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Author(s): Reza Mahjub, Moojan Radmehr, Farid Abedin Dorkoosh, Seyed Naser Ostad, and Morteze Rafiee-Tehrani
Article title: Lyophilized insulin nanoparticles prepared from quaternized N-aryl derivatives of chitosan as a newstrategy for oral delivery of insulin: in vitro, ex vivo and in vivo characterizations
Article no: LDDI_A_841187
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Prefix Given name(s) Surname Suffix
1 Reza Mahjub
2 Moojan Radmehr
3 Farid Abedin Dorkoosh
4 Seyed Naser Ostad
5 Morteze Rafiee-Tehrani
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http://informahealthcare.com/ddiISSN: 0363-9045 (print), 1520-5762 (electronic)
Drug Dev Ind Pharm, Early Online: 1–15! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.841187
ORIGINAL RESEARCH PAPER
Lyophilized insulin nanoparticles prepared from quaternized N-arylderivatives of chitosan as a new strategy for oral delivery of insulin:in vitro, ex vivo and in vivo characterizations
Reza Mahjub1, Moojan Radmehr1, Farid Abedin Dorkoosh1, Seyed Naser Ostad2, and Morteze Rafiee-Tehrani1,3
1Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran, 2Department of Pharmacology and
Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran, and 3Nanotechnology Research Center, Faculty of Pharmacy,
Tehran University of Medical Sciences, Tehran, Iran
Abstract
Objective: The purpose of this research was the development, in vitro, ex vivo and in vivocharacterization of lyophilized insulin nanoparticles prepared from quaternized N-arylderivatives of chitosan.
Q4 Methods: Insulin nanoparticles were prepared from methylated N-(4-N,N-dimethylaminobenzyl),methylated N-(4 pyridinyl) and methylated N-(benzyl). Insulin nanoparticles containing non-modified chitosan and also trimethyl chiotsan (TMC) were also prepared as control. The effectsof the freeze-drying process on physico-chemical properties of nanoparticles were investigated.The release of insulin from the nanoparticles was studied in vitro. The mechanism of the releaseof insulin from different types of nanoparticles was determined using curve fitting. Thesecondary structure of the insulin released from the nanoparticles was analyzed using circulardichroism and the cell cytotoxicity of nanoparticles on a Caco-2 cell line was determined.Ex vivo studies were performed on excised rat jejunum using Frantz diffusion cells. In vivostudies were performed on diabetic male Wistar rats and blood glucose level and insulin serumconcentration were determined.Results: Optimized nanoparticles with proper physico-chemical properties were obtained. Thelyophilization process was found to cause a decrease in zeta potential and an increase in PdI aswell as and a decrease in entrapment efficiency (EE%) and loading efficiency (LE%) butconservation in size of nanoparticles. Atomic force microscopy (AFM) images showed non-aggregated, stable and spherical to sub-spherical nanoparticles. The in vitro release studyrevealed higher release rates for lyophilized compared to non-lyophilized nanoparticles.Cytotoxicity studies on Caco-2 cells revealed no significant cytotoxicity for preparednanoparticles after 3-h post-incubation but did show the concentration-dependent cytotoxicityafter 24 h. The percentage of cumulative insulin determined from ex vivo studies wassignificantly higher in nanoparticles prepared from quaternized aromatic derivatives ofchitosan. In vivo data showed significantly higher insulin intestinal absorption in nanoparticlesprepared from methylated N-(4-N, N-dimethylaminobenzyl) chitosan nanoparticles comparedto trimethyl chitosan.Conclusion: These data obtained demonstrated that as the result of optimized physico-chemicalproperties, drug release rate, cytotoxicity profile, ex vivo permeation enhancement andincreased in vivo absorption, nanoparticles prepared from N-aryl derivatives of chitosan can beconsidered as valuable method for the oral delivery of insulin.
Keywords
Caco-2 cell cytotoxicity, circular dichroism,ex vivo and in vivo studies, insulin oraldelivery, lyophilized nanoparticles,methylated N-(4-N,N-dimethylaminobenzyl) chitosan,methylated N-(4 pyridinyl) chitosan,methylated N-(benzyl) chitosan, transportefficiency
History
Received 4 May 2013Revised 3 August 2013Accepted 29 August 2013Published online 2 2 2
Introduction
Insulin, like most peptides, is classified as a hydrophilic andmacromolecular drug that obviously considered as low perme-able and inadequate stable compound in gastrointestinal (GI)tract1. Therefore, parentral way is believed to be the main route
of administration of insulin so far. The parentral administrationhas several disadvantages including low patient compliance,high risk of infection, pain, trauma and also high cost ofpreparation2. In addition, considering the fact that liver andportal circulation are the first target sites of insulin, it has beenshown that parentral administration of exogenous insulin fails tosimulate the physiological fate of endogenously secreted insulindue to accumulation of the drug in peripheral circulation ratherthan portal circulation3–5.
A non-invasive route of administration of insulin is inhalationthat considered as one of the alternative routes for subcutaneous
Q5 Address for correspondence: Morteza Rafiee-Tehrani, School of Phar-macy, Tehran University of Medical Sciences, P.O. Box 14395/459,Tehran Postal Code 14, Tehran, Iran. Tel: +98 21 66964209, +98 912309-2832. E-mail: [email protected]
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delivery. It has been proven that insulin molecules can beabsorbed to the blood stream after inhalation from the alveoli6.Exubera� is considered as the first pulmonary insulin that wasintroduced to the market in 2006 but due to some failures, it hasbeen withdrawn from the market in 2007. Technosphere� insulinor Afreeza� is an inhaled insulin product that is currently inclinical development7. Afreeza� is considered as the ultra rapidacting insulin8. There are some concerns for inhalation deliveryof insulin including dose–response characteristics of inhaledinsulin, intra-subject variability, long term safety, rising titersof antibodies, diminished lung functions in some patients, lowbioavailability and high cost7–9. Insulin is such an expensivesubstance and therefore it is important to increase the efficacy ofdelivery system to reduce the costs.Q1
Therefore, among the other routes of administration, oraldelivery of insulin is the most preferred issue. The major obstaclesfor oral delivery of insulin are summarized as: harsh acidic mediaof stomach, hydrolytic enzymatic activity in the different regionsof GI such as stomach, intestinal lumen and intestinal brushborders cause degradation the peptides, the presence of tightjunctions in intestinal epithelium that act as a barrier forparacellular permeation and also the low mean residence time(MRT) of chemicals through intestinal lumen that decrease thetime of exposure of insulin to absorption site10. Several strategieshave been reported to overcome these problems includingapplication of permeation enhancers, enzyme inhibitors, chemicalmodification, mucoadhesive polymers and development of lipidbase carriers including liposomes, niosomes, microparticles andalso solid lipid nanoparticles11,12.
The application of nanoparticles for enhancing the permeabil-ity and consequently bioavailability of non-parentral delivery ofhydrophilic macromolecular drugs such as peptides and proteinshas been widely investigated.
Chitosan as the non-toxic, biocompatible and biodegradablecationic polysaccharide poses high positive charge density at pHvalues 55.5, thus it can adhere to negatively charged surfacesincluding mucus membranes which results in mucoadhesiveproperties of the polymer13–15. Chen et al.16 have reported theenhancing effect of chitosan on intestinal absorption.
Considering the Pka of 6.4, in neutral and basic environments,chitosan molecules lose their positive charge and becomeinsoluble and percipitate. In order to overcome this majordrawback of chitosan, quaternized derivatives of this polymerhave been synthesized. These derivatives pose permanent positivecharge due to quaternized amino functional group, and cause thepolymer be soluble in wide range of pH values17. Several studieshave reported the application of quaternized derivatives ofchitosan for delivery of peptides and proteins18–20.
Some studies have reported higher transfection efficiency ofaromatic derivatives of chitosan for gene delivery purposes toHuh 7 cell lines in comparison to chitosan and TMC21,22. Theyshowed that by increasing the degree of aromatic substitution asthe hydrophobic group, the transfection efficiency of the deliverysystem would be increased suggesting the presence of hydropho-bic interaction between polymer and cell surface in addition toelectrostatic interaction of the positively charged polymer onnegatively charged sites of the cell membranes21–23. Kowapraditet al.24 have reported the high transport efficiency across Caco-2cell monolayer using amino-benzyl chitosan as free-solublepolymer. Based on these finding, it was assumed that preparationof nanoparticles from N-aryl derivatives can enhance paracellularabsorption of insulin across intestinal epithelium.
The main aim of the present study was preparation of noveland effective insulin oral delivery system suing nanoparticlesprepared form quaternized N-aryl derivatives of chitosan. In thisstudy, the N-aryl derivatives of chitosan were used for preparation
of insulin nanoparticles; nanoparticles were characterized andeffect of lyophilization process on physico-chemical properties ofnanoparticles and also the release profile of insulin fromnanoparticles have been investigated; The release pattern weremathematically modeled; the released insulin from nanoparticleswere studied by circular dichroism (CD) for detection of anychanges in secondary structure of insulin during the formulationprocesses such as the electrostatic interaction between polymerand insulin and lyophilization process; the cytotoxicity ofnanoparticles was investigated by MTT cell cytotoxicity assayon Caco-2 cell culture; ex vivo studies were performed on excisedrat jejunum for determination of transport efficiency of preparednanoparticles and also in vivo studies were done on male wistarrats for investigation of the absorption of insulin nanoparticles.
Materials and methods
Materials
Chitoclear� chitosan [viscosity, 1% (w/v) solution in acetic acid,22 mPa s, molecular weight value of 120 KDa] was purchasedfrom Primex (Siglufjordur, Iceland). Crystaline recombinanthuman insulin was provided from Lilly (France). Human coloncarcinoma cell line (Caco-2) was obtained from American TypeCulture Collection (Rockville, MD). Culture media includingRPMI 1640, Dulbecco’s modified Eagle medium (DMEM), FetalBovine Serum (FBS) were purchased from Gibco (Grand Island,NY). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-ditetrazolium bromide(MTT) was purchased from Sigma–Aldrich (France). Sucrose wasprovided from NP Pharm (Bazainville, France). Mannitol wasobtained from Helm AG (Hamburg, Germany). Glucose wasprovided from Nanjing Zelang Medical Technology Co. (Nanjing,China). Trehalose was obtained from Omicron Biochemicals (IN).Simulated intestinal fluid (SIF) was prepared using potassiumdihydrogen phosphate (0.05 M) and sodium hydroxide (0.2 M)adjusted to pH value of 6.8. Phosphate buffered saline (PBS) wasprepared using sodium chloride (137 mM), potassium chloride(2.7 mM), disodium hydrogen phosphate (10 mM) and potassiumdihydrogen phosphate (2 mM) adjusted to pH value of 7.4.
The quaternized aromatic derivatives of chitosan includingmethylated N-(4-N,N-dimethylaminobenzyl) chitosan, methylatedN-(4-pyridinyl) chitosan and methylated N-(benzyl) chitosan thatwere designated as methylated (aminobenzyl) chitosan, methy-lated (pyridinyl) chitosan and methylated (benzyl) chitosan,respectively, with the degree of aromatic substitution of 37%,42%, 34%, degree of N-aliphatic quaternization of 46%, 43% and52% and degree of N-aromatic quaternization of 17%, 4% and 0%,respectively, were prepared and characterized in our laboratory asreported previously25. Tri-Methyl Chitosan (TMC) has beensynthesized in our lab according to previous reports26 and thedegree of N-quaternization were determined by 1H-NMRSpectroscopy and reported to be 43%. Further characterizationsof synthesized derivatives have been shown on Table 1.
All other reagents were pharmaceutical or analytical grade andwere used as received.
Preparation and characterization of nanoparticles
Insulin nanoparticles were prepared by the polyelectrolyte com-plexation (PEC) method with 5 ml of insulin solution (1 mg/ml,pH¼ 8.0) added drop wise to an equal volume of polymersolution under gentle magnetic stirring. For improved curing ofthe particles, the obtained opalescent colloidal nano-suspensionwas continually stirred for 20 min. The suspension was centri-fuged at 15 000 rpm for 20 min at 4 �C. The separated nanopar-ticles were reconstituted in freshly prepared distilled water andthe supernatant was discarded. The obtained nanoparticles were
2 R. Mahjub et al. Drug Dev Ind Pharm, Early Online: 1–15
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characterized using a Zetasizer 3000 HS (Malvern instrument,Worcestershire, UK) at 25 �C.
Insulin content in nanoparticles
The reconstituted nanoparticles were immersed in 1 N HClaqueous solution for 7 min. The particles were degraded andtheir total content of insulin was released to the acidic aqueousmedia as the previous reports27. The total insulin content of theparticles was determined by detection of insulin using HPLC.
Determination of entrapment efficiency (EE%) and load-ing efficiency (LE%)
The direct method was used to determine the EE% and LE%. Inthis method the insulin content in nanoparticles was determinedand calculated according to following equations:
EE% ¼ Insulin content in nanoparticle
Total amount of Insulin� 100 ð1Þ
LE% ¼ Insulin content in nanoparticle
Total weight of nanoparticle� 100: ð2Þ
HPLC analysis
Insulin was analyzed using the Agilent� 1260 infinity equippedwith 1260 Quat pump VL, 1260 ALS auto sampler and 1260 DADVL detector that was set at 214 nm.Q1 MZ� analytical Perfect SilTarget� ODS�3 (150 mm� 4.6 mm, 5mm) column was used forliquid chromatography.Q1 The mobile phase was consisted of buffer:acetonitrile (70:30). The buffer was contained monobasic potas-sium phosphate (0.1 M) and triethylamine (5% v/v). The pH of thebuffer was adjusted to 2.7 using ortho-phosphoric acid(Darmstadt, Merck). The flow rate was set at 0.8 ml/min.
Optimization of nanoparticles by D-optimal responsesurface methodology
As previously reported by our group, prepared nanoparticles fromaromatic derivatives of chitosan were optimized by D-Optimalresponse surface methodology25. The nanoparticles were char-acterized by their size, zeta potential, PdI and also entrapmentefficiency (EE%). Design-Expert� software (V. 7.0.0, Stat Ease,Inc., Minneapolis) has been used for modeling and optimization.
The nanoparticles composed of TMC and also non-modifiedchitosan were not applied to D-Optimal response surface and wereoptimized using changing one separate factor at the time.
Lyophilization of the nanoparticles
Before lyophilization, optimized nanoparticles were freezed at�20 �C for overnight. Lyophilization was preceded by usingLyotrap Plus (LTE Scientific Ltd, Oldham, UK). For freezedrying, as previous reports, samples were dried for 48 h at aworking pressure of 0.07 mbar at condenser temperature of�46 �C28. Freeze drying was performed using different lyopro-tectants including sucrose, mannitol, glucose and treholose with
various concentrations (i.e. 1%, 2%, 5%, 7% and 10% (w/v). Afterlyophilization, the lyophilized particles were reconstituted indistilled water and the size of particles was measured usingMalvern� instrument. Q1For each colloidal suspension, the conser-vation ratio was calculated by dividing the size of particles afterlyophilization to the size of particles before lyophilization. Allmeasurements were performed in triplicate. Based on thesescreening studies, sucrose (5% w/v) was used as the lyoprotectantfor freeze drying of nanoparticles in further studies.
Determination of morphology of particles
For determination of the morphology of the nanostructures, theprepared particles were re-suspended in freshly prepared Milli-Qwater. The morphology of the dried samples was analyzed byatomic force microscopy (AFM). The topographical images wereacquired and analyzed by Dual-Scope� (V. 2.1.1.2, Danish MicroEngineering, Copenhagen, Denmark).
For further investigation on the morphology, the reconstitutedlyophilized nanoparticles were studied by transmission electronmicroscopy (TEM) using a CEM 902A (Zeiss, Oberkochen,Germany). In this technique, the size of nanoparticles wasdetermined by direct observation.
In vitro release studies
The in vitro release of insulin from optimized nanoparticles wasstudied in simulated intestinal fluid (SIF) adjusted to pH value of6.8 as the simulated medium of apical side of the intestinalepithelium and phosphate buffered saline (PBS) adjusted to pHvalue of 7.4 representing the basolateral side.
For the freeze drying process, the appropriate amounts oflyophilized or non-lyophilized nanoparticles (equivalent to200 mg of insulin) were collected and incubated in 100 ml ofbuffer using a water bath shaker (Memmert�, Germany) whilemaintaining the temperature at 37� 1 �C and an agitation rate of100 rpm in a sink conditioned medium. At scheduled timeintervals, 1 ml of the medium was collected and immediatelyreplaced by preheated freshly prepared buffer. The collectedsamples were centrifuged at 15 000 rpm for 20 min and theamount of insulin in the supernatant was detected by highperformance liquid chromatography (HPLC).
Circular dichroism analysis
For determination of the effects of formulation procedures onsecondary structure of insulin, Circular Dichroism (CD) studieswere performed on insulin released from nanoparticles. The CDspectra were measured at room temperature using a J-810spectropolarimeter from Jasco, Inc. (Easton, MD). CD spectrawere compared with spectrum of standard insulin.
Release kinetic studies
Various kinetic models were used to fit the in vitro release datausing mathematical analysis.
Table 1. Characteristics of chitosan and synthesized derivatives.
PolymerDegree of aromatic
substitution (%)
Degree ofquaternization(Aliphatic) (%)
Degree ofquaternization(Aromatic) (%)
Recovery(%)
Mw(kg/mol)
Zeta potential(mean� SD)
Methylated(amino-benzyl) chitosan 37 46 17 83 138 56.1� 1.68Methylated (pyridynyl) chitosan 42 43 4 71 140 49.7� 2.27Methylated (benzyl) chitosan 34 52 – 78 138 36.7� 1.42Chitosan – – – – 142 23� 1.21
DOI: 10.3109/03639045.2013.841187 Insulin nanoparticles from N-aryl derivatives 3
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In order to perform curve fitting, all experimentally obtaineddata were fitted to various mathematical models including zero-order kinetic model, first order kinetic model, Higuchi’s squareroot of time equation, Korsmeyer–Peppas’ power law equationand Hixson–Crowell’s cube root of time equation. In order toselect the best-fitted model, the correlation coefficient (R2) andalso adjusted correlation coefficient (adj-R2) for each proposedmodel were determined.
The zero-order kinetic can be considered in pharmaceuticalformulations which do not disintegrate and release the drugslowly and constantly29,30. This condition can be expressed byEquation (3).
Mt
M1 ¼ K0t: ð3Þ
Where Mt/M1 is the fractional amount of drug release,t onsidered to be the time and K0 s the kinetic constant that isdetermined by fitting the proposed model.
The first order kinetic is assumed in systems in which the rateof drug release is dependent on the amount of drug remaining inthe matrix31,32.
The first order release kinetic can be represented by theEquation (4), considering that K1 s the first order proportionallyconstant.
Mt
M1 ¼ 1� eð�K1tÞ: ð4Þ
The Hixson–Crowell release kinetic describes a drug deliverysystem that their dimensions diminish proportionally in a mannerthat the geometric shape of the dosage form stays constant as drugrelease is occurred33,34. In this kinetic behavior, the rate of drugrelease can be mathematically express as the Equation (5).
ffiffiffiffiffiffiffi
M03p
�ffiffiffiffiffiffi
Mt3p
¼ Khc � t: ð5Þ
Where M0 s the initial amount of the drug in the delivery systemMt s the amount of drug released at determined time t nd Khc s aconstant incorporating the surface-volume relation.
In Higuchi release theory, it is assume that a powdered drug ishomogenously dispersed throughout the matrix of an erodiblepolymer. The drug will dissolve in the polymer matrix and diffuseout from the surface of the device35. In order to study the releasepattern from such systems the relation obtained as Equation (6)can be applied while Kh s assumed to be the Higuchi dissolutionconstant.
Mt
M1 ¼ Kh �ffiffi
tp: ð6Þ
The Korsmeyer–Peppas equation represents the power lawrelease kinetic that relates the drug release to elapsed time and hasbeen described in Equation (7).
Mt
M1 ¼ Kp � tn: ð7Þ
Where Kp s a constant incorporating structural and geometriccharacteristic of the delivery system and n s the releaseexponent36,37.
Caco-2 cell culture
Caco-2 cells were obtained from the American Type CultureCollection (passage number of 5). Cells were cultured on 25 cm2
Nunc plastic flasks (Roskilde, Denmark). The medium consistedof 50% v/v of RPMI 1640, 34% v/v of Dulbecco’s modified Eaglemedium (DMEM) supplemented with 1% v/v non-essential aminoacids, 15% v/v of fetal bovine serum (FBS) and 1% v/v ofpenicillin-streptomycin (100 U/ml). Cells were incubated in a
humidified atmosphere containing 5% CO2 and 95% air at 37 �C.The culture medium was changed every second day. Cells werepassaged after 7 days when reaching the desired confluency.Cells with passage numbers of 30–45 were used for cytotoxicityassay studies.
Evaluation of cytotoxicity using MTT assay
The cytotoxic effects of synthesized polymers in free-soluble formand also prepared nanoparticles were studied on Caco-2 cell lineby the MTT cell cytotoxicity assay at the cell density of 1� 104
cells per well on 96-well microplate. After formation ofmonolayer, the cells were exposed to the synthesized aromaticpolymers in free-soluble form or the prepared nanoparticles atvarious concentration of 0.01–5 mM of polymer in Hank’sbalanced salt solution (HBSS) buffered with n-(2-hydroxyethyl)piperazine-n-(2-ethanosulfonic acid) (HEPES) at pH 7.4 for 3 or24 h. As the polymers including amino-benzyl, pyridinyl andbenzyl chitosan are considered as quaternized derivatives ofchitosan, the studied polymers were soluble in cell culturemedium with pH value of 7.4. Non-modified chitosan wasexcluded for the cytotoxicity assay due to insolubility andprecipitation in the stated pH.
The non-treated cells are considered to be as the control andtheir related viability was assumed to be 100%. The related IC50,the drug concentration at which the inhibition of 50% cell growthwill occur, was calculated by the curve fitting of the cell viabilitydata using Prism� software (V. 4.0 Graphpad, San Diego, CA).
Evaluation of cytotoxicity using lactate dehydrogenase(LDH) assay
Cells were seeded on 96-well micro plates (Nunc, Denmark)at the cell density of 1� 105 cells per well. After formation of themonolayer, the cells were incubated samples including preparednanoparticles at various concentration of 0.01–3.00 mM ofpolymer in HBSS/HEPES for the period of 24 h. The freshmedium was used as control. After specified time, the supernatantwas collected for detection of LDH released from damaged cellsusing a Cytotoxicity detection kit (Roche Diagnostics, Manheim,Germany) according to the manufacturer specifications.
Ex vivo studies on excised rat jejunum
All animal procedures were approved by an ethical committee forthe use of animals in research and were in full compliance withthe standard international ethical guidelines.
A segment of duodenal intestine was removed from maleWistar rats under phenobarbital anesthesia. Fresh rat jejunum wasplaced between the donor and acceptor chamber of Frantzdiffusion cells and were thermostated at 37 �C.
The colloidal suspension of prepared nanoparticles wasdiluted 1:2 with Hanks Balanced Salt Solution (HBSS) bufferedat pH of 6.0. Each sample was placed in the donor chamber whilea blank solution of HBSS buffered at pH 7.4 was used in theacceptor phase.
After 3 h of incubation, samples were taken from the acceptorchamber and the amount of insulin in the collected samplesdetermined using HPLC. The results were reported as thecumulative percentage of insulin transported across the jejunum.
In vivo studies
All experiments were undertaken in accordance with theFederation of European Laboratory Animal ScienceAssociation. Male Wistar rats (200–250 g) were housed undercontrolled temperature conditions and fed laboratory animalstandard diet with water provided ad libitum.
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Prior to the study, the rats were rendered diabetic by a singleintraperitoneal injection of 60 mg/kg of streptozocine in isotoniccitrate buffer. After 4 days, rats with frequent urination andfasted blood glucose level 4250 mg/dL were randomly selectedand separated into four groups (n¼ 6). The selected rats werefasted for 24 h before the experiments but were provided freeaccess to water.
Lyophilized nanoparticles were administered orally to ratsusing pre-clinical capsules (Capsugel�, Austria). The capsuleshad been previously enteric the coated using Acryl-EZE� aqueousenteric system (Colorcon, Idstein, Germany). Freeze driednanoparticles prepared from aminobenzyl chitosan and trimethylchitosan were administrated at an equivalent dose 30 U/kg ofinsulin. The positive and negative control groups were adminis-tered subcutaneously injected insulin (5 U/kg) and orally admin-istered free form insulin in enteric coated capsules(30 U/kg),respectively.
At predetermined time intervals, blood samples (0.3 ml) weretaken from a tail vein. Samples were separated into two equalportions for the separate measurement of plasma glucose leveland serum insulin concentration, separately. Samples were takenat time zero, just prior to administration of test samples, toestablish base glucose and insulin levels.
The plasma glucose level was determined using a glucometer(Abbot, Portugal); serum insulin concentration was measuredusing an enzyme-linked immunosorbent assay (ELISA) test kit(Mercoddia, Uppsala, Sweden). The data obtained were plottedagainst time and were used to determine any treatmentdifferences.
Statistical analysis
All experiments were performed in triplicate and the relatedvalues were reported as mean� SD. For better analysis, in vivostudies were performed in six times (n¼ 6). Statistical signifi-cance of differences was evaluated by one-way analysis ofvariance (ANOVA) with appropriate post hoc tests in the case ofcomparison between several groups or Student’s independentsamples t-test in the case of comparison between just two groupsusing SPSS� (V. 19.0.0, IBM Statistics, NY). The differenceswere considered significant when p50.05.
Results
Preparation and characterization of nanoparticles
The prepared particles were optimized by using the D-Optimalresponse surface methodology. Table 2 illustrates the optimizedformulation characteristics for the preparation of the nanoparti-cles. The 3-D response surface graph of desirability versuspolymer pH and concentration ratio of polymer/insulin is shownin Figure 1(a–c). The physico-chemical properties of theoptimized nanoparticles including size, zeta potential, PdI, EE%and LE% are summarized in Table 3.
The size of the particles is identified as one the maincharacteristics of the nanoparticles. It has been demonstrated that
by decreasing the size of the particles, the permeability across theintestinal epithelium will be increased38,39.
Table 3 shows the zeta potential results. The chitosannanoparticles showed the lowest zeta potential value of all theother types of nanoparticles at 16.1� 1.31 mV. The zeta potentialof the other nanoparticles were in descending order 28.5� 1.98,26.7� 1.10 and 21.8� 2.62 mV for methylated (amino-benzyl),methylated (pyridinyl) and methylated (benzyl) chitosan,respectively.
As shown in Table 3, nanoparticles prepared from methylated(pyridinyl) chitosan showed the highest EE% and LE% while thelowest levels were observed in non-modified chitosan nanopar-ticles. The order of decreasing the EE% and LE% of particleswere as:
methylated pyridinylð Þchitosan4methyalted
amino� benzylð Þchitosan4
methylated benzylð Þchitosan4 tri�methyl chiotsan TMCð Þ4 chitosan
Freeze drying of the nanoparticles
The effect of different lyoprotectants in conservation of the size ofprepared nanoparticles has been shown on Figure 2(a–c). Asshown in the figure, using sucrose (5% w/v) as lyoprotectantduring freeze drying, the minimum conservation ratio in all typesof nanoparticles was observed.
Table 4 illustrates the physico-chemical related properties ofnanoparticles after lyophlization. Statistical analysis showedsignificant differences in zeta potential, PdI, EE% and LE% inall types of nanoparticles before and after lyophilization(p50.05). Particle size showed a slight increase after lyophiliza-tion but it was not significant (p40.05). The ratios of the size ofnanoparticles after lyophilization to the size beforehand rangedbetween 1.05 and 1.37, clearly indicating good conservation ofparticle size during the lyophilization process.
In vitro release of insulin from nanoparticles
The in vitro release of insulin from optimized nanoparticles,before and after freeze drying, was studied in SIF adjusted to pHvalue of 6.8 and PBS adjusted to pH value of 7.4. The releaseprofile of different types of prepared nanoparticles is shown inFigure 3(a and b). Comparing the release profile of nanoparticlesbefore and after lyophilization (shown on Figure 3a and b), it canbe clearly observed that the lyophilized nanoparticles achievehigher rate of drug release than insulin nanoparticles beforelyophilization. This, accords well with previous studies40,41.Statistical analysis has shown significant differences in thepercentages of insulin released from nanoparticles before andafter lyophilization in all types of nanoparticles (p50.05) exceptin the case of insulin released from non-modified chitosan whichshows significant differences in burst release (p50.05) but a non-significant difference in the total amount of release (p40.05).
The effect of pH on the amount of insulin released fromnanoparticles has been shown in Figure 3(b and c). Independentsample t-test statistical analysis has shown that the burst release ofinsulin from nanoparticles was significantly higher in PBScompared to SIF (p50.05). The same results were observedin the statistical analysis of the total percentage of insulin releaseat 600 min.
Visualization of nanoparticles
In this study, AFM was used for acquiring images from thesurface of the nanoparticles. In order to determine the
Table 2. Optimized formulations of the prepared nanoparticles.
Optimized independent variables
Polymer typePolymer
pHConcentration
ratio
Methylated (amino-benzyl) chitosan 5.0 0.61Methylated (pyridinyl) chitosan 3.9 1.02Methylated (benzyl) chitosan 4.0 1.09Chitosan 4.5 1.00
DOI: 10.3109/03639045.2013.841187 Insulin nanoparticles from N-aryl derivatives 5
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Figure 1. 3-D response surface plot of desirability function versus polymer pH and concentration ratio: (a) methylated N-(4,N,N-dimethylaminobenzyl) chitosan, (b) methylated N-(4-pyridinyl) chitosan and (c) methylated N-(benzyl) chitosan.
6 R. Mahjub et al. Drug Dev Ind Pharm, Early Online: 1–15
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morphology of the particles, topographical images within a scanarea of 10 mm� 10mm were acquired. Figure 4(a–c) shows thenon-aggregated, spherical to sub-spherical homogenously dis-persed nanoparticles from quaternized aromatic derivatives ofchitosan were obtained. The particles were observed to beseparated from each other with no signs of aggregation, suggest-ing the preparation of stable poly-electrolyte complexes. Fordetermination of the size of particles, cross-section analysis wasperformed on AFM images. As shown in Figure 5(a–c), the sizesof 371, 312 and 343 nm were calculated for nanoparticlesprepared from amino-benzyl chitosan, pyridinyl chitosan andbenzyl chitosan, respectively.
As well as AFM images, images acquired using TEM shownon-aggregated, spherical particles (Figure 6). As shown in thefigures, the sizes of the particles are in nano scale range.
In vitro release kinetics
The results of curve fitting indicated that the insulin releaseobserved fitted best to the Korsmeyer–Peppas model innanoparticles prepared from aromatic derivatives; as well asTMC with R2 values ranging from 0.9729 to 0.9875. Innanoparticles prepared from non-modified chitosan, the insulinrelease was best fitted to the first order kinetics with relativeR2 values of 0.9460 (data not shown).
The data and a comparison of the proposed models for drugrelease indicate that freeze drying does not change the mechanismof drug release.
Circular dichroism (CD) study
The secondary structure of in vitro released insulin fromnanoparticles prepared from quaternized aromatic derivatives ofchitosan has been studied using circular dichroism (CD). Thespectrum of a 0.1 mg/ml insulin solution in phosphate buffer wasanalyzed as standard. Figure 7 shows that the spectrum ofstandard insulin shows two minima at 209 and 222 nm, repre-senting the a-helix structure of the protein42,43. The far-UV CDspectra of insulin released from the freeze dried nanoparticlesprepared from methylated (amino-benzyl), methylated (pyridinyl)and methylated (benzyl) chitosan showed minor differences tothe standard insulin solution but revealed no significant changesin secondary structure of the insulin due to the electrostaticinteraction of the protein and polymer or the freeze dryingprocess.
Cytotoxicity assay using MTT
The cytotoxicity of synthesized derivatives and the preparednanoparticles on Caco-2 cell cultures were investigatedusing MTT.
The results showed that all the synthesized quaternized N-arylderivatives of chitosan polymers in free solution form, and also asprepared nanoparticles; showed no signs of cytotoxicity in Caco-2cells incubated for 3 h with IC50 values45 mM.
As shown in Figure 8(a and b), all of the studied derivatives aswell as the appropriate nanoparticles, showed a concentrationdependent cytotoxicity in Caco-2 cells incubated for 24 h. IC50values are shown on Tables 5 and 6.
Statistical analysis has shown significant differences betweenIC50 values for the different polymers and for the different typesof nanoparticles (p50.05).
The results showed a higher cytotoxicity for nanoparticlescompared to synthesized derivatives (p50.05). It seems thatpolymeric nanoparticles have a greater ability to permeate cellscompared to corresponding free-soluble polymers. This increasedpermeation results in nanoparticles causing higher cytotoxicitycompared to the free-soluble polymer.
Cytotoxicity assay using LDH
The cytototxicity of prepared nanoparticles was examined usinglactate dehydrogenase (LDH) cytotoxicity assay. The LDHactivity of damged cells after exposure to different concentrationof nanoparticles has been shown on Figure 9. As illustrated in thefigure, all nanoparticles exhibit dose dependent cytotoxicity inwell-accordance with MTT results. Statistical analysis by one-wayanalysis of variance showed no significant difference in LDHactivity for control and nanoparticles samples with the polymerconcentration in the range of 0.01–0.05 in all types ofnanoparticles (p40.05). By increasing the concentration ofpolymer, the nanoparticles showed significant dose-dependentcytotoxocity (p50.05). The obtained results from LDH assayconfirm the data provided by MTT assay.
Ex vivo studies
The cumulative amount of insulin transported across excised ratjejunum was used as the indicator of transport efficiency and wasdetermined for freeze dried insulin nanoparticles prepared fromamino-benzyl, pyridinyl, benzyl chitosan as quaternized N-arylderivatives, TMC as quaternized aliphatic derivative, non-modified chitosan and also for insulin in free form.
As illustrated in Figure 6, after 180-min incubation of thediffusion cells at 37 �C, the cumulative percentage of insulintransported across the intestinal barrier using insulin nanoparti-cles prepared from amino-benzyl chitosan (42.1� 2.86%) wassignificantly higher than for insulin nanoparticles prepared fromother polymer types (p50.05). Corresponding values for othertypes of nanoparticles were 31.6� 1.85%, 26.7� 3.64%,18.4� 2.7%, 9.76� 1.3% and 1.6� 0.19% for insulin nanoparti-cles prepared from pyridinyl chitosan, benzyl chitosan, TMC,non-modified chitosan and insulin in free form, respectively.Nanoparticles prepared from all types of quaternized N-arylderivatives showed significantly higher transport efficiencycompared to TMC (p50.05) (Figure 9). In accordance withprevious reports, nanoparticles prepared from TMC showedhigher transport efficiency than nanoparticles prepared fromchitosan. The higher positive charge density of nanoparticlesprepared from TMC and consequently higher ability to open tight
Table 3. Physico-chemical characteristics of nanoparticles, before lyophilization (n¼ 5).
Physico-chemical characteristics (Mean� SD)
Polymer type Size (nm) Zeta potential (mV) PdI EE (%) LE (%)
Methylated (amino-benzyl) chitosan 339� 37 28.5� 1.98 0.208� 0.03 70.3� 3.27 23.9� 2.93Methylated (pyridinyl) chitosan 315� 49 26.7� 1.10 0.279� 0.02 84.5� 4.45 27.5� 1.76Methylated (benzyl) chitosan 279� 25 21.8� 2.62 0.340� 0.03 68.1� 3.30 15.8� 2.51Tri-methyl chiotsan (TMC) 264� 37 19.4� 1.67 0.253� 0.04 65.7� 4.21 13.1� 1.43Chitosan 326� 31 16.1� 1.31 0.325� 0.05 56.8� 3.74 11.4� 2.06
DOI: 10.3109/03639045.2013.841187 Insulin nanoparticles from N-aryl derivatives 7
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junctions might explain the observed increase in transportefficiency of these nanoparticles.
The results show that the transport efficiency of nanoparticlesincreases in the order of: chitosan5TMC5benzyl chitosan5pyridinyl chitosan5amino-benzyl chitosan.
In vivo studies
Figures 10 and 11 show an in vivo comparison of the hypogly-cemic effects of insulin loaded nanoparticles prepared frommethylated N-(4-N, N-dimethylaminobenzyl) chitosan with insu-lin nanoparticles composed of TMC (well-known, extensivelydescribed oral drug delivery system) at the loading equivalentdose level of 30 U/kg, subcutaneously injected insulin at 5 U/kgand enteric coated capsules filled with free form of insulin at
30 U/kg. Figure 11 depicts the changes in blood glucose level withFigure 12 showing the serum insulin concentration profile. Q3
Out of three types of N-aryl derivatives of chitosan, insulinnanoparticles prepared from amino-benzyl chitosan was selectedfor in vivo studies as the result of their higher transport efficiencyin ex vivo experiment.
As expected, there was no significant reduction in blood glucoselevel following administration of free form insulin, demonstratinglow oral absorption of insulin in free form (Figure 10).
Subcutaneous administration of insulin caused a significantand rapid decrease in blood glucose level, reaching a valueof 25.8� 6.24% of the base level 3-h post-administration.After that, the blood glucose level gradually increased till avalue of 68.77� 13.59% of the base level obtained 8-h post-administration.
Figure 2. Effect of different lyoprotectants on conservation in size for lyophilized nanoparticles prepared from: (a) methylated N-(4,N,N-dimethylaminobenzyl) chitosan, (b) methylated N-(4-pyridinyl) chitosan and (c) methylated N-(benzyl) chitosan.
Table 4. Physico-chemical characteristics of nanoparticles, after lyophilization (n¼ 5).
Physico-chemical characteristics (Mean� SD)
Type of nanoparticles Size (nm) Zeta potential (mV) PdI EE (%) LE (%)
Methylated (amino-benzyl) chitosan 358� 31 23.4� 1.31 0.351� 0.04 55.9� 2.66 16.4� 3.78Methylated (pyridinyl) chitosan 405� 49 17.6� 2.43 0.388� 0.02 64.1� 3.38 22.7� 1.92Methylated (benzyl) chitosan 341� 32 15.2� 2.28 0.443� 0.04 55.4� 4.03 10.6� 1.74Tri-methyl chiotsan (TMC) 394� 45 13.6� 3.2 0.468� 0.03 47.3� 3.85 8.54� 2.57Chitosan 447� 31 11.4� 2.37 0.501� 0.03 41.2� 3.17 5.8� 2.14
8 R. Mahjub et al. Drug Dev Ind Pharm, Early Online: 1–15
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Figure 3. In vitro release profile of insulin from nanoparticles: (a) before lyophilization (pH¼ 6.8), (b) after lyophilization (pH¼ 6.8) and (c) afterlyophilization (pH¼ 7.4) (n¼ 3).
Figure 4. Atomic force microscopy images of: (a) methylated N-(4,N,N-dimethyl aminobenzyl) chitosan, (b) methylated N-(4-pyridinyl) chitosan and(c) methylated N-(benzyl) chitosan.
DOI: 10.3109/03639045.2013.841187 Insulin nanoparticles from N-aryl derivatives 9
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Comparing the oral administration of enteric coated capsulescontaining freeze dried nanoparticles prepared from TMC andnanoparticles prepared from amino-benzyl chitosan, it was shownthat lyophilized nanoparticles prepared from the quaternizedaromatic derivative of chitosan, resulted in significantly greaterhypoglycemic effect (p50.05) with the blood glucose level 4-hpost-administration reported as 42.10� 10.91% of the base level.Lyophilized insulin nanoparticles prepared from TMC caused areduction of blood glucose levels to 65.4� 14.12% of the baselevel in the same post administration time.
As shown in Figure 8, the corresponding serum insulinconcentration time profile agreed well with the data obtainedfrom the blood glucose levels time profiles that werementioned previously. Rats subcutaneously administered freeform insulin showed a sharp increase in serum insulinconcentration 1-h post-administration with a rapid decreaseafterwards (Figure 11).
No detectable plasma insulin concentration was found follow-ing the oral administration of enteric coated capsules containingthe free form of insulin, while the oral administration of enteric
Figure 5. Cross-section analysis of nanoparticles prepared from: (a) methylated N-(4,N,N-dimethyl aminobenzyl) chitosan, (b) methylated N-(4-pyridinyl) chitosan and (c) methylated N-(benzyl) chitosan.
10 R. Mahjub et al. Drug Dev Ind Pharm, Early Online: 1–15
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coated capsules containing lyophilized nanoparticles caused aslower, but more prolonged, increase in serum insulin level.
Administration of lyophilized nanoparticles prepared fromamino-benzyl chitosan caused a significantly higher serum insulinconcentration compared with lyophilized nanoparticles preparedfrom TMC (p50.05) (Figure 11).
Discussion
In order to investigate the effects of derivatives of chitosancontaining various aromatic residues on physic chemical
properties and permeation enhancing properties, insulin nanopar-ticles using quaternized aromatic derivatives of chitosan includemethylated N-(4-N,N-dimethylaminobenzyl), methylated N-(4pyridinyl) and methylated N-(benzyl) chitosan were preparedusing the PEC method. In this method, the particles are formedusing an electrostatic interaction between the positively chargedpolymer and negatively charged insulin. The insulin carries twonegative charges per molecule in a pH above its isoelectric point(i.e. pH¼ 6.1). By increasing the pH, the negative charge densityof the insulin molecule increases leading to consequentialincrease in electrostatic interaction between the polymer and
Figure 6. TEM images of prepared nanoparticles: (a) methylated N-(4,N,N-dimethyl aminobenzyl) chitosan, (b) methylated N-(4-pyridinyl) chitosanand (c) methylated N-(benzyl) chitosan. Each centimeter represents 500 nm.
Figure 7. Circular dichroism spectra of insu-lin released from different types ofnanoparticles.
DOI: 10.3109/03639045.2013.841187 Insulin nanoparticles from N-aryl derivatives 11
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insulin molecules which results in the formation of stable, highlyentrapped insulin nanoparticles. It has been shown that theoptimized insulin nanoparticles can be prepared followingadjusting the pH of insulin to 8.027.
One of the major problems in the formulation of nanoparticlesis their stability. This problem can be overcome through thedevelopment of freeze-dried solid nanoparticles using lyophiliza-tion40,44. In order to prevent the aggregation, precipitation andalso degradation of the entrapped proteins, the application oflyoprotectants is necessary during the freeze drying process41.
Conservation of size is considered as one the most importantfactors in a successful lyophilization process of nanoparticles.Disaccharides such as sucrose and trehalose show a higher level ofconservation with regards to the physico-chemical properties ofnanoparticles compared to monosaccharides, such as mannitoland glucose. Crystallization of the lyoprotectant may limit theformation of hydrogen bonds between the lyoprotectant and polarfunctional groups on the surface of nanoparticles and maydestabilize the nanoparticles as a consequence. The crystallizationof lyoprotectant may occur when using monosaccharides, butstudies have shown an amorphous state of disaccharides after thelyophilization process45. Based on the screening studies, sucrose(5% w/v) was selected as the lyoprotectant. Previous studies have
shown that the application of the proper lyoprotectant has a keyrole in conservation of the size of the particles46.
With regards to the decrease in zeta potential after lyophiliza-tion, the hydrogen bonding between the hydroxyl functionalgroup of sucrose, applied as a cryoprotectant and the surfaceof the nanoparticles can mask their positive zeta potential.Therefore, a significant decrease in zeta potential will be observedafter lyophilization. This finding correlates well with previousreports47.
The decrease in EE% and LE% can be explained through twomechanisms. The lyophilization process may cause the migrationof the drug to the surface of the nanoparticles and, consequently,the leakage of the entrapped drug from the nanoparticles48.Alternatively, as discussed previously, the zeta potential of theparticles might have been decreased, resulting in a reductionin electrostatic interaction between the insulin and polymermolecules, consequently decreasing the EE% and LE% of theparticles47.
The increased release rate of entrapped insulin from thenanoparticles after freeze-drying can be explained by severalmechanisms. As discussed in the previous section, the leakage ofthe entrapped drug might have occurred during lyophilization,with the drug becoming concentrated on the surface of thenanoparticles. As a result, it can release faster than a drugentrapped inside the nanoparticles. Alternatively, the significantdecrease in surface charge of the particles after lyophilization may
Figure 8. Cell viability of Caco-2 cells after 24 h exposure to: (a) free-soluble polymerþ insulin and (b) insulin nanoparticles (n¼ 3).
Table 5. Cytotoxicity studies on Caco-2 cells for 3 and 24 h. Free-solublepolymerþ insulin.
Polymer type
IC50 (mM)at 3 h
Mean� SD
IC50 (mM)at 24 h
Mean� SD
Methylated (pyridinyl) chitosan 45 0.854� 0.026Methylated (aminobenzyl) chitosan 45 0.621� 0.0560Methylated (benzyl) chitosan 45 0.360� 0.012
Table 6. Cytotoxicity studies on Caco-2 cells for 3 and 24 h, insulinnanoparticles (n¼ 3).
Polymer type
IC50 (mM)at 3 h
Mean� SD
IC50 (mM)at 24 h
Mean� SD
Methylated (pyridinyl) chitosan 45 0.765� 0.039Methylated (aminobenzyl) chitosan 45 0.503� 0.029Methylated (benzyl) Chitosan 45 0.275� 0.025
Figure 9. Activity of LDH released from damaged cells after incubationwith nanoparticles (n¼ 3).
12 R. Mahjub et al. Drug Dev Ind Pharm, Early Online: 1–15
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weaken the electrostatic interactions between insulin and thepolymer within the electrolyte complexes thus increasing therelease rate.
The observed increase in insulin in vitro release rate fromnanoparticles in PBS (pH¼ 7.4) is thought to occur because of theinstability of nanoparticles in higher pHs due to the low zetapotential of particles, and also the low solubility of chitosan andtheir related derivatives in high pH media49.
The data obtained from in vitro release studies revealed thehigh burst effect for insulin nanoparticles prepared from non-modified chitosan; other types of nanoparticles, prepared fromquaternized aromatic derivatives; show a lower burst effect.As discussed before, the surface charge density of quaternizedaromatic derivatives of chitosan were observed to be significantlyhigher than the zeta potential of non-modified chitosan. Theresultant stronger electrostatic interaction between insulin andpolymer found in synthesized derivatives, therefore explains thelow burst effect observed in the in vitro release profile of insulinnanoparticles prepared from quaternized, aromatic derivatives.
The release exponent (n) which determines the drug releasemechanism in the Korsmeyer–Peppas equation (Equation 7) hasbeen identified by fitting the data to appropriate model and hasbeen found to be 50.5 in all cases related to Kosrmeyer–Peppasmathematical model, suggesting the diffusion as the probablerelease mechanism from prepared from amino-benzyl, pyridinyl,benzyl and trimethyl chitosan.
The power law Fickian drug release mechanism found innanoparticles prepared from quaternized aromatic derivatives, andalso TMC, is a drug transport mechanism occurring through theusual molecular diffusion of the drug as a result of a chemicalpotential gradient. The first order release mechanism observed innon-modified chitosan nanoparticles showed that the drug isreleased from a swelled porous polymeric matrix. These findingsare explained by considering the fact that at a physiological pH of6.8, the quaternized derivatives of chitosan are protonated and therelease kinetic is probably diffusion while the non-derivatedchitosan is in an un-protonated form and may develop a swelledporous gel like matrix.
The obtained data from ex vivo and in vivo studies clearlydemonstrated that oral administration of insulin nanoparticlesprepared from quaternized N-aryl derivatives of chitosan result inhigher transport efficiency across intestinal epithelium andconsequently higher oral absorption compared to insulin nano-particles prepared from TMC. This could be due to the presenceof hydrophobic moieties of aromatic residues in the structure ofthe derivatives that can enhance the opening of the tight junctionsand consequently paracellular permeation of macromoleculessuch as insulin in a result of development of hydrophobicinteraction of polymer-cell membrane. In addition to the positivecharge of chitosan and related quaternized derivatives, thehydrophobicity is considered as the one of the effective mech-anisms for mucoadhesion. Although the permanent positivecharge may enhance the mucoadhesive properties, the amphiphilicnature of the cell membrane is important for developing thehydrophobic–hydrophobic interaction between the cell membraneand chitosan derivatives that contain hydrophobic residues. Basedon the facts mentioned above, it is suggested that bothhydrophobicity and cationic charge play the important role inmucoadhesive properties of quaternized chitosan derivatives.Kowapradit et al.24 have reported higher transport efficiency ofmethylated N-(4-N,N-dimethyl aminobenzyl) chitosan compare tonon-modified chitosan across Caco-2 cells. The higher tranfectionefficiency of methylated N-(4-pyridinyl) chitosan was reportedin hepatoma cell lines23. Sajomsang et al.50 suggested thatdelocalized positive charge in aromatic ring and hydrophobicproperties are considered as the main mechanisms for enhancingparracellular permeability of quaternized aromatic derivatives ofchitosan. In N-aryl chitosan derivatives, the polymer wassubstituted with N-aryl groups to provide hydrophobic interactionwith the amphiphilic mucus membrane. The derivatives werequaternized to overcome the solubility problems of the polymer.Consequently, using quaternized form of derivatives of chitosanresiding hydrophobic moieties cause more favorable cell mem-brane–polymer interaction.
Among three different quaternized aromatic derivatives,nanoparticles prepared from methylated amino-benzyl chitosan
Figure 10. Cumulative percentage of insulintransported across excised rat jejunum fordifferent types of nanoparticles (n¼ 3).
DOI: 10.3109/03639045.2013.841187 Insulin nanoparticles from N-aryl derivatives 13
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showed higher permeability in ex vivo studies on excised ratjejunum. It was assumed that methylated amino-benzyl chitosantightly bind to cell membranes more than methylated pyridinylchitosan. It would be suggested that the steric hindrance of N-pyridinyl group shield the positive charges of quaternary ammo-nium group on the quaternized derivative and consequently causeweaker cell membrane–polymer interaction. In addition, due toresonance effect, the positive charge on the pyridinyl aromaticring would be delocalized. In this study, no significant differenceswere found in ex vivo permeation studies between nanoparticlesprepared from methylated pyridinyl and methylated benzylchitosan (p40.05). The studied N-aryl derivatives of chitosandue presence of hydrophobic aromatic ring, can pose favorablehydrophilic–hydrophobic balance and consequently can interactmore efficiently with intestinal mucosa. This can describe thesignificant higher transport efficiency of nanoparticles preparedfrom quaternized N-aryl derivatives of the chitosan.
In vivo experiments reported lower blood glucose level andhigher serum insulin concentration following oral administrationof nanoparticles prepared from methylated amino-benzyl chitosanrather than TMC. The results showed that although the activity oforal nanoparticulate system is slower than subcutaneous insulindelivery but it provides prolonged absorption of insulin thatmakes this system suitable for providing basal insulin level.
Conclusion
In this study, nanoparticles were prepared from methylated N-(4-N, N-dimethyl aminobenzyl) chitosan, methylated N-(4-pyridinyl)chitosan and methylated N-(benzyl) chitosan. The obtained datarevealed that nanoparticles with the desired physico-chemicalproperties including size, zeta potential, PdI, EE% and LE% hadbeen prepared. The addition of sucrose (5% w/v) The size ofnanoparticles, as one the most important characteristics, posegreat during the lyophilization procedure as a lyoprotectantimproved the conservation of the sizes of nanoparticles which isregarded as one the most important factors in the process.
The in vitro release profile of insulin from nanoparticles showsa low burst effect in SIF, indicating strong electrostatic interactionbetween insulin and synthesized derivatives while nanoparticlesprepared from non-modified chitosan exhibit a high burst effectdue to weak electrostatic interaction.
The mechanism of insulin release from quaternized aromaticchitosan nanoparticles and also nanoparticles prepared from TMCwas found to be diffusion according to curve fitting of the releasedata to the Korsmeyer–Peppas power law equation while therelease kinetic of nanoparticles prepared from non-modifiedchitosan was found to be the first order.
Thorough the use of circular dichroism technique, it wasdemonstrated that the lyophilization and also preparation of thenanoparticles did not affect the secondary structure of insulin.
The cytotoxicity studies on Caco-2 cells revealed no signifi-cant cytotoxicity for prepared nanoparticles after 3 h of incubationbut a concentration dependent cytotoxicity was found after 24 h ofincubation.
Ex vivo studies on excised rat jejunum demonstrated thatinsulin nanoparticles prepared from quaternized N-aryl deriva-tives of chitosan would show greater transport efficiencycompared to previously reported insulin nanoparticles preparedfrom TMC and also non-modified chitosan.
In vivo experiments produced data demonstrating an increasedabsorption of nanoparticles prepared from methylated N-(4-N, N-dimethyl aminobenzyl) chitosan compared to nanoparticlesprepared from TMC.
These findings show that the preparation of nanoparticlesusing N-aryl derivatives of chitosan can be considered a novelapproach for delivery of the insulin across intestinal mucosa withgreater transport efficiency and bioavalibility compared to TMC.
Declaration of interest
This study was financially supported by Deputy of Research, TehranUniversity of Medical Sciences. The authors reported no declaration ofinterests.
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DOI: 10.3109/03639045.2013.841187 Insulin nanoparticles from N-aryl derivatives 15
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