The aim of this study was to investigate an ability of chitosan nanoparticles for tissue delivery of the peptide glutathione. Formulations composed of chitosan or chitosan plus cyclodextrin-beta comlex were prepared. Reduced glutathione was loaded and delivered to mucosal layer of small intestine after ischemia-reperfusion injury more efficiently in chitosan/cyclodextrin-beta nanoparticles. From the data obtained, we believe that chitosan / cyclodextrin nanoparticles can be used for the oral administration of glutathione and other small peptides.
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Rotar, O.V.

Rotar, V.I.

SocioEconomic Challenges Journal (Sumy State University)

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  
NANOMATERIALS: APPLICATIONS AND PROPERTIES 
Vol. 2 No 4, 04NABM07(2pp) (2013) 
 
 
2304-1862/2013/2(4)04NABM07(2) 04NABM07-1  2013 Sumy State University 
Application of Chitosan / Cyclodextrin Nanoparticles for Tissue Glutathione Delivery 
 
O.V. Rotar, V.I. Rotar 
 
Bukovinian State Medical University, 2, Teatralna Sq., 58000 Chernivtsi, Ukraine 
 
(Received 19 May 2013; published online 30 August 2013) 
 
The aim of this study was to investigate an ability of chitosan nanoparticles for tissue delivery of the 
peptide glutathione. Formulations composed of chitosan or chitosan plus cyclodextrin-beta comlex were 
prepared. Reduced glutathione was loaded and delivered to mucosal layer of small intestine after ischemia-
reperfusion injury more efficiently in chitosan/cyclodextrin-beta nanoparticles. From the data obtained, we 
believe that chitosan / cyclodextrin nanoparticles can be used for the oral administration of glutathione 
and other small peptides. 
 
Keywords: Glutathion, Nanoparticles, Cyclodextrin, Chitosan. 
 PACS number: 87.85.Rs 
 
 
1. INTRODUCTION  
 
Glutathione (GSH) is the predominant intracellular 
non-protein thiol in a wide range of cells. It has important 
functions in the detoxification system of almost every cell 
type. The reversible oxidation of GSH provides electrons 
for the GSH-peroxidase catalyzed reduction of hydrogen 
peroxide or organic peroxides [1]. However, the 
application of GSH as a functional food ingredient has 
been limited because of its low bioavailability due to its 
poor cellular uptake and its instability. Under conditions 
of oxidative stress, the GSH thiol group can be easily 
oxidized to glutathione disulfide, which loses its 
antioxidant activity based on its radical scavenging 
activity. Nanoparticle (NP) prototypes from biodegradable 
polymers have been proposed for local drug delivery [2]. 
Among them, NPs based on the polysaccharide chitosan 
(CS) have shown particularly promising results due to 
their intrinsic properties including biocompatibility, 
mucoadhesion and ability to transiently open the tight 
junctions of the intestinal barrier [3]. 
 
2. MATERIAL AND METHODS 
 
2.1 Materials 
 
Chitosan (CS, Mw 600 kDa, degree of deacetylation 
90 %) is a commercial product of Bioprogress (Russian 
Federation). Sodium tripolyphosphate (TPP) was 
purchased from Sigma (USA). Reduced glutathione and 
cyclodextrin-beta (CD) were purchased from Applichem 
(Germany). All other chemicals were of analytical 
grade and used as received. The water used in all 
experiments was double distilled. 
 
2.2 Preparation of Nps 
 
Preparation of NPs was based on the ionic gelation 
of CS with TPP anions [4] in 1st group (CS NPs) and CS 
with TPP and CD (CS / CD NPs) in 2nd group [5]. In all 
groups CS nanoparticles were prepared with CS 
concentration of 1 mg/ml and CS : TPP ratio 3 : 1. In 
both groups, CS was first dissolved in acetic acid 
aqueous solution. The concentration of acetic acid in 
the aqueous media was 1.75 times that of CS. To 9 ml 
of CS 2 ml aqueous solution of 0,5 % w/v of GSH or 2 ml 
of mixture of GSH and CD in ratio 1 : 1 as 1.0 % w/v 
(2nd group) were added and mixed 45 min under 
magnetic stirring. Than TPP aqueous solution was 
added drop wise under magnetic stirring onto the 
corresponding amount of the CS solution at room 
temperature.  
 
2.3 Encapsulation Efficiency of Nps 
 
The prepared Nps suspension containing the GSH 
was centrifugated at 9,000 rpm for 90 min and free 
GSH in the supernatant was quantified by 
spectrophotometric method [6].  
 
2.4 In Vivo Determination of Ability of NPs for 
Tissue GSH Delivery 
 
To determine the ability of NPs deliver GSH to 
tissue intestinal ishemia-reperfusion injury were 
applied on 60 Wistar rats by clamping superior 
mesenteric artery for 60 min with 4 hours of 
reestablishing of blood circulation under ketamine 
anastesia. CS or CS / CD NP loaded with 3 mg of GSH 
were infused into jejunum just before ischemia period 
in two groups of animals and appropriate volume of 
normal saline in control animals (n  15 in every 
group). After reperfusion period animals were killed by 
sodium thiopental overdosing and concentration of 
GSH, malone dialdehyde (MDA), diene conjugates (DC) 
and catalase (CAT) in mucosal layer of small intestine 
were determined. Morphological examination of the NP 
was performed by transmission electron microscopy 
(TEM). 
 
3. RESULTS  
 
Regular and spherical morphology was revealed by 
TEM both for CS and CS / CD NPs with similar 
diameters. The capacity of the different NP systems to 
load GSH was determined: CS NPs presented 
encapsulation efficiency near 10 % and CS / CD NPs – 
25 %. GSH was delivered to mucosal tissue in rat 
model of intestinal ischemia-reperfusion injury by 
CS / CD NPs on 34,9 % (p  0,05) higher level than CS 
NPs (Table 1), which was followed by decreasing of 
concentration of oxidant injury markers. 
 O.V. ROTAR, V.I. ROTAR PROC. NAP 2, 04NABM07 (2013) 
 
 
04NABM07-2 
Table 2 – Concentration Of Markers of Oxidant Injury and Antioxidants In Mucosal Layer Of Small Intestine 
 
 
Groups 
CS NPs, 
(n  15) 
CS / CD NPs, 
(n  15) 
Control, 
(n  15) 
Sham-operated, 
(n  15) 
GSH, mol/g 1,22  0,16c 2,32  0,14a,b 0,92  0,17c 2,62  0,08 
MDA, nmol/g 7,61  0,44 6,11  0,31a 8,88  0,29c 5,61  0,21 
DC, nmol/g 10,14  0,31c 9,04  0,31a 11,43  0,41c 8,13  0,21 
CAT, mol/min/g 375  21,4c 445  21,4 305  31,4c 542  11,4 
a) p  0,05 with control group; b) p  0,05 between CS and CS / CD NPs; c) p  0,05 with control group 
 
4. DISCUSSION 
 
CS and CS / CD NPs were prepared by ionic 
gelation in the presence of TPP, as described in the 
methodology section. The mechanism of formation of 
the nanosystems combines the electrostatic interaction 
between CS and CDs, which are oppositely charged, 
with the ability of CS to undergo a liquid-gel transition 
due to its ionic interaction with TPP. The low affinity of 
GSH to CS NPs matrix might be explained by the low 
molecular weight of the peptide and to the presence of 
only one net negative charge in its chemical structure. 
Formation of CD GSH complex before it incorporation 
in CS matrix increased encapsulation effeiciency of 
NPs system thus enhancing GSH delivery to tissue. 
CS has mucoadhesive properties so its nanosystems 
may be used for sustained drug delivery to mucosal 
layer of intestine [4]. CS / CD NPs were only 25 % 
loaded with GTH but their oral administration 
diminished ischemia-reperfusion injury of small 
inteatinal mucosa: concentration of MDA decreased on 
31,2 % (p  0,05), DC – on 20,9 % (p  0,05), but CAT 
increased on 45,9 (p  0,05). 
 
5. CONCLUSION 
 
CS NPs prepared by TPP gelation alone have low 
GSH capacity. Formation of CS / CD NPs increased 
affinity to GSH, enhanced its delivery to tissue and 
diminished intestinal mucosa ischemia-reperfusion 
injury. 
 
 
REFERENCES 
 
1. E. Valencia, A. Marin, G. Hardy, Nutrition 783 (2001).  
2. G. Sandri, M.C. Bonferoni, S. Rossi, et al., Eur. J. Pharm. 
Biopharm. 68, 65 (2007).  
3. S. Chopra, S. Mahdi, J. Kaur, et al., J. Pharmacy 
Pharmacology 58, 1021 (2006).  
4. P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso, 
Pharmacol. Res. 14, 1431 (1997).  
5. S.K.H. Khalil, G.S. El-Fekyb, S.T. El-Bannaa, W.A. Khalil, 
Carbohydrate Polymers 90, 1244 (2012).  
6. G. Ellman, Arch. Biochem. Biophysics, 32, 7130 (1959). 
 


English

Application of Chitosan / Cyclodextrin Nanoparticles for Tissue Glutathione Delivery

Electronic Sumy State University Institutional Repository

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  NANOMATERIALS: APPLICATIONS AND PROPERTIES Vol. 2 No 4, 04NABM07(2pp) (2013)   2304-1862/2013/2(4)04NABM07(2) 04NABM07-1  2013 Sumy State University Application of Chitosan / Cyclodextrin Nanoparticles for Tissue Glutathione Delivery  O.V. Rotar, V.I. Rotar  Bukovinian State Medical University, 2, Teatralna Sq., 58000 Chernivtsi, Ukraine  (Received 19 May 2013; published online 30 August 2013)  The aim of this study was to investigate an ability of chitosan nanoparticles for tissue delivery of the peptide glutathione. Formulations composed of chitosan or chitosan plus cyclodextrin-beta comlex were prepared. Reduced glutathione was loaded and delivered to mucosal layer of small intestine after ischemia-reperfusion injury more efﬁciently in chitosan/cyclodextrin-beta nanoparticles. From the data obtained, we believe that chitosan / cyclodextrin nanoparticles can be used for the oral administration of glutathione and other small peptides.  Keywords: Glutathion, Nanoparticles, Cyclodextrin, Chitosan.  PACS number: 87.85.Rs   1. INTRODUCTION   Glutathione (GSH) is the predominant intracellular non-protein thiol in a wide range of cells. It has important functions in the detoxification system of almost every cell type. The reversible oxidation of GSH provides electrons for the GSH-peroxidase catalyzed reduction of hydrogen peroxide or organic peroxides [1]. However, the application of GSH as a functional food ingredient has been limited because of its low bioavailability due to its poor cellular uptake and its instability. Under conditions of oxidative stress, the GSH thiol group can be easily oxidized to glutathione disulﬁde, which loses its antioxidant activity based on its radical scavenging activity. Nanoparticle (NP) prototypes from biodegradable polymers have been proposed for local drug delivery [2]. Among them, NPs based on the polysaccharide chitosan (CS) have shown particularly promising results due to their intrinsic properties including biocompatibility, mucoadhesion and ability to transiently open the tight junctions of the intestinal barrier [3].  2. MATERIAL AND METHODS  2.1 Materials  Chitosan (CS, Mw 600 kDa, degree of deacetylation 90 %) is a commercial product of Bioprogress (Russian Federation). Sodium tripolyphosphate (TPP) was purchased from Sigma (USA). Reduced glutathione and cyclodextrin-beta (CD) were purchased from Applichem (Germany). All other chemicals were of analytical grade and used as received. The water used in all experiments was double distilled.  2.2 Preparation of Nps  Preparation of NPs was based on the ionic gelation of CS with TPP anions [4] in 1st group (CS NPs) and CS with TPP and CD (CS / CD NPs) in 2nd group [5]. In all groups CS nanoparticles were prepared with CS concentration of 1 mg/ml and CS : TPP ratio 3 : 1. In both groups, CS was ﬁrst dissolved in acetic acid aqueous solution. The concentration of acetic acid in the aqueous media was 1.75 times that of CS. To 9 ml of CS 2 ml aqueous solution of 0,5 % w/v of GSH or 2 ml of mixture of GSH and CD in ratio 1 : 1 as 1.0 % w/v (2nd group) were added and mixed 45 min under magnetic stirring. Than TPP aqueous solution was added drop wise under magnetic stirring onto the corresponding amount of the CS solution at room temperature.   2.3 Encapsulation Efﬁciency of Nps  The prepared Nps suspension containing the GSH was centrifugated at 9,000 rpm for 90 min and free GSH in the supernatant was quantiﬁed by spectrophotometric method [6].   2.4 In Vivo Determination of Ability of NPs for Tissue GSH Delivery  To determine the ability of NPs deliver GSH to tissue intestinal ishemia-reperfusion injury were applied on 60 Wistar rats by clamping superior mesenteric artery for 60 min with 4 hours of reestablishing of blood circulation under ketamine anastesia. CS or CS / CD NP loaded with 3 mg of GSH were infused into jejunum just before ischemia period in two groups of animals and appropriate volume of normal saline in control animals (n  15 in every group). After reperfusion period animals were killed by sodium thiopental overdosing and concentration of GSH, malone dialdehyde (MDA), diene conjugates (DC) and catalase (CAT) in mucosal layer of small intestine were determined. Morphological examination of the NP was performed by transmission electron microscopy (TEM).  3. RESULTS   Regular and spherical morphology was revealed by TEM both for CS and CS / CD NPs with similar diameters. The capacity of the different NP systems to load GSH was determined: CS NPs presented encapsulation efﬁciency near 10 % and CS / CD NPs – 25 %. GSH was delivered to mucosal tissue in rat model of intestinal ischemia-reperfusion injury by CS / CD NPs on 34,9 % (p  0,05) higher level than CS NPs (Table 1), which was followed by decreasing of concentration of oxidant injury markers.  O.V. ROTAR, V.I. ROTAR PROC. NAP 2, 04NABM07 (2013)   04NABM07-2 Table 2 – Concentration Of Markers of Oxidant Injury and Antioxidants In Mucosal Layer Of Small Intestine   Groups CS NPs, (n  15) CS / CD NPs, (n  15) Control, (n  15) Sham-operated, (n  15) GSH, mol/g 1,22  0,16c 2,32  0,14a,b 0,92  0,17c 2,62  0,08 MDA, nmol/g 7,61  0,44 6,11  0,31a 8,88  0,29c 5,61  0,21 DC, nmol/g 10,14  0,31c 9,04  0,31a 11,43  0,41c 8,13  0,21 CAT, mol/min/g 375  21,4c 445  21,4 305  31,4c 542  11,4 a) p  0,05 with control group; b) p  0,05 between CS and CS / CD NPs; c) p  0,05 with control group  4. DISCUSSION  CS and CS / CD NPs were prepared by ionic gelation in the presence of TPP, as described in the methodology section. The mechanism of formation of the nanosystems combines the electrostatic interaction between CS and CDs, which are oppositely charged, with the ability of CS to undergo a liquid-gel transition due to its ionic interaction with TPP. The low afﬁnity of GSH to CS NPs matrix might be explained by the low molecular weight of the peptide and to the presence of only one net negative charge in its chemical structure. Formation of CD GSH complex before it incorporation in CS matrix increased encapsulation effeiciency of NPs system thus enhancing GSH delivery to tissue. CS has mucoadhesive properties so its nanosystems may be used for sustained drug delivery to mucosal layer of intestine [4]. CS / CD NPs were only 25 % loaded with GTH but their oral administration diminished ischemia-reperfusion injury of small inteatinal mucosa: concentration of MDA decreased on 31,2 % (p  0,05), DC – on 20,9 % (p  0,05), but CAT increased on 45,9 (p  0,05).  5. CONCLUSION  CS NPs prepared by TPP gelation alone have low GSH capacity. Formation of CS / CD NPs increased affinity to GSH, enhanced its delivery to tissue and diminished intestinal mucosa ischemia-reperfusion injury.   REFERENCES  1. E. Valencia, A. Marin, G. Hardy, Nutrition 783 (2001).  2. G. Sandri, M.C. Bonferoni, S. Rossi, et al., Eur. J. Pharm. Biopharm. 68, 65 (2007).  3. S. Chopra, S. Mahdi, J. Kaur, et al., J. Pharmacy Pharmacology 58, 1021 (2006).  4. P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso, Pharmacol. Res. 14, 1431 (1997).  5. S.K.H. Khalil, G.S. El-Fekyb, S.T. El-Bannaa, W.A. Khalil, Carbohydrate Polymers 90, 1244 (2012).  6. G. Ellman, Arch. Biochem. Biophysics, 32, 7130 (1959).  

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Application of Chitosan / Cyclodextrin Nanoparticles for Tissue Glutathione Delivery

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