly associated with invasion and local
or distant metastases, impacting on the overall outcome [].
HA and its derivatives have been also employed as anticancer
Hindawi Publishing Corporation
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Volume 2014, Article ID 921549, 7 pages
BioMed Research International
F : Functional groups of HA subjected to chemical modica-
drug carriers because of their ability to be recognized by
specic cellular receptors overexpressed on tumor cells mem-
brane [,].
Although properties such as high biocompatibility,
biodegradability, high hydrophilicity, and viscoelasticity led
to considerable use of HA both in medicine and in cosmetics,
in particular, in the treatment of joint problems and in
the “tissue augmentation” [], its solubility in water, rapid
absorption, and short residence time in situ limit its appli-
cation [,]. Moreover, exogenous HA is quickly degraded
in vivo (half-life of – h) by hydrolytic enzymes, that
is, hyaluronidases (HAses). To ensure greater permanence
in situ, the HA is generally subjected to chemical changes
such as derivatization or, especially, cross-linking processes
[], which decrease the solubility in water and increase
its resistance to enzymatic degradation. Currently on the
market, there are several “stabilized” (cross-linked) and
biocompatible gels based on HA [,]andtheresearchis
continuously engaged in the development of new derivatives
that have advantages over those already in use, in terms of
both degradation times and native polymer biocompatibility
In this paper, we reported the preparation of the acetyl
ester of HA (HA-Acet) (Figure ), with the aim to prolong
the eect and improve its radical scavenging, antioxidant
properties and bioavailability in vitro. We have also evaluated
the HA and HA-Acet cytotoxicity in three cellular lines, that
is, murine monocyte/macrophage cell line (JA.), murine
brosarcoma cells (WEHI-), and human epithelial kidney
cells (HEK-), and, aer that, the HA-Acet inhibition of
NO release from JA. murine macrophages has been
studied in comparison with the free HA form.
2. Material and Methods
2.1. Chemistry. Unless stated otherwise, all reagents and
its molecular weight ( kDa) was determined by GPC (gel
permeation chromatography). GPC analysis of the sample
was made at C using a tool, equipped with UV detector,
refractive index detector, and a set of four PPS columns
(made of polystyrene) having pore dimensions, respectively,
of 5˚
A, 4˚
A, 3˚
A, and 2˚
A and particles size of  m.
It was used as a solvent tetrahydrofuran at a rate of ow
of . mL/min. For the determination of the molecular
weight a calibration curve was obtained. e progress of
the reaction was controlled by thin-layer chromatography
(TLC), performed on a . mm layer of silica gel  PF
Merck. e nal product (MW kDa) was puried by
column chromatography with silica gel (Merck silica gel) and
characterized by 1H NMR ( MHz) and the spectrum was
recorded on Bruker  spectrometer.
2.2. Procedure for the Acetylation of Hyaluronic Acid (HA-
Acet). To a stirred cold solution (C) of  mg of sodium
hyaluronate in  ml of toluene were added a catalytic amount
of -dimethylaminopyridine (DMAP) and an excess of acetic
anhydride. e mixture was stirred at reux, under nitrogen,
e solid residue was puried by silica gel chromatography
using dichloromethane and methanol ( : ) as eluent, obtain-
ing the pure compound as white solid [](Scheme ). 1H
NMR (CDCl3): .(s,H,OCOCH3); .–. (m,
H, CH2OCOCH3,HNCOCH3); .–. (m, H,  CH2);
.–. (m, H, CH); .–. (br, H, OH); .–. (br,
H, NH).
2.3. Determination of Scavenging Eect on ABTS Rad-
ical Cation. e scavenging activity of native HA and
HA-Acet towards the hydrophilic ABTS (,󸀠-azinobis-(-
ethylbenzothiazoline--sulfonic acid)) radical cation was
assessed according to the literature with slight modications
[]. ABTS was dissolved in water to a  mM concentration;
radical cation (ABTS∙+)wasproducedbyreactingABTS
stock solution with . mM potassium persulfate (nal
concentration) and allowing the mixture to stand in the dark
at room temperature for – h before use. Because ABTS
and potassium persulfate react stoichiometrically at a ratio of
 : ., this will result in incomplete oxidation of the ABTS.
Oxidation of the ABTS commenced immediately, but the
absorbance was not maximal and stable until more than h
had elapsed. e concentration of the resulting blue-green
ABTS∙+ solutionwasadjustedtoanabsorbanceof0.970 ±
0.020at  nm. e radical was stable in this form for more
In the present study,  mg of each sample was mixed
from light, were incubated in a water bath at Cformin.
e decrease of absorbance at  nm was measured at the
endpoint of  min. e antioxidant activity was expressed as a
percentage of scavenging activity on ABTS radical according
Inhibition (%)=0−1
0×100, ()
where 0istheabsorbanceofastandardpreparedinthe
same conditions, but without any sample, and 1is the
absorbance of the hyaluronic acid samples. All samples were
assayed in triplicate and data expressed as means (±SD).
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OH Tol u e n e
S : Acetylation of hyaluronic acid.
2.4. Nitric Oxide Radical (NO)ScavengingAssay. e anti-
inammatory activities of native HA and HA-Acet were
evaluated by performing the in vitro nitric oxide radical
scavenging assay, in which NOgenerated from sodium
nitroprusside (SNP) was measured spectrophotometrically
according to the method reported in literature with slight
modications [].  mg of each sample was incubated with
. mL of the reaction mixture, containing SNP (mM) in
phosphate-buered saline (pH .), at C for  h in front
of a visible polychromatic light source ( W tungsten lamp).
e generated NOradical interacted with oxygen to produce
the nitrite ion (NO2) which was assayed at  min intervals
by mixing the incubation mixture with mL of Griess
reagent (% sulfanilamide in % phosphoric acid and .%
naphthylethylenediamine dihydrochloride). e absorbance
of the chromophore (purple azo dye) formed during the dia-
zotization of nitrite ions with sulphanilamide and subsequent
coupling with naphthylethylenediamine dihydrochloride was
measured at  nm. e anti-inammatory activity was
expressed as a percentage of scavenging activity according to
(). Each experiment was performed in triplicate and the data
presented as average of three independent determinations.
2.5. In Vitro Bioavailability Studies. In vitro bioavailability
studies were carried out in simulated gastric and intestinal
uids by performing a slight modied version of the dialysis
tubing procedure [,] with the aim to simulate the oral
intake of native and acetylated hyaluronic acid. e dialysis
tubing method is characterized by two consecutive enzymatic
digestions: pepsin and pancreatin digestion, respectively.
ese steps are described as follows.
Pepsin Digestion. A  mg amount of each sample was mixed
with . mL of a . N HCl solution containing  U of
porcine pepsin per mL. e obtained mixture was introduced
into a dialysis bag (Spectrum Laboratories Inc., MWCO: –
, Dalton, USA), which was then carefully closed and
immersed inside a ask containing mL of a . N  HCl
solution (pH .). e ask was then incubated in a shaking
water bath at C to simulate the human body temperature
conditions for  h.
Pancreatin Digestion. At the end of the  h pepsin digestion,
the dialysis bag was opened and  mg of amylase,  mg of
esterase, and . mL of a . M NaHCO3solution containing
. mg porcine pancreatin/mL were added to the peptic
digesta. Aer the digesta and enzyme solution were well
mixed, the dialysis bag was sealed at each end with clamps
.. e ask was incubated in the shaking water bath at C
for a further  h. Aer the pancreatin incubation time, the
hydrolyzed hyaluronic acid was determined spectrophoto-
metrically according to the literature []. Each experiment
was performed in triplicate.
2.6. Cell Lines and Cultures. e murine monocyte/
macrophage cell line (JA.), murine brosarcoma cells
(WEHI-), and human epithelial kidney cells (HEK-)
were obtained from American Tissue Culture Collection
(ATCC). Dulbeccos modied Eagle’s medium (DMEM),
penicillin/streptomycin HEPES, glutamine, fetal calf serum
(FCS), and horse serum were from Euroclone (Euroclone-
Celbio, Pero, Milan, Italy). J.A were grown in adhesion
on Petri dishes and maintained at Caspreviously
described []. WEHI- and HEK- were maintained
in adhesion on Petri dishes with DMEM supplemented
with % heat-inactivated FCS,  mM HEPES,  u/mL
penicillin, and  g/mL streptomycin.
2.7. Cell Viability Assay. J.A, WEHI-, and HEK-
(. ×4cells/well) were plated on -well microtiter plates
and allowed to adhere at Cina%CO
for  h. ereaer, the medium was replaced with  L
of fresh medium and  L aliquot of serial dilution of
each test compound was added and then the cells were
incubated for  h. Serial dilution of -mercaptopurine (-
MP) was added, as reference drug. In some experiments,
HA or HA-Acet were added only to JA. macrophages for
 h. Mitochondrial respiration, an indicator of cells viability,
was assessed by the mitochondrial-dependent reduction
of [-(,-dimethylthiazol--yl)-,-phenyl-H-tetrazolium
bromide] (MTT) to formazan and cells viability was assessed
as previously reported [].
Briey,  L of MTT ( mg/mL) was added and the cells
were incubated for an additional h. ereaer, cells were
solution containing % (v:v) N,N-dimethylformamide and
% (w:v) SDS with an adjusted pH of . []. e optical
density (OD) of each well was measured with microplate
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spectrophotometer (Titertek, Multiskan MCC/) equipped
with a  nm lter. e viability of each cell line in response
to treatment with testedcompounds and -MP was calculated
as % dead cells =  (OD treated/OD control) ×. IC50
values (concentration that causes % growth inhibition)
were determined [].
2.8. NO2Release from J774A Cells. Nitrite content (NO2),
index of NO released by cells in the culture supernatant, was
measured in JA. cells. To stimulate nitric oxide (NO)
release from macrophages, E. coli lipopolysaccharide (LPS,
×3u/mL) was used []. Macrophages (. ×4
cells/well) were plated on -well microtiter plates and
2atmosphere for
 h. HA and HA-Acet (.– mol/L) were added for  h
to cells and then coexposed to  g/mL LPS for further
 h. NO2amounts were measured by Griess reaction.
Briey,  Lofcellculturemediumwasmixedwith
 LofGriessreagentequalvolumesof%(w:v)sul-
phanilamide in % (v:v) phosphoric acid and .% (w:v)
naphthylethylenediamine-HCl—and incubated at room tem-
perature for  min, and then the absorbance was measured
at  nm in a microplate reader Titertek (Dasit, Cornaredo,
Milan, Italy). e amount of NO2(as mol/L) in the samples
was calculated from a sodium nitrite standard curve.
2.9. Data Analysis. Data are reported as mean ±standard
error mean (s.e.m.) values of independent experiments,
or more independent observations. Statistical analysis was
performed by Student’s -testoranalysisofvariancetest,and
multiple comparisons were made by Bonferroni’s test. A
value less than . was considered signicant.
3. Results and Discussion
3.1. Evaluation of the HA and HA-Acet Antioxidant and Anti-
Inammatory Activity. In the aim to establish the antioxi-
dant and anti-inammatory activities of HA and HA-Acet,
their reactivity towards ABTS and nitric oxide (NO)was
evaluated. ABTS is a preformed stable organic radical with
absorption maximum at  nm; nitric oxide (NO)isa
pivotal proinammatory mediator []anditscontribution
to oxidative damage is due to the reaction with superoxide
to form the peroxynitrite anion, which is a potential strong
oxidantthatcandecomposetoproduceOH and NO2[]. In
the present study, nitroprusside (SNP) was employed as a NO
radical donor in the aim to evaluate the anti-inammatory
properties of native acid and acetylated hyaluronic acid. NO
released from SNP, indeed, has a strong NO+character which
nents. e scavenger ability of each sample (HA or HA-Acet)
was evaluated in terms of radical reduction and data have
been expressed as inhibition (%) and reported in Tab l e  .Both
samples were found to have good and comparable scavenging
properties towards the selected radicals conrming that the
acetylation of native HA does not aect the biological activity
of this polysaccharide.
T : Antioxidant and anti-inammatory activity and in vitro
bioavailability of HA and HA-Acet. Statistical analysis was per-
formed using Students -test.
Sample Inhibition (%) Bioavailability (%)
HA 35±0.7 77± 0.9 8±0.7
HA-Acet 34±1.0 75±1.1 48±0.3
indicates 𝑃 < 0.001 of HA-Acet versus HA.
T  :  e I C  ,expressedasmol/L, value is the concentration
of compound that aords a % reduction in cell growth (aer a
 h incubation). J.A = murine monocyte/macrophage cell lines.
HEK- = human epithelial kidney cell lines. WEHI- = murine
brosarcoma cell lines. -MP = -mercaptopurine.
cmp IC (M)
J.A HEK- WEHI-
HA-Acet > > >
HA > > >
-MP  . .
3.2. Bioavailability Studies. Dialysis tubing procedure is a
fast and low cost method to evaluate the bioavailability of
dierent kinds of compounds and, in this study, it was
used in the aim to evaluate the bioavailability of native HA
and HA-Acet. Bioavailability was dened as the percentage
of tested HA and HA-Acet recovered in the bioaccessible
fraction, aer in vitro digestion, in relation to the original
nondigested samples. is value can be calculated by the
following equation:
bioaccessible content
total content ×100. ()
In the present study, we supposed that the chemical
modication of native HA by introducing acetyl groups can
improve the bioavailability of this biopolymer. e obtained
data (Tab l e  ) conrmed our supposition showing that the
acetylation of native polysaccharide increases its bioavail-
ability of six times. is higher value could be ascribable to
backbone more lipophilic.
3.3. In Vitro Cytotoxicity and Anti-Inammatory Experiments.
We next evaluated the in vitro cytotoxic activity of HA and
HA-Acet on three dierent cell lines (J.A, WEHI-,
and HEK-). Our results clearly showed a low cytotoxicity,
compared to -MP, on all the three used cell lines and also at
the concentration range (i.e., .– mol/L) used for NO
release determination in JA. cell line; HA or HA-Acet
treatments did not elicit antiproliferative eects, as evidenced
by the IC50 results shown in Ta b l e  .
e anti-inammatory properties observed in previous
experiments have been conrmed, as well, by testing HA and
HA-Acet on JA. murine macrophages. e latter were
stimulated with LPS ( g/mL), in the presence or absence
of HA or HA-Acet (.– mol/L), to determine whether
these compounds were able to modulate NO release. JA.
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Ctrl 12.5 25 50 100 Ctrl 12.5 25 50 100
∗∗ ∗∗∗ ∗∗∗
∗∗ ∗∗∗ ∗∗∗
Ctrl 12.5 25 50 100 Ctrl 12.5 25 50 100
F : Eect of HA (Panel (a)) and HA-Acet (Panel (b)) on NO release from JA. macrophages stimulated with LPS for  h. ∗∗∗ and
∗∗ denote < 0.001and < 0.01, respectively, versus LPS alone.
cells challenged with LPS exhibited a high increase of NO
accumulation, evaluated as nitrite, as shown in Figure ,
panels (a)-(b), whereas HA or HA-Acet, per se, did not
aect basal NO production at the tested concentrations
(.– mol/L). Conversely, a signicant reduction in NO
release was detected in LPS-treated macrophages in presence
of HA or HA-Acet at all tested concentrations, with a slight
increase of HA-Acet ability in decreasing NO release from
cells. We believe that this feature is most probably due to
the better bioavailability and higher stability of HA-Acet with
respect to the free HA form.
It is noteworthy that HA is usefully employed for the
preparation of several derivatives which have been used
as vector or delivery system for many molecules used in
therapy mostly, but not only, for cancer treatment, due to
the observation that HA-binding receptors such as clus-
ter determinant  (CD), receptor for hyaluronic acid-
mediated motility (RAHMM), and lymphatic vessel endothe-
lial receptor- (LYVE-) are dramatically overexpressed in
cancer cells []. Under this point of view, acetylation
is a simple and suitable technique which allows increasing
the hydrophobicity without impairing the ability of HA-
receptors to interact with the acetylated-HA []. Moreover, it
should be considered that generation of ROS (reactive oxygen
species) plays a key role in human diseases and aging process
and that HA is involved in the activation and modulation of
the inammatory response, including a scavenging activity
OH). On the other
hand, inhibition of tumor cells and protection of tissue
from free radical damage have also been attributed to a
mixture of hyaluronic acid fragments and, in recent years,
several reports described that HA exerts antiageing eect
with potential antioxidant properties both in vitro and in
vivo []. e ecacy ohese considerable properties is
related to many factors and, more strictly, to the catabolism of
HA in the considered biologic environment. e major actors
involved in its degradation are hyaluronidases, which would
diminish its presence in the extracellular environment, so that
most importantly without altering the interaction with its
receptors, has been pursued over time in order to increase
its stability, bioavailability, and, lastly, its eects. Our results
are promising for further studies addressed to a better
understanding of the interactions of HA-Acet with biological
4. Conclusions
In this study we reported the synthesis of an acetylated
HA derivative which exhibited a better bioavailability and
stability with respect to the HA free form. ese features
release inhibition from murine monocyte/macrophage cell
lines (J.A). HA-Acet showed a low cytotoxicity in all the
three cell lines, at least at the drug doses used in the experi-
ments and, moreover, a slight but signicant increased anti-
inammatory activity, dose-dependent, has been evidenced.
Our results bring a new contribution to the studies focused
on the several biological properties and therapeutic uses of
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Authors’ Contribution
Carmela Saturnino and Maria Stefania Sinicropi equally
contributed to this work.
is work was supported by the Programma Operativo
Nazionale (PON) Ricerca e Competivit`
BioMed Research International
Convergenza, /-CCI: ITPO, to AC and
OIP, and by Commissione Europea, Fondo Sociale Europeo
(FSE /-PROGRAMMA ARUE), and Regione Cal-
abria to DI.
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... Liang et al. [22] investigated and compared the oral bioavailability of resveratrol and its acetylated derivative, 3,5,4'-tri-O-acetylresveratrol (TARES), via pharmacokinetic tests, and they found that the AUC value of TARES is greater than that of resveratrol, indicating that acetylated resveratrol has a higher absorption in rats. Moreover, acetylation enhanced the radical scavenging and anti-inflammatory activity of hyaluronic acid via its improved bioavailability in murine monocyte/macrophage cells [44]. ...
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A kind of hydroxylated polymethoxyflavone (PMFs) existing in the citrus genus, 5-Demethyltangeretin (5-DTAN), has been reported to possess several bioactivities in vitro and in vivo. The aim of this study was to investigate whether acetylation could enhance the anticancer activity and oral bioavailability of 5-DTAN. PC-3 human prostate cancer cells were treated with tangeretin (TAN), 5-DTAN, and 5-acetylated TAN (5-ATAN), and the results showed that the cytotoxic effect 5-ATAN (IC50 value of 5.1 �M) on the cell viability of PC-3 cells was stronger than that of TAN (IC50 value of 17.2 �M) and 5-DTAN (IC50 value of 11.8 �M). Compared to 5-DTAN, 5-ATAN treatment caused a more pronounced DNA ladder, increased the sub-G1 phase population, and induced G2/M phase arrest in the cell cycle of PC-3 cells. We also found that 5-ATAN triggered the activation of caspase-3 and the progression of the intrinsic mitochondrial pathway in PC-3 cells, suggesting the induction of apoptosis. In a cell wound healing test, 5-ATAN dose-dependently reduced the cell migration, and the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) was decreased after 48 h of 5-ATAN treatment. Moreover, oral administration of 5-ATAN showed a significantly stronger inhibitory effect on tumor size and tumor weight in tumor-bearing nude mice than those of vehicle or the 5-DTAN group (p < 0.05). Furthermore, pharmacokinetic results showed that single-dose oral administration of 5-ATAN exhibited a higher maximum concentration (Cmax) and area under the curve (AUC) of 5-DTAN in plasma than that of 5-DTAN. More extensive distribution of 5-DTAN to most tissues of mice was also observed in mice treated with 5-ATAN for 7 days. In conclusion, acetylation strongly enhances the anticancer activity and oral bioavailability of 5-DTAN and could be a promising strategy to promote the potential bioactivities of natural products.
... Similar results were reported by Saturnino et al., where the non-scavenged • NO by HA was measured in the form of nitrite. 43 Furthermore, a possibility still exists for the reaction of HA chains with peroxynitrite (ONOO •− ), which is generated as a downstream RNS, produced in the buffer as in reaction 2, and induces luminol oxidation (reaction 3). 44 Moreover, ONOO •− has been reported to interact with HA causing partial fragmentation of its chain; 26,45 however, the probability of these reactions is low due to the short period of the assay ...
Full-text available
Nitric oxide (•NO) is one of the prominent free radicals, playing a pivotal role in breast cancer progression. Hyaluronic acid (HA) plays an essential role in neutralizing free radicals in tumor tissues. However, its interactions with nitric oxide have not been thoroughly investigated. Hence, this study attempts to understand the mechanism of these interactions and the different effects on the intracellular •NO levels and migration of breast cancer cells. The affinity of HA to scavenge •NO was investigated alongside the accompanying changes in specific physicochemical properties and the further effects on the •NO-induced attachment and migration of the breast cancer cell lines, MDA-MB-231 and HCC1806. The reaction of the nitrogen dioxide radical, formed via •NO/O2 interactions, with HA initiated a series of oxidative reactions, which, in the presence of •NO, induce the fragmentation of the polymeric chains. Furthermore, these interactions were found to hinder the NO-induced migration of cancer cells. However, the NO-induced HA modification/fragmentation was inhibited in the presence of hemin, a NO-scavenging compound. Collectively, these results help toward understanding the involvement of HA in the •NO-induced cell migration and suggest the possible modification of HA, used as one of the main materials in different biomedical applications.
... Others have pursued improving topical humectant performance by modifying HA through covalently bonded cross-linking struc- tures and covalently bonded hydrophobic modifications (e.g., AcHA). 19,20,22,23,34,35 Although these altered forms of HA do offer superior hydration benefits compared to traditional linear HA, they do not combine both properties of improved water binding by cross-linking with improved affinity to the skin's sur- face through hydrophobic modifications. However, we theorized greater water binding and improved affinity for skin deposition could be achieved through the development of an IPN. ...
Full-text available
Background Hyaluronic acid (HA) has been considered the gold standard ingredient for improving skin hydration and combating age‐related effects, however it is an inefficient polymer with inconsistent results partially due to its poor skin penetration, surface deposition, and rapid degradation. Herein we report the synthesis and in vitro characterization of a newly developed, topical super‐humectant with the goal of attracting and binding water molecules more efficiently than traditional, cosmetic‐grade forms of HA. Methods A modified interpenetrating polymer network (IPN) was developed using three polymers into a three‐dimensional formation (3D3P) for entrapping HA and water. This 3D3P‐IPN functions as a super‐humectant, attracting and binding water molecules more efficiently than the traditional cosmetic‐grade forms of HA. We compare 3D3P‐IPN serum samples to a traditional commercial benchmark product of similar ingredients using microscopic analysis, rheology, Karl Fischer (KF) titration, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), and dynamic vapor sorption (DVS) techniques. Results The 3D3P‐IPN samples appeared to bind water tighter than the benchmark sample as evidenced by maximum endpoints of endotherms occurring at significantly higher temperatures. The DVS results further confirm this speculation as the 3D3P‐IPN samples lost approximately 10% less water up to 35% RH than the benchmark. The 3D3P‐IPN samples also absorbed more water as the humidity level increased,demonstrating superior humectant properties. KF titration indicated that all three samples had similar water concentrations; however, TGA results demonstrated that the benchmark (a viscous, humectant‐rich hydrating masque) did not have much bound water. Conclusion Through the synthesis of a 3D3P‐IPN using simplified methods, we were able to increase the water‐binding and HA‐delivery capabilities of a thin serum. This 3D3P‐IPN serum has potential to deliver more hydration to the skin's surface compared to traditional HA formulations.
... ) was assessed; if Nitric Oxide is present in high concentrations, indeed, it can be considered a proinflammatory mediator [29]. NO is able to react with superoxide and thus produce peroxynitrite anion, a potential strong oxidant that triggers oxidative damage [30]. For all of these reasons, the ability to scavenge Nitric Oxide is used to investigate in vitro the anti-inflammatory properties of several natural samples. ...
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Lycium barbarum (Goji) fruits are mainly cultivated in northwestern China and are well known for their beneficial and healthy effects. In this work, the biological and functional properties of Calabrian Goji extract, obtained from Goji berries cultivated in the Sibari Plain (in the Italian region of Calabria), were demonstrated. In order to evaluate the use of this extract as a food supplement for cognitive and mental disorders, the quantification of Carotenoids as Zeaxanthin equivalents was made. The antioxidant activity was investigated by evaluating the scavenging properties against 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals and by performing the ORAC (Oxygen Radical Absorbance Capacity) assay. The inhibition of lipid peroxidation was quantified by bleaching test and the ability to inhibit acetylcholinesterase enzyme and to scavenge nitric oxide radical was also evaluated. All the results were compared to those obtained from a Chinese Goji extract used as a reference. Based on the reported data, Calabrian Goji might be used as a food supplement with a possible application in cognitive disorders, mental impairments and other neurodegenerative diseases, due to its biological properties and the high levels of Carotenoids.
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This study demonstrated both adipose-derived stem cells (ASCs) in vitro and in vivo combined with three-dimensional (3D) porous sponge matrices on implant wound healing. Sponge matrices were created from hyaluronic acid (HA), collagen (Col), and gelatin (Gel), constructing two types: HA-L (low content) and HA-H (high content), to be cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Fourier transform infrared spectroscopy method verified carboxyl groups of HA and amino groups of Col and Gel reacting between the raw materials and scaffolds to identify the successive cross-linking. The swelling ratios of two types of sponge matrices were analyzed by water absorption capabilities, and the results displayed both over 30-fold dry scaffold weight enhancements. In biodegradation tests, matrices were hydrolyzed over time by three cutaneous enzymes, hyaluronidase, lysozyme, and collagenase I. ASCs from rats were cultured within the HA-H scaffold, demonstrating higher antioxidative abilities and secretions on related genes and proteins compared to the other two groups. The ASC HA-H matrix promoted cell proliferation to stimulate capillary angiogenesis inducer secretions, including vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β). In vivo histological examinations showed ASCs from implanted HA-H implant transported into the subcutis, and rat skin cells also infiltrated into the original matrix zone to increase the extracellular matrix (ECM) reconstructions. Our experimental data revealed that the ASC HA-H sponge implant was effective in improving wound repair.
Thermogels that undergo temperature-dependent sol-gel transition have recently attracted attention as a promising biomaterial for injectable tissue engineering. However, conventional thermogels usually suffer from poor physical properties and low cell binding affinity, limiting their practical applications. Here, a simple approach for developing a new thermogel with enhanced physical properties and cell binding affinity is proposed. This thermogel (AcHA/HGC) was obtained by simple blending of a new class of polysaccharide-based thermogel, N-hexanoyl glycol chitosan (HGC), with a polysaccharide possessing good cell binding affinity, acetylated hyaluronic acid (AcHA). Gelation of AcHA/HGC was initially triggered by the thermosensitive response of HGC and gradually intensified by additional physical crosslinking mechanisms between HGC and AcHA, resulting in thermo-irreversible gelation. Compared to the thermos-reversible HGC hydrogel, the thermo-irreversible AcHA/HGC hydrogel exhibited enhanced physical stability, mechanical properties, cell binding affinity, and tissue compatibility. These results suggest that our thermo-irreversible hydrogel is a promising biomaterial for injectable tissue engineering.
Construction of scaffolds which are similar to natural niches regarding both biochemical composition and mechanical characteristics has gained great attention in the field of tissue engineering. However, application of natural polymers, such as hyaluronic acid, is challenging in construction of scaffolds due to physicochemical properties, difficult to use in electrospinning and low cell adhesion rate. In this study, HA was acetylated to make it soluble in high polarity solvent and blended with PCL for construction of nanofibrous composite (ac-HA/PCL) scaffolds. Chondroinductivity of the constructed scaffolds was investigated using human mesenchymal stem cells (hADSCs). The presence of acetyl groups, as well as morphology and biocompatibility of the composite scaffolds were characterized by HNMR, FTIR, SEM and MTT assay respectively. Expression of cartilage-specific genes (SOX9, Col II and Aggrecan) was monitored by Real-time PCR. Significant increase in expression of Sox9 and Col II as the markers of chondrogenic differentiation as well as the results of Alcian blue staining, indicated the chondro-inductive potential of HA/PCL nanofibrous scaffolds. Acetylated HA was biocompatible with chondroinductivity features, therefore it not only had the positive characteristics of natural HA, but also enhanced the cellular attachment and application potential.
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Sericin is a natural protein that has been used in biomedical and pharmaceutical fields as raw material for polypeptide-based drug delivery systems (DDSs). In this paper, it has been employed as pharmaceutical biopolymer for the production of sunitinib–polypeptide conjugate. The synthesis has been carried out by simple click reaction in water, using the redox couple l-ascorbic acid/hydrogen peroxide as a free radical grafting initiator. The bioconjugate molecular weight (50 kDa < Mw < 75 kDa) was obtained by SDS-PAGE, while the spectroscopic characteristics have been studied in order to reveal the presence of grafted sunitinib. In both FT-IR and UV/Vis spectra, signals corresponding to sunitinib functional groups have been identified. Since sunitinib is an anticancer drug characterized by low bioavailability and low permeability, the bioconjugation aimed at their enhancement. In vitro studies demonstrated that bioavailability has been increased to almost 74%, compared with commercial formulation. Also cell membrane permeability has been augmented in in vitro tests, in which membrane models have been used to determine the lipid membrane/physiological fluid partition coefficient (Kp). The log(Kp) value of the bioconjugate was increased to over 4. This effect resulted in a three-fold decrease of IC50 value against MCF-7 cells.
Background Hyaluronic acid (HA) injection is widely used in the treatment of temporomandibular joint (TMJ) osteoarthritis (OA). Proteoglycan 4 (PRG4) is another joint lubricant that protects surface of articular cartilage. But few studies had explored the role of HA in regulation of PRG4 expression in TMJ OA. In this study, the effects of HA on the expression of PRG4 in osteoarthritic TMJ synovial cells were investigated in hypoxia, which was similar to the TMJ physiologically.Methods Synovial cells were isolated from the TMJ OA patients and were treated with or without HA under normoxia or hypoxia for indicated time periods. The proliferation of synovial cells was measured using Cell Counting Kit-8 (CCK-8). The gene expression of HAS2, VEGF, and PRG4 was detected by quantitative real-time PCR, and the secretion of PRG4 and VEGF was assayed by enzyme-linked immunosorbent assay (ELISA). Immunofluorescence was used to examine the protein expression of hypoxia-induced factor-1α (HIF-1α).ResultsHyaluronic acid markedly increased the proliferation of osteoarthritic synovial cells in hypoxia. The expression of HAS2 and PRG4 mRNA of osteoarthritic synovial cells under hypoxia was enhanced by HA treatment. However, HA had no effect on reducing the VEGF and HIF-1α expression in synovial cells in hypoxia.Conclusions Hyaluronic acid could promote the expression of HAS2 and PRG4, but could not modulate HIF-1α and VEGF expression of TMJ osteoarthritic synovial cells in hypoxia.
Full-text available
In this work a new functionally enhanced food was prepared by using Maca flour as powerful ingredient. Bread with improved antioxidant and anti-inflammatory properties was prepared and characterized by performing different assays. For the characterization of Maca enhanced bread, in vitro tests, such as 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Oxygen radical absorbance capacity (ORAC), Folin-Ciocalteu and Deoxyribose assays were performed in order to evaluate the antioxidant properties, while the anti-inflammatory activity was investigated in terms of peroxynitrite scavenging ability. Furthermore, in vitro enzymatic assays were performed in the aim to evaluate the efficiency of the proposed functional food in the reduction of sugar intake. The obtained data confirmed the high potential applicability of prepared bread in human nutrition.
Full-text available
Endocannabinoids are produced during the organ damage caused by different pathological situations. The most abundant endocannabinoid is 2-Arachidonoylglycerol (2-AG) present in large amounts within the brain. The endogenous levels of 2-AG are linked to the activities of two enzymes: diacylglycerol lipase (DAGL) and monoacylglycerol lipase (MAGL). In this paper we reported the synthesis of different oleylphosphonates substituted that should be specifically recognized from DAGL and MAGL of the endocannabinoid system.
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Low molecular weight organogels are semisolid systems structured by the assembly of molecules that crystallise under suitable process conditions. The inner microstructure of organogels is made up of a 3-D network, in which both an organic liquid solvent and other dispersed particles can be entrapped. In this work, olive oil organogels, structured from policosanol (a nutraceutical mixture of fatty alcohols), were studied in order to obtain the best formulation for producing a support for delivery of lipophilic agents (namely ferulic Acid) via oral administration. A rheological optimisation of the olive oil-policosanol organogel was first of all performed with Step Shear Rate Temperature Ramp tests. This provided important information on the policosanol fraction to be added to the system and on the onset of crystallisation temperature, an indirect measurement of the system melting point. It was found that a policosanol weight percentage of 0.03 was suitable to obtain contemporary semisolid organogels, consistent enough and thermally stable for human ingestion. In vitro tests on organogels loaded with ferulic acid were also carried out in order to simulate the oral intake of the nutraceutical compound. This evidenced a release mechanism determined by both erosion and diffusion; a good performance of gels and their ability to control the release rate through the degree of structuration were also observed.
Resistance against the cytotoxic actions of tumor necrosis factor-alpha (TNF) is an active process requiring the synthesis of TNF-inducible proteins. The specific TNF-induced proteins so far identified (manganese superoxide dismutase and plasminogen activator inhibitor type 2) as having a role in resistance against TNF cytotoxicity are able to confer only partial protection to cells, suggesting that other genes are involved. A20 is a TNF-induced primary response gene which encodes a novel zinc ringer protein. In this report we demonstrate that A20 protein is induced by TNF in a variety of cells. A survey of A20 expression in human breast carcinoma cell lines that are either sensitive or resistant to TNF cytotoxicity revealed increased expression of A20 message and protein in TNF-resistant cells. Constitutive expression of A20 after stable transfection of NIH 3T3 and WEHI 164 cells results in significant, but partial, resistance to TNF cytotoxicity. This work gives additional support to a role for TNF-induced immediate early response genes in protecting cells from TNF-induced death.
A convenient synthesis of novel functionalized 1,4-dimethylcarbazole derivatives containing 3,4,5-trimethoxybenzamido-ureido or N-(3,4,5-trimethoxyphenyl)ureido group starting from their corresponding indole derivatives is reported. Three derivatives prepared (5g, 6c, and 6g) were active against leukemia cell lines HL60. Both 5g and 6g showed potent antiproliferative activity against KB cell lines, likely associated with the inhibition of tubulin polymerization.
background.: Several biomaterials are available for the purpose of soft tissue augmentation, but none of them has all the properties of the ideal filler material. The recent development of hyaluronic acid gels for dermal implantation give the physician new possibilities of effective treatment in this field. objective.: This study provides a clinical and histological evaluation of safety and efficacy of a cross‐linked stabilized non‐animal hyaluronic acid gel (Restylane, Q‐Med, Uppsala, Sweden) to determine its characteristics, advantages, disadvantages, and side‐effects. methods.: 158 patients were treated with facial intradermal implant of hyaluronic acid gel for augmentation therapy of wrinkles and folds, and for lip augmentation and/or recontouring. The results were evaluated in all patients by subjective judgement by the physician and the patient, and by photographic method at time 0 and after 1, 2, 4 and 8 months from the procedure. In addition, a smaller histological study was carried out in five volunteer patients for a term of 52 weeks to determine the interaction and duration of the material in human healthy skin. results.: Clinically, both the physicians' and patients' evaluations revealed very satisfactory results, with a global 78.5% and 73.4% respectively of moderate or marked improvement after eight months, independent of the treated area. The photographic evaluation revealed even better results with a 80.4% of moderate or marked improvement after 8 months. The safety evaluation showed a 12.5% of postoperative immediate adverse events, that were localized and transient. There was no evidence of major systemic side effects. Histologically, the product was shown to be long‐lasting and well tolerated as judged by histological techniques. conclusions.: Stabilized, non‐animal, hyaluronic acid gel is well tolerated and effective in augmentation therapy of soft tissues of the face. This material presents several advantages in comparison to previously used injectable biomaterials and expands the arsenal of therapeutic tools in the field of soft tissue augmentation.
Recently a series of chiral N-(phenoxyalkyl)amides have been reported as potent MT1 and MT2 melatonergic ligands. Some of these compounds were selected and tested for their antioxidant properties by measuring their reducing effect against oxidation of 2′,7′-dichlorodihydrofluorescein (DCFH) in the DCFH-diacetate (DCFH-DA) assay. Among the tested compounds, N-[2-(3-methoxyphenoxy)propyl]butanamide displayed potent antioxidant activity that was stereoselective, the (R)-enantiomer performing as the eutomer. This compound displayed strong cytoprotective activity against H2O2-induced cytotoxicity resulting slightly more active than melatonin, and performed as Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor, too.