Department of Irradiation Technology, Nuclear Research Center of Algiers, BP-399 Algiers, AlgeriaUSTHB, Faculty of Biological Sciences, Laboratory of Cellular and Molecular Biology, BP 32 El-Alia, Bab Ezzouar, Algiers, Algeria
a r t i c l e i n f o
rticle history:eceived 31 December 2012eceived in revised form 28 February 2013ccepted 28 March 2013vailable online xxx
Insulin is mainly administered via subcutaneous route by injection which is the cause of painful and pos-sible infections. Oral insulin administration would present a more convenient form of application becauseit is less invasive. Oral delivery of insulin to the gastrointestinal tract is one of the most challenging issues,because it numerous barriers to overcome in order to create an effective system for insulin delivery. In thepresent study, insulin-loaded alginate/chitosan blend gel beads were prepared with different mass ratios.Chitosan was depolymerized by gamma irradiation at a dose of 80 kGy reducing its molecular weight forideal blend with sodium alginate. The homogeneous solution of alginate and chitosan was dripped intoCaCl2 solution (2%), the resultant calcium crosslinked beads were dipped in glutaraldehyde (2%) solutionsequentially to prepare dual crosslinked beads with improved mechanical properties so as to withstandthe simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Morphological structure, FTIR analy-sis, thermogravimetry analysis, specific surface area, gel fraction, swelling kinetics in SGF and SIF, loadingefficiency, insulin release behavior, mucoadhesivity of the alginate/chitosan beads were investigated. Thecumulative insulin release of pure alginate beads (10:0) reached as maximum level 100% in 3 h after they
were dipped in SIF. Concerning the beads Alg/Chi (8:2), Alg/Chi (7:3) and Alg/Chi (6:4) the cumulativerelease of insulin reached 90.5%, 89.2% and 70.2%, respectively in 6 h. The rate of 100% was reachedafter 24 h for Alg/Chi (8:2), Alg/Chi (7:3) and after 73 h for Alg/Chi (6:4). The presence of chitosan in theblend beads decreased the cumulative insulin release in gastric media and enhanced behavior of algi-nate/chitosan beads in intestinal medium due to the crosslinking. The alginate/chitosan beads crosslinked
e con
by glutaraldehyde may b
. Introduction
Diabetes represents a sanitary grave threat that is considered ashe main cause of blindness, renal insufficiency, myocardial infarc-ion, cerebral vascular accident and amputation. In the world, morehan 300 million are affected by diabetes during the last decay [1].
Insulin is the most effective drug used to control blood glucoseevels. However, in clinical therapy, insulin is mainly administeredia subcutaneous route which is the cause of pain and possiblenfections, thus, leading to a higher patient compliance. The dis-omfort associated with this type of administration has led diabeticatient to neglect and even to give up the therapy. For this reason,
lternative procedures of insulin administration using differentoutes as buccal/sublingual [2], nasal [3], pulmonary [4–6], ocular7], rectal [8], transdermal [9,10] and oral [11] have been tested.
Oral insulin administration would present more convenientform of application, when compared to the other ways as it is lessinvasive. Furthermore, insulin would be directly transported to theliver after oral administration preventing the patient from periph-eral hyperinsulinemic side effects. However, several problems areencountered for the development of oral insulin delivery systemsto the blood stream, among which the degradation of insulin byproteolytic enzymes and acidic environment in stomach and thelow penetration of insulin across the lining of the intestine into theblood stream are the most constraining [12]. Several methods havebeen proposed to improve oral bioavailability and gastrointestinaluptake of poorly absorbable insulin, among them encapsulationwhich represents a promising concept using synthetic or naturalpolymers. Natural polymers (polysaccharides) have been chosenpredominantly for encapsulation of insulin because of many advan-tages of biodegradability, biocompatibility, non toxic effects and
safety. In this respect, the alginate an anionic and chitosan a cationicagent have been successfully used [13–16].
Alginate contains two uronic acids, �-(1-4) linked d-mannuronic acid (M) and �-(1-4) linked l-guluronic acid (G),
nd is composed of homopolymeric blocks M–M or G–G, andlocks with an alternating sequence of M–G blocks [17]. In addi-ion, sodium alginate has a unique property of crosslinking in theresence of multivalent cations, which is rather complex with–G sequences in the polymer chain to form the egg box junctions
18]. Alginate is a pH sensitive, stable in acidic pH of stomach, butt swells and starts dissolving in the intestinal alkaline pH [19].lginate has been considered as the most suitable biopolymer forncapsulation material allowing the entrapment of a wide rangef substances such as insulin [20].
Chitosan is a polysaccharide composed of mainly repeatingnits of � (1-4) linked N-acetyl-d-glucosamine that is derived fromhitin, a major component of the crustacean exoskeletons. It is a nonoxic and biocompatible cationic polysaccharide produced by par-ial deacetylation of chitin. These properties provide high potentialor many applications [21]. Chitosan is well known for its mucoad-esive properties, which may allow a prolonged interaction of theelivered drug with the epithelia membrane, facilitating more effi-ient absorption [22].
The major inconvenients of the use of pure alginate beads as arug delivery system are the rapid release of the loaded moleculesnd low drug encapsulation efficiency [23], which are attributedo the gel porosity that is large enough to cause leakage of theoaded drugs and further drug diffusion from the gel networko the CaCl2 solution [24]. In order to modify the encapsulationarameters and release profiles from pure alginate beads, severalethod and different polymers have been used in combinationith sodium alginate, such as chitosan [20,25], dextran sulphate
26] and pectin [27]. The polyelectrolyte complex between alginatend chitosan has been widely used in order to obtain microcapsulesontaining magnetic nanoparticles for controlled release of insulin25], other types of microspheres composed of alginate and dex-ran sulphate have been prepared by ionotropic gelation [26]. Inhese systems, also, the efficiency of encapsulation and release areirectly dependent on pore dimension of the polymeric network.asparakis and Bouropoulos have reported that coating of the cal-ium alginate beads with chitosan caused significant reduction oficro-cracks and macroscopic pores observed on the surface and,
hus, a decrease of its permeability [28].The aim of this study is to prepare a stable insulin loaded
lginate beads by reinforcement of the calcium alginate matrixith chitosan crosslinked by glutaraldehyde in order to improveechanical properties so as to withstand the simulated gastric fluid
SGF) and simulated intestinal fluid (SIF) with prolonged releaserofile of insulin in intestinal media.
. Materials and methods
.1. Materials
Sodium alginate and chitosan powder with initial moleculareight of 173 kDa and 324 kDa, respectively were purchased from
igma–Aldrich. The deacetylation degree of chitosan determinedy infrared spectroscopy method [29] was 75.5%. Glutaralde-yde (GLA) of analytical grade was purchased from Fluka. Human
nsulin (INSUDAL®, 100 IU/ml) was purchased from Saidal Com-any (Algeria). Male Albino Wistar rats were obtained from theharmaceutical Products Control Laboratory (Algeria).
.2. Preparation of blend gel beads
To ensure good mixing of the blend components, the molecu-ar weight of the chitosan was reduced by radiolysis using gammaadiation of Co-60. This was achieved by exposing the chitosan inhe solid state and in presence of air, to gamma rays at the dose rate
ical Macromolecules 58 (2013) 160– 168 161
of 59.11 Gy/min. At the absorbed dose of 80 kGy, the initial molec-ular weight of 324 kDa was reduced to 141 kDa. The blend solutioncontaining sodium alginate 2% (w/v) and chitosan was preparedwith different mass proportions of (6:4); (7:3); (8:2); (10:0). A givenamount of sodium alginate was dissolved in 40 ml distilled water atroom temperature under mechanical stirring. The chitosan powderwas added into the solution and mixed homogeneously. Chitosanwas dissolved by adding acetic acid 0.4 ml into the mixture; thepH was adjusted to 5. An homogeneous blend solution of the twobiopolymers was formed under stirring at room temperature for60 min. Firstly, the blend solution was dripped through an injec-tion needle into the solution of calcium chloride 2% (w/v). Smoothand spheric beads were formed under mechanical stirring for 4 h.Then, the gel beads were intensively rinsed with distilled water toremove any remaining calcium chloride. Secondly, the wet beadsobtained were suspended in glutaraldehyde solution (2%) for 48 h atroom temperature. Then, the beads were intensively washed withhot distilled water, air-dried, and stored at 4 ◦C.
2.3. Characterization of the alginate/chitosan beads
2.3.1. Beads size measurementThe size of the crosslinked dried alginate/chitosan beads was
calculated as the average value of the size of 300 beads using imageanalysis software Image J.
2.3.2. FTIR spectroscopyKBr pellets were prepared with alginate/chitosan beads (1%),
infrared spectra were measured using an FTIR Nicolet 380 spec-trophotometer. Spectra were recorded as an accumulation of 256scans at 4 cm−1 resolution.
2.3.3. Scanning electron microscopy (SEM)The morphological structure of the beads was examined under a
scanning electron microscopy (Philips XL 30 ESEM). Prior to obser-vation, samples were dried at room temperature and then coatedwith thin layer of gold.
2.3.4. Thermogravimetry analysisThe thermograms were obtained using a thermogravimeter
Instrument (SETARAM). Dried beads (40 mg) were crimped in astandard aluminum pan and heated from 30 ◦C to 400 ◦C at a heat-ing constant rate of 10 ◦C/min under constant purging with drynitrogen at 20 ml/min. All samples were run in duplicate.
2.3.5. Specific surface area (BET)The specific surface area of the gel beads was measured using a
BET Instrument. An amount of 300 mg of dried beads were degassedunder nitrogen atmosphere at 80 ◦C for 6 h.
2.3.6. Gel fractionThe beads were first dried at room temperature for 72 h, then
in vacuum oven at 60 ◦C until a constant weight (Wi) was reached.The sol fraction was extracted by autoclaving the beads for 2 h in 5%acetic acid solution at 120 ◦C and 1 bar pressure. Then, the extractedbeads were dried at 60 ◦C to a constant weight (WF). The gel fraction(G%) was determined as the ratio of the dry gel weight before (Wi)and after (WF) autoclaving.
The swelling studies of 100 g of alginate/chitosan beads werecarried out in 10 ml SGF pH 1.2 during 2 h at 37 ◦C [30], then
162 D. Tahtat et al. / International Journal of Biological Macromolecules 58 (2013) 160– 168
F ) Wetc
tdu
S
Sib
2
2s4
2
dppttuw
2
psSsTti
2
Ro
TC
ig. 1. Photographs of the typical beads at different steps of their preparation. (arosslinked with glutaraldehyde. (c) Dry alginate/chitosan beads.
ransferred in SIF pH 6.5 at 37 ◦C for 4 h [31]. All experiments wereone in triplicate. The swelling rate of the beads was calculatedsing the formula 2 [13]:
% = Wt − Wo
Wo× 100 (2)
% is the swelling rate, Wt is the weight of the beads at appropriatentervals in saline buffer and Wo is the weight of absolutely driedeads.
.4. Loading of insulin by diffusion filling method
An amount of 500 mg of alginate/chitosan beads was dropped in0 ml human insulin (100 IU/ml) and kept for 4 h at 4 ◦C under mildtirring. Excess insulin was filtered off and the beads were dried at◦C [19].
.5. Insulin content and loading efficiency
To determine the insulin content in the beads, 20 mg ofry insulin loaded beads were completely dissolved in 10 ml ofhosphate buffer at pH 7.4, under magnetic stirring at room tem-erature. After full dissolution of the beads, the insulin content inhe filtered supernatant was assayed at 272 nm. Blank beads (con-rol) were also dissolved, and then the filtered supernatants weresed as blanks in order to avoid interference [2]. All the samplesere analyzed in triplicate.
.6. Release of insulin in vitro
One hundred milligrams (100 mg) of insulin loaded beads werelaced in 20 ml of release medium and incubated at 37 ◦C undertirring. The release media was SGF (pH 1.2) for 2 h and then inIF (pH 6.5) for 4 h [19]. At appropriate time intervals, 2 ml of theolution was removed for analysis and replaced by fresh medium.he amount of insulin released from the beads was evaluated spec-rophotometrically at 272 nm [30]. All the samples were analyzedn triplicate.
.7. Estimation of in situ mucoadhesivity
Bioadhesivity was tested with in situ method as described byao and Buri [32] with minor modifications. A freshly cut long piecef small intestine (10 cm) of a rat was cleaned by washing it with
able 1haracteristics of alginate/chitosan beads as a function of components mass ratio.
Alginate/chitosan mass ratio Mean size (mm) Specific surface are
(10:0) 1.03 ± 0.22 2.17
(8:2) 1.10 ± 0.15 2.11
(7:3) 1.56 ± 0.25 1.54
(6:4) 1.48 ± 0.17 1.09
alginate/chitosan beads crosslinked with CaCl2. (b) Wet alginate/chitosan beads
isotonic saline. Twenty beads were placed in contact within themucosal surface. Intestine piece was maintained at 37 ◦C and 80%relative humidity for 120 min. The intestine piece was taken out andflows phosphate buffers (pH 6) go through the intestine piece for2 min at 20 ml/min. The perfusate was collected to get no adherentbeads.
2.8. Statistical analysis
The obtained results were treated statistically by the applica-tion of the t-test for a comparison between the various arithmeticmeans and by the calculation of the central tendency and dispersionparameters.
3. Results and discussion
3.1. Morphology and size
Spherical and oval beads were obtained with sodium algi-nate/chitosan solution at 2%. After the first step that consistsof alginate crosslinking, white and opaque beads were obtained(Fig. 1a). After the second step, the beads became of a light browncolor, due to the crosslinking of chitosan with glutaraldehyde(Fig. 1b). The diameter of beads increased progressively with theincrease of the concentration of chitosan in the blend. The bead wasrelatively homogenous in size. The coefficient of variation betweenmean size and standard deviation was less than 0.33 (Table 1).
The comparison of mean size between beads Alg/Chi (10:0) andother mass ratio was done by the calculation of the variable test (ε)that has given superior values at εa = 1.96, which indicates that thedifference was significant, due to the presence of chitosan in blend.The difference is also significant between Alg/Chi (8:2) and Alg/Chi(6:4), whereas it was not significant between Alg/Chi (8:2)–Alg/Chi(7:3) and between Alg/Chi (7:3)–Alg/Chi (6:4), probably due to alow difference in the concentration of chitosan in the blend.
3.2. FTIR spectrum
Infrared spectra of alginate, chitosan, and alginate–chitosan
blend are illustrated in Fig. 2. The FTIR spectrum of alginatebeads showed carboxyl peaks near 1603 cm−1 (symmetric COOstretching vibration) and 1427 cm−1 (asymmetric COO stretchingvibration) [20] (Fig. 2a). The peaks at 1084 cm−1 and 1031 cm−1
a (m2/g) Micropore volume (×10−4 cm3/g) Gel fraction (%)
8.00 41.007.93 64.325.46 74.014.27 82.80
D. Tahtat et al. / International Journal of Biological Macromolecules 58 (2013) 160– 168 163
orresponded to stretching vibration of C O of uronic acids Gnd M. The main characteristic peaks of chitosan at 2877 cm−1
C H stretch), 1665 cm−1 (N H bend), 1319 cm−1 (C N stretch),155 cm−1 (bridge O stretch) and 1074 cm−1 (C O stretch) arehown in Fig. 2b. In the spectrum of alginate/chitosan beadsroadened and slightly shifted from 1603 cm−1 to 1615 cm−1
nd 1427 cm−1 to 1417 cm−1 after complexation with chitosanFig. 2c). New absorption peaks were also observed at 2875.2 cm−1
ttributed to stretching vibration of CH2 group of chitosan. Thencrease in the absorption bands of the amino, carboxyl, and amideroups can be attributed to the ionic interaction between the car-onyl group of alginate and the amino group of chitosan [33].
In the spectrum of insulin loaded alginate/chitosan beadsFig. 2d) and insulin subtracted from insulin loaded Alg/Chi6:4) beads as shown in Fig. 2e, several peaks were observed at852 cm−1 (C H stretch), 1725 cm−1 associated with (C O)tretching, 1472.8 cm−1 and 1383.9 cm−1 (C O stretch),284.8 cm−1 and 1246.1 cm−1 (C O carbonyl compounds),80.5 cm−1, 927.4 cm−1 and 781.1 cm−1 (C H stretch), whichorrespond to characteristic interaction peaks between insulinnd alginate/chitosan beads. Insulin contains several ionizableroups, due to six amino acid residues capable of attaining aositive charge of amine group of chitosan and 10 amino acidesidues capable of attaching a negative charge of carboxylic groupf alginate. This could be responsible for the entrapment of insulinnto the beads [34].
.3. Scanning electron microscopy (SEM)
Pure alginate beads Alg/Chi (10:0) showed a relatively smoothurface and had a regular shape, homogenous and opaque withome wrinkles and fissures (Fig. 3). After reinforcing the struc-ure with chitosan, the alginate/chitosan beads revealed a texture
odification. The beads showed a heterogeneous surface and anrregular shape with less wrinkles and fissures. These morpholog-
cal changes indicated that chitosan has interacted well with thelginate, modifying the bead initial textures by physicochemicalnteractions. During the preparation of the alginate/chitosan blendeads, the pH of the solution was adjusted to 5, which increased
the interaction between carboxylic groups of alginate and aminegroups of chitosan [35]. The texture of the alginate/chitosan beadswas finally a result of electrostatics interactions.
3.4. DTA and DSC analysis
The thermograms of derivative thermal analysis of algi-nate/chitosan beads with different mass ratio are shown inFig. 4a. Three peaks ranging between 45–170 ◦C, 170–254 ◦C, and254–360 ◦C were observed. The first two peaks of Alg/Chi (8:2),Alg/Chi (7:3) and Alg/Chi (6:4) overlap with peaks of Alg/Chi (10:0),but the weight loss (mg/min) increased with the decrease of chi-tosan ratio in the blend. Whereas the 3rd peak was shifted by about40 ◦C from 295 to ≈251 ◦C compared to the control Alg/Chi (10:0).This decrease of temperature was attributed to the presence ofchitosan in the beads.
The thermograms of alginate/chitosan beads show initialbroaden endothermic peaks at 125 ◦C and 200 ◦C and higherexothermic peaks at 300 ◦C for Alg/Chi (10:0) (Fig. 4b). The increaseof the mass ratio of chitosan in the beads induced the shift ofendothermic peaks values from 125 ◦C to 100 ◦C, and exothermicpeaks values from 300 ◦C to 244 ◦C, 256 ◦C and 270 ◦C for Alg/Chi(6:4), Alg/Chi (7:3) and Alg/Chi (8:2), respectively. This could repre-sent the coalescence of both isolated endothermic and exothermicpolymer peaks, resulting from individual contribution of alginateand chitosan [20,36]. The endothermic peaks are correlated withthe loss of water associated to the hydrophilic groups of polymers,while the exothermic peaks resulted from the degradation of poly-electrolytes due to dehydration and depolymerization reactions,most probably to the partial decarboxilation of the protonatedcarboxylic groups and oxidation reactions of the polyelectrolytes[36–38].
Thermograms of insulin loaded alginate/chitosan beads areshown in Fig. 5. The endothermic peaks values were broadenedand shifted from 108 ◦C to 100 ◦C, and the exothermic peaks val-
ues from 270 ◦C to 259 ◦C, but the endothermic peak at 208 ◦C wasunchanged. It was reported that the thermogram pattern of insulinshowed two endothermic peaks at 62.3 ◦C and 83 ◦C, and a tinyand broad exothermic peak at 251.1 ◦C [39]. The two endothermic
164 D. Tahtat et al. / International Journal of Biological Macromolecules 58 (2013) 160– 168
0), (d
pgbaitslTip
Fig. 3. SEM micrographs of alginate/chitosan beads. (a–c) Alg/Chi (10:
eaks attributed to insulin became indistinct in a broaden sin-le peak after the insulin was loaded into the alginate/chitosaneads, and reached this endothermic condition at lower temper-ture values about 100 ◦C compared to unloaded beads, thus, annteraction between the protein from insulin and the polyelec-rolytes from alginate and chitosan may be occurred [20]. Theame observation was made for the exothermic peak of insulinoaded alginate/chitosan beads compared to the unloaded beads.
he peak shifted and started at 259 ◦C, due to the presence ofnsulin, where the decomposition of insulin started at a lower tem-erature 251.1 ◦C compared to the unloaded beads.
The BET specific surface area and micropore volume of algi-nate/chitosan beads are summarized in Table 1. The value of theBET specific surface area of alginate beads (10:0) was significantlyhigher than that of the other formulations. The increase of themass ratio of chitosan in the blend has led to the decrease ofboth the specific surface area and the micropore volume of the
alginate/chitosan beads. This observation denotes that the purealginate beads (10:0) are characterized by homogeneous, regularand porous matrix. After reinforcing the structure with chitosan,
0 50 100 150 200 250 300 350 400 450
-60
-50
-40
-30
-20
-10
0
10
20
30
40
300°C
200°C
125°C
(b)
Exo (
µV
)
Temperature (°C)
Alg/Chi (10:0)
Alg/Chi (8:2)
Alg/Chi (7:3)
Alg/Chi (6:4)
san beads (a) DTA and (b) DSC.
D. Tahtat et al. / International Journal of Biological Macromolecules 58 (2013) 160– 168 165
0 50 100 150 200 250 300 350 400 450
-30
-20
-10
0
10
270 °C259 °C
108 °C
100 °C200 °C
Exo
(µ
V)
Alg/Chi (8:2)
Insuli n lo ade d A lg/Chi (8:2)
tbse
3
mpttttTt
tatttetcsw
3
a6apgliwoc
eas
0 2 4 6 8 10 12 14 16 18 20 22 24
0
100
200
300
400
500
600
700
800
SIF (pH=6.5)
SGF
(pH=1 .2)
De
gre
e o
f sw
elli
ng
(%
)
Time ( hours)
Alg/Chi (10:0)
Alg/Chi (8:2)
Alg/Chi (7:3)
Alg/Chi (6:4)
Temperature (°C)
Fig. 5. Thermograms of insulin loaded alginate/chitosan beads.
he beads matrix became irregular and less porous, due to the distri-ution of chitosan within the alginate gel, which reduces its specificurface area and porosity [26]. These results confirm the scanninglectron microscopy (SEM) observations.
.6. Gel fraction
The variation of gel fraction of the beads as a function of theass ratio is shown in Table 1. The gel fraction is the insoluble
art of the polymer resulted by crosslinking. It was observed thathe gel fraction increased with the increase of chitosan content inhe blend beads. The latter led to a significant increase in gel frac-ion, due to an increase of crosslinking density of chitosan, thus,he structure becomes insoluble whatever the pH of the solution.he crosslinking enhances the mechanical strength, chemical resis-ance and stability of the beads in acidic and alkali media.
The mechanism of chemical crosslinking of chitosan chains withhe bifunctional glutaraldehyde occurs by a Schiff’s reaction ofldehyde groups on glutaraldehyde with amine groups on the chi-osan biopolymer chain [40]. The increase of chitosan content inhe blend promotes the crosslinking process. As a consequence,he gel fraction increases. The presence of chitosan seems to influ-nce significantly the gel fraction. The Alg/Chi beads (6:4) showedhe highest gel fraction (about 83%), thus, a very high density ofrosslinking which induced the decrease of pores size, and specificurface area. These results are in agreement with those obtainedith BET and SEM characterization.
.7. The swelling behavior of the beads in SGF and SIF
The swelling kinetics of alginate/chitosan beads in SGF and SIFre shown in Fig. 6. The swelling ratio increased during the first
h, and then slightly decreased. This decrease which was observedfter 24 h then after 72 h could be attributed to the effect of theH on the structure of the beads. Indeed at pH ∼ 7 starts a pro-ressive decomplexation of the blend, i.e. the dissociation of theinks between the amino groups and hydroxyl groups. This resultsn a progressive dissociation of chitosan molecules from alginate,
hich leads to the disintegration of the beads. The swelling degreef alginate/chitosan beads decreased with the increase of chitosanontent in the blend.
The beads with the lowest chitosan content of Alg/chi (8:2)xhibited the highest degree of swelling. It is well known that thelginate bead is stable in acidic pH of stomach, but it swells andtarts dissolving in the intestinal alkaline pH > 6 [19]. The addition
Fig. 6. Swelling kinetics of alginate/chitosan beads in SGF and SIF at 37 ◦C.
of chitosan in alginate beads aims at reinforcing their structure,thus, increasing their stability in the intestinal alkaline pH. Thebead swells by the absorption of water, which is kept in the net-work structure of polymer. Water uptake is the highest when thenetwork is connected by a relatively low number of intermolecularbonds and it decreases with crosslinking density increase, due tothe formation of covalent links between macromolecular chains ofalginate and chitosan, which induces the formation of dense threedimensional networks.
In SGF medium, the swelling degree of pure alginate beadsAlg/Chi (10:0) increased considerably versus time from 100% to200%. The t-test showed that there is a significant difference in thedegree of swelling between the first hour and the second hour. Forthe beads Alg/chi (8:2), Alg/Chi (7:3) and Alg/Chi (6:4), the swellingdegree increased slightly from 100% to 120% during the first hour,and stabilized during the second hour due to the crosslinked chi-tosan which conferred a dense structure to the beads. In acidicmedia (SGF), 95% of amino groups exist in chitosan as positivelycharged NH3
+ [41] and all carboxylic groups exist in alginate as neg-ative charges COO−, in pure alginate beads these charged groups(COO−) along the chains exert repulsive force, which, consequently,extends the network and loosen up the structure of the polymer.Therefore, it can absorb more water. In addition, the increase in theion concentration in the matrix makes water easy to diffuse intothe structure due to the thermodynamic driving forces, leading to ahigher swelling ratio which is higher in the acidic solution [42]. Butthe blend of chitosan and alginate, allows an increase of ionic inter-action between amine groups of chitosan and carboxylic groups ofalginate, which contributes to the decrease of the swelling rate. Thisfact is attributed to a more rigid network of the blend beads formedby inter and intra-polymer chain bonds that have been formed byionic interaction and chitosan crosslinking.
In SIF media, pure alginate beads Alg/Chi (10:0) dissolvedrapidly, due to the chelating action of sodium ions in sodiumhydrogenocarbonate (NaHCO3) buffer solution with pH 6.5. Theaffinity of sodium for calcium is higher than that of alginate [13].Swelling rate of the beads Alg/Chi (8:2), Alg/Chi (7:3) and Alg/Chi(6:4) increased considerably to reach after 6 h values of 550%, 220%and 200%, respectively, then decreased slightly due to the decom-plexation of the beads after 24 h except for Alg/Chi (6:4) beads. Asexpected, the beads with the highest chitosan content showed the
lowest swelling ratio. The obtained result is in agreement with thegel fraction result.
In Fig. 7 are shown the gel fraction and the degree of swelling ofalginate/chitosan beads as a function of the component mass ratio.
166 D. Tahtat et al. / International Journal of Biological Macromolecules 58 (2013) 160– 168
(10:0) (8:2) (7:3) (6:4)150
200
250
300
350
400
450
500
550
600
650
700 Degree of swelling in SIF
Gel frac tion
Alginate/c hit osan mass ratio
De
gre
e o
f sw
elli
ng
(%
)
30
40
50
60
70
80
90
Ge
l fractio
n (%
)
Ff
IitwmattsA
3
iipisIbiabw
F4
0 2 4 6 8 10 12 14 16 18 20 22 24
10
20
30
40
50
60
70
80
90
100
110
120
130
SIFSGF
Insu
lin c
um
ula
tive
re
lea
se
(%
)
Time (hours)
Alg/Chi (10:0)
Alg/Chi (8:2)
Alg/Chi (7:3)
Alg/Chi (6:4)
ig. 7. Gel fraction and degree of swelling in SIF of alginate/chitosan beads as aunction of the mass ratio.
t was observed that the swelling equilibrium decreased with thencreasing chitosan content in the blend beads, but the gel frac-ion increased. The higher chitosan content in the beads, the loweras the swelling degree and the higher was gel fraction. The defor-ations produced by water pressure on macromolecular chains
re less important; therefore the diffusion of water into the struc-ure decreased consequently, the swelling degree decreased. The-test showed that there is a significant difference in the degree ofwelling in SIF between the beads Alg/Chi (8:2), Alg/Chi (7:3) andlg/Chi (6:4).
.8. Insulin loading efficiency
The insulin loading efficiency of the alginate/chitosan beadss shown in Table 2. The highest retention value was observedn the beads Alg/Chi (6:4) the lowest value was observed in theure alginate beads. The entrapment of insulin increased with the
ncrease of chitosan content in the beads. Efficiency of entrapmenteemed to be independent from the beads swelling behavior.n Fig. 8 is shown the swelling degree of the alginate/chitosaneads in phosphate buffer at 4 C◦ and pH 7.2, simulating the
nsulin loading condition by the diffusion filling method. The purelginate beads (10:0) presented the highest degree of swellingut the lowest entrapment, whereas the alginate/chitosan beadsith mass ratio (6:4) showed a low degree of swelling and high
0 1 2 3 4 5 6 7 8
0
500
1000
1500
2000
2500
Sw
elli
ng
de
gre
e (
%)
Time (hours)
Alg /Chi (10:0)
Alg /Chi (8:2)
Alg /Chi (7:3)
Alg /Chi (6:4)
ig. 8. Swelling degree of the beads alginate/chitosan in phosphate buffer pH 7.2 at◦C.
Fig. 9. Insulin cumulative release of alginate/chitosan beads.
entrapment. The addition of chitosan into alginate beads led toa higher retention of insulin rather than in pure alginate beads.The physical entrapment of insulin in the beads structure isenhanced by the high crosslinking density favored by the presenceof chitosan. The electrostatic interaction between the protein andthe polyanion might also favor the retention of insulin within thebeads; insulin monomer contains many ionizable groups, due tosix amino acid residues capable of attaining a positive charge and10 amino acid residues capable of attaching a negative charge[34]. These properties are, therefore, possibly responsible for theentrapment of insulin into alginate/chitosan beads [20].
For better appreciation of the amount of insulin encapsulated,the conversion to IU/100 mg of beads was necessary, 1 IU cor-responds to 0.035 mg of insulin, the appropriate insulin dosage(0.5–1 IU/kg per day) (Table 2). For many type 2 patients, the timecourse of insulin action requires two or three injections per day tomeet glycemic goals. For an average weight body of 60 kg, insulinadministration dosage will be 30–60 IU per day, which means,10–20 IU per administration. Insulin entrapped by different massratio of beads Alg/Chi corresponds to this dosage, which can achievethe individual appropriate insulin dosage recommended by thePharmacopeia.
3.9. Insulin release behavior in SGF and SIF
The insulin release behavior of alginate/chitosan beads wasstudied in SGF and SIF. The obtained results are represented inFig. 9. It can be observed that the cumulative release of insulinincreased progressively with time increasing. For the pure alginatebeads Alg/Chi (10:0) the cumulative release reached 100% in 3 hafter being dipped in SIF due to the full disintegration of the beadsin pH 6.5. This result was predicted in the swelling study where thetotal solubilization of the pure alginate beads was observed.
For the beads Alg/Chi (8:2), Alg/Chi (7:3) and Alg/Chi (6:4), thecumulative release reached 90.5%, 89.2% and 70.2%, respectively in6 h. The rate 100% was reached after 24 h for Alg/Chi (8:2), Alg/Chi(7:3) and 73 h for Alg/Chi (6:4), which is in agreement with theobservation made in the swelling study in SGF and SIF. The cumu-lative release of insulin seems to be proportional to the swellingrate of the beads.
In SGF media, after 2 h, the cumulative release of insulin was51.9%, 36.4%, 39.2%, and 44% for Alg/Chi (10:0), Alg/Chi (8:2),Alg/Chi (7:3) and Alg/Chi (6:4), respectively. Except for pure algi-nate beads (10:0), the cumulative release was relatively less than
D. Tahtat et al. / International Journal of Biological Macromolecules 58 (2013) 160– 168 167
Table 2Loading efficiency and cumulative release of insulin of alginate/chitosan beads.
Alginate/chitosanmass ratio
Loading efficiency(mg/100 mg beads)
Insulin concentration(IU/100 mg beads)
Cumulative releasein SGF and SIF (IU)
Releasein SIF (IU)
(10:0) 0.81 23.07(8:2) 1.86 53.06
(7:3) 2.12 60.57
(6:4) 2.16 61.71
Table 3Percentage of adhered beads into rat intestine.
Alginate/chitosan mass ratio Adherence percentage (%)
(10:0) 35 ± 7(8:2) 90 ± 0
4ntr
iA(
iTpAtttgt
3
arTtcccthermtdacdot
4
min
[
[
[[
[
[
[
[[
[
[
[
(7:3) 90 ± 14(6:4) 95 ± 7
5% which allows the release of the remaining insulin in the intesti-al media. The presence of chitosan in the blend beads decreaseshe cumulative release in gastric media due to the crosslinkingeaction.
In SIF media, after 6 h, the cumulative release of insulinncreased with time, the highest release level was observed withlg/Chi (8:2) beads, the lowest release was obtained with Alg/Chi
6:4) beads.The conversion of cumulative release in IU allowed an appreciat-
ng of the quantity of insulin released in the intestinal media in 6 h.he obtained results are shown in Table 2. The Alg/Chi (7:3) beadsresented the highest amount of cumulative release 30.29 IU, whilelg/Chi (6:4) beads showed the lowest release 16.11 IU. The quan-
ity of insulin released (IU) in the intestinal medium (SIF) withouthe quantity released in the gastric medium (SGF) was enough tohe recommendation of the appropriate insulin dosage to meet thelycemic goals ranging between 10 IU and 20 IU per administra-ion.
.10. Mucoadhesion estimation
Bioadhesion is defined as the ability of a material to adhere to biological tissue for an extended period of time. The obtainedesults for the bioadhesion of alginate/chitosan beads is shown inable 3. The adherence percentage of alginate/chitosan beads tohe mucosal surface of the rat intestine was between 90% and 95%ompared to the pure alginate beads which represent a low per-entage of adherences (35%). The increase of mass ratio of chitosanontent in the blend of beads has enhanced the bioadhesivity ofhe beads alginate/chitosan. Chitosan is well known for its bioad-esive nature. The mechanism of bioadhesion of chitosan could bexplained by the fact that in the cationic form, the d-glucosamineesidue of chitosan could interact with the sialic acid residues ofucin (mucosal surface of the small intestine of the rat) by elec-
rostatic interaction. This allows a prolonged interaction of theelivered insulin with the membrane epithelia; thus, facilitating
more efficient absorption [19]. The prolonged mucoadhesion ofhitosan could increase the absorption of insulin at mucosal sitesue to a prolonged interaction with the membrane epithelia or thepening of the tight junctions between the cells to facilitate theransport of the insulin [22].
. Conclusion
In this study alginate/chitosan blend gel beads with differentass ratio were prepared, loaded with insulin and tested for the
nsulin release. The beads were prepared by dripping an algi-ate/chitosan blend solution into calcium chloride, transferring the
[[
[
48.02 28.7054.03 30.2943.32 16.11
calcium crosslinked gel beads into glutaraldehyde 2% to crosslinkthe chitosan in view to strengthen their mechanical propriety inSGF and SIF. In vitro, insulin release studies showed for the purealginate beads (10:0) the cumulative release reached 100% in 3 hafter it was dipped in SIF whereas for the beads Alg/Chi (8:2),Alg/Chi (7:3) and Alg/Chi (6:4), the cumulative release reached90.5%, 89.2% and 70.2% respectively in 6 h. The rate of 100% wasreached after 24 h for Alg/Chi (8:2), Alg/Chi (7:3) and 73 h forAlg/Chi (6:4). The cumulative quantity of insulin released (IU) inthe intestinal media (SIF) was enough to meet the glycemic goals.The beads Alg/Chi (8:2), Alg/Chi (7:3) and Alg/Chi (6:4) could beused with a wide range of insulin dosages and at different times ofrelease. The alginate/chitosan beads crosslinked by glutaraldehydemay be considered as potential oral insulin carriers.
Acknowledgements
The authors would like to acknowledge the help of Y. Ham-mache, D. Haddad, S. Khemaïsia and A. Azzouz from NuclearResearch Center of Draria for DTA, DSC analysis and BET measure-ments.
References
[1] R. Williams, L. Van Gaal, C. Lucioni, Diabetologia 45 (7) (2002) 13–17.[2] A. Portero, T. Osorio, M.J. Alonso, C. Remunan-Lopez, Carbohydrate Polymers
68 (2007) 617–625.[3] S. Yu, Y. Zhao, F. Wu, X. Zhang, W. Lu, H. Zhang, Q. Zhang, International Journal
of Pharmaceutics 281 (2004) 11–23.[4] J. Farr Stephen, A. McElduff, E. Mather Laurence, J. Okikawa, M.E. Ward, I. Gonda,
V. Licko, R.M. Rubsamen, Diabetes Technology and Therapeutics 2 (2) (2000)185–197.
[5] J.S. Patton, J. Bukar, S. Nagarajan, Advanced Drug Delivery Reviews 35 (2/3)(1999) 235–247.
[6] H. Schultz, Pharmaceutical Science & Technology 1 (1998) 336–344.[7] D.R. Owens, Nature Reviews Drug Discovery 1 (7) (2002) 529–540.[8] O. Yoshinori, M. Mariko, T. Kozo, T. Sinji, C. Yoshiyuki, I. Koichi, N. Tsuneji,
International Journal of Pharmaceutics 198 (2) (2000) 147–156.[9] R. Rastogi, S. Anand, A.K. Dinda, V. Koul, Drug Development and Industrial
Pharmacy 36 (8) (2010) 993–1004.10] W. Chien Yie, O. Siddiqui, W.M. Shi, P. Lelawongs, J.C. Liu, Journal of Pharma-
ceutical Sciences 78 (5) (1989) 376–383.11] Y. Zhang, W. Wei, L.V. Piping, W. Lianyan, M. Guanghui, European Journal of
Pharmaceutics and Biopharmaceutics 77 (2011) 11–19.12] V.H.L. Lee, A. Yamamoto, Advanced Drug Delivery Reviews 4 (1990) 171–207.13] Y. Xu, C. Zhan, L. Fan, L. Wang, H. Zheng, International Journal of Pharmaceutics
336 (2007) 329–337.14] C.M. Silva, A.J. Ribeiro, I.V. Figueiredo, A.R. Gonc alves, F. Veiga, International
Journal of Pharmaceutics 311 (2006) 1–10.15] S. Honary, M. Maleki, M. Karami, Tropical Journal of Pharmaceutical Research
8 (1) (2009) 53–61.16] S. Maitil, S. Ranjitl, B. Sa, International Journal PharmTech, Research 2 (2) (2010)
1350–1358.17] A. Haug, B. Larsen, O. Smidsrod, Carbohydrate Research 32 (1974) 217–225.18] B. Thu, P. Bruheim, T. Espevik, O. Smidsrød, P. Soon-Shiong, G. Skjåk-Bræk,
Biomaterials 1711 (1996) 1069–1079.19] T. Manoj Kumar, W. Paul, C.P. Sharma, M.A. Kuriachan, Trends in Biomaterials
& Artificial Organs 18 (2) (2005) 198–202.20] B. Sarmento, D. Ferreira, F. Veiga, A. Ribeiro, Carbohydrate Polymers 66 (2006)
25] P.V. Finotelli, D. Da Silva, M. Sola-Penna, A. Malta Rossi, M. Farina, L.R. Andrade,A.Y. Takeuchi, M.H. Rocha-Leão, Colloids and Surfaces B 81 (2010) 206–211.
26] S. Martins, B. Sarmento, B.S. Eliana, C.F. Domingos, Carbohydrate Polymers 69(2007) 725–731.
27] J. Bajpai, A. Bajpai, S. Mishra, Journal of Macromolecular Science. Part A: Pureand Applied Chemistry 43 (2006) 165–186.
28] G. Pasparakis, N. Bouropoulos, International Journal of Pharmaceutics 323(2006) 34–42.
29] D. Tahtat, C. Uzun, M. Mahlous, O. Guven, Nuclear Instruments and Methods inPhysics Research Section B 265 (2007) 425–428.
30] Y.C. Nho, S.E. Park, H.I. Kim, T.S. Hwang, Nuclear Instruments and Methods inPhysics Research Section B 236 (2005) 283–288.
31] I.M. Barmpalia-Davs, I. Geornaras, P.A. Kendall, J.N. Sofos, Applied and Environ-ment Microbiology 74 (2008) 5563–5567.
32] K.V. Ranga Rao, P. Buri, International Journal of Pharmaceutics 52 (1989) 265.33] A.J. Ribeiro, C. Silva, D. Ferreira, F. Veiga, European Journal of Pharmaceutical
Sciences 25 (1) (2005) 31–40.
[[
[
ical Macromolecules 58 (2013) 160– 168
34] J. Brange, Galenics of Insulin: The Physico-chemical and PharmaceuticalAspects of Insulin and Insulin Preparations, Springer, Berlin, 1987.
35] J. Berger, M. Reist, J.M. Mayer, O. Felt, R. Gurny, European Journal of Pharma-ceutics and Biopharmaceutics 57 (2004) 35–52.
36] T. Gazori, M.R. Khoshayand, E. Azizi, P. Yazdizade, A. Nomani, I. Haririan, Car-bohydrate Polymers 77 (2009) 599–606.
37] T. Mimmo, C. Marzadori, D. Montecchio, C. Gessa, Carbohydrate Research 340(2005) 2510–2519.
