Research Article Acetylated Hyaluronic Acid: Enhanced Bioavailability and Biological Studies

Abstract

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).

Full text

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
1DepartmentofPharmacy,UniversityofSalerno,ViaGiovanniPaoloII132,84084Fisciano,Italy
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; saturnino@unisa.it and Maria Stefania Sinicropi; s.sinicropi@unical.it
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
cited.
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
andfacilitatinganincreaseinproinammatorycytokines
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
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BioMed Research International
Volume 2014, Article ID 921549, 7 pages
http://dx.doi.org/10.1155/2014/921549

BioMed Research International
O
O
O
O
HO
HO
HO
HO
NH
O
OH
F : Functional groups of HA subjected to chemical modica-
tion.
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
conservation.
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
compoundsusedwereobtainedfromSigma-Aldrich(Milan,
Italy).esynthesiswasmadeusingsodiumhyaluronateand
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,
forhoursandthenconcentratedunderreducedpressure.
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
thantwodayswhenstoredinthedarkatroomtemperature.
In the present study,  mg of each sample was mixed
withmLofABTSradicalsolution.emixtures,protected
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
to
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).

BioMed Research International 
OO
O
O
O
O
O
NH
O
O
O
O
O
O
O
O
O
O
HO
HO
HO
HO
HO
NH
O
OH Tol u e n e
Ac2O, D MAP
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 NO∙generated 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 NO∙radical 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
andplacedinaaskwithmLofbuersolutionatpH
.. 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). Dulbecco’s 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
2atmosphere
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
lysedandthedarkbluecrystalssolubilisedwithLofa
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. NO2−Release 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
allowedtoadhereat
∘Cina%CO
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. NO2−amounts were measured by Griess reaction.
Briey,  Lofcellculturemediumwasmixedwith
 LofGriessreagent—equalvolumesof%(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,
whichweredoneatleastthreetimes,eachtimewiththree
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
oxidantthatcandecomposetoproduce∙OH 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
canalterthestructureandfunctionofmanycellularcompo-
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 Student’s -test.
Sample Inhibition (%) Bioavailability (%)
ABTS NO∙
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  ,expressedasmol/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
thepresenceofacetylmoietieswhichmakethepolymeric
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
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
+LPS
NO2
−(𝜇M)
−LPS
∗∗
∗∗ ∗∗∗ ∗∗∗
HA
(𝜇M)
(a)
NO2
−(𝜇M)
−LPS
∗∗
∗∗ ∗∗∗ ∗∗∗
Ctrl 12.5 25 50 100 Ctrl 12.5 25 50 100
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
+LPS
HA-Acet
(𝜇M)
(b)
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
towardsROS,suchashydroxylradical(
∙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
thestrategytochemicallymodifyHA(e.g.,byacetylation),
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
molecules.
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
havebeenconrmed,aswell,bytheevaluationoftheNO
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
HA.
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.
Acknowledgments
is work was supported by the Programma Operativo
Nazionale (PON) Ricerca e Competivit`
aperleRegionidella

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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