International Journal of Biological Macromolecules 70 (2014) 1–9
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
j ourna l h o mepa ge: www.elsev ier .com/ locate / i jb iomac
ydrolyzed polyacrylamide grafted maize starch based microbeads:pplication in pH responsive drug delivery
. Mallikarjuna Settya, Anand S. Deshmukhb,∗, Aravind M. Badigerc
Department of Pharmaceutics, The Oxford College of Pharmacy, 6/9, 1st cross Begur Road, Hongasandra, Bangalore 560068, IndiaDepartment of Pharmaceutics, Shree Dhanvantary Pharmacy College, Kim, Kudsad Road, Surat, Gujarat 394110, IndiaBDR Pharmaceuticals International Private Limited, Vadodara, Gujarat 390 001, India
r t i c l e i n f o
rticle history:eceived 28 April 2014eceived in revised form 30 May 2014ccepted 11 June 2014vailable online 24 June 2014
The present study details the synthesis, characterization and pharmaceutical application of hydrolysedpolyacrylamide grafted maize starch (HPam-g-MS) as promising polymeric material for the develop-ment of pH responsive microbeads. Different grades of graft copolymer were synthesized by changingthe net microwave irradiation time, while keeping all other factors constant. Acute oral toxicity studyperformed in rodents ensured the bio-safety of graft copolymer for clinical application. Various batches ofaceclofenac loaded microbeads were prepared by ionic gelation method using synthesized graft copoly-mers and evaluated for formulation parameters. FTIR spectroscopy confirmed the chemical compatibilitybetween drug and graft copolymer. Results of in vitro release study (USP type-II) carried out in two dif-ferent pH media (pH 1.2 acid buffer and pH 7.4 phosphate buffer) showed that release rate of drug from
developed microbeads was a function of both: (a) surrounding pH and (b) the matrix composition. Thedrug release was relatively higher at alkaline pH as compared to acidic pH and this feature is desirablefrom viewpoint of site specific drug delivery. A direct correlation was observed between percentagegrafting and microbeads performance and it presents a scope for further research on application andoptimization of HPam-g-MS based microbeads as drug delivery carriers.
Along the entire length of human gastrointestinal tract (GI tract),H varies widely i.e. acidic in stomach to alkaline in the lower partf intestine. Exploitation of this differing pH (as a trigger) at vari-us locations of GI tract using stimuli responsive polymeric carrierintelligent material) has become one of the major strategies inhe design and development of site specific drug delivery system.n other words, this type of delivery system can release enclosedrug from the matrix more specifically at predetermined site (suchs at alkaline pH of intestine while avoiding/minimizing release intomach) at desirable rate under predetermined conditions [1].
Native starch irrespective of its source is undesirable for manyharmaceutical applications due to its inability to withstand var-
ous processing conditions such as extreme temperature, diverse
∗ Corresponding author at: Department of Pharmaceutics, Shree Dhanvantaryharmacy College, Near Kim Railway Station, Kudsad Road, Kim (E), Surat, Gujarat94110, India. Tel.: +91 9726609206; fax: +91 2621231077.
pH, high shear rate, and freeze thaw variations. To overcome this,chemical modification of starch such as etherification, esterifica-tion, cross linking, oxidation, cationization and grafting are usuallydone. It incorporates unique physicochemical and functional char-acteristics into starch molecules, making it suitable (as promisingpolymeric material) for diverse applications [2,3]. Among varioustypes of chemical modification, grafting of polyacrylamide chainson the backbone of starch is quite simple and economic. Both con-ventional [4,5] and microwave assisted [6,7] grafting techniquesare reported.
Comparatively, grafting with the help of microwave radiationis cleaner, straightforward, eco-friendly and highly reproducibletechnology which conforms to all the accepted norms of greenchemistry. Further in case of microwave irradiation based method,the process is very simple and if all other factors (wattage of radi-ation, polysaccharide concentration and monomer concentration)are kept constant, then the percentage grafting can be preciselycontrolled in terms of net irradiation time, which being regulated
electronically by the inbuilt digital timer of the microwave oven,tends to be highly accurate and reproducible [1,8]. From experi-mental viewpoint, our intention is to take the advantage of thisfact for the synthesis of different matrix compositions (percentage
rafting) of polyacrylamide grafted maize starch, wherein the per-entage grafting will be controlled precisely by changing the netrradiation time alone, keeping all other factors constant.
The potential of polyacrylamide grafted starch is thoroughlynvestigated in areas like industrial flocculant [6], in algal biomassarvesting [7] and in mud drilling fluid [9] but its application
n drug delivery has been scantily studied, up to now, only aingle study is reported, where conventionally synthesized poly-crylamide grafted starch (a neutral copolymer) was used as simpleydrogel base for controlled delivery of ceftriaxone sodium [5]. Anduch data from Al-Karawi et al. proved the feasibility of the use oftarch graft copolymer for drug delivery application.
Upon alkaline hydrolysis, the amide functional groups of graftedolyacrylamide chains acquire a polyelectrolyte (anionic) charac-er and it greatly enhances the functionality of copolymer. Duringhe hydrolysis reaction, a CONH2 group of polyacrylamide chainsould be converted to a COOH group resulting in incorporation ofionic property’, which show rapid response to the changing envi-onment such as pH and ionic strength [10]. Recently, the synthesisf pH responsive microparticles from such ionic copolymers haseen the main focus of research in the area of site specific drugelivery [10,11]. The non covalent ionotropic network formationy aqueous in-liquid curing process between polyionic chains ofolysaccharide with multivalent counter ions such as Ca2+, Al3+,e3+ etc. could be used to obtain spherical matrix or microbeads.urthermore, this process offers a simple and mild processing con-itions (i.e. organic solvent-free environment), which can avoid theossible toxicity of reagents and other undesirable effects.
With this fact in mind, our hypothesis is that by partial alkalineydrolysis of polyacrylamide grafted starch; a polyionic characteran be introduced upon it, which could be then used as a new andromising polymeric matrix for the development of ionically cross
inked pH responsive microbeads. To the best of our knowledge,o studies have been reported so far that describes the formationf hydrolyzed (ionic) polyacrylamide grafted maize starch (HPam--MS) based microbeads in the design and development of pHesponsive drug delivery system.
In the present research therefore we aims to: (a) synthesize a dif-erent graft compositions of polyacryalamide grafted maize starchy modulating the net time of microwave irradiation, (b) performn alkaline hydrolysis of graft copolymers and its characterization,c) develop the pH responsive microbeads of ionic (hydrolyzed)raft copolymers by eco-friendly ionotropic gelation technique andd) study the effect of percentage grafting (matrix composition) onoth in vitro drug release profiles and overall performance of theicrobeads.Aceclofenac, a newer derivative of diclofenac, is a promising
SAID that possess remarkable anti-inflammatory, analgesic andnti-pyretic property; and causes minimum or no gastrointesti-al complications [12,13]. In the present study, it is selected asodel drug for two reasons: (a) it is an ideal candidate for sustained
elease [12] and (b) it is characterized with poor water solubility14] and this feature is desirable because excessive water solu-ility increase the leaching of drug during the ionotropic gelationethod.
– 0 0 0.92 0
2. Materials and methods
2.1. Materials
Aceclofenac was obtained as a gift sample from Rantus PharmaPvt Limited, Hyderabad, India. Maize starch (amylose to amy-lopectin ratio was 20:80), analytical grade ammonium persulphateand hydroquinone were purchased from E.Merck Limited, Mumbai,India. Acrylamide monomer was purchased from Finar ChemicalsLimited, Ahemedabad, India. HPLC grade acetone was supplied byS.D. Fine Chemicals, Mumbai, India. All other materials of reagentgrade were purchased from S.D. Fine Chemicals, Mumbai, India andused as received. Double distilled water was used throughout thestudy.
2.2. Synthesis
2.2.1. Microwave assisted synthesis of polyacrylamide graftedmaize starch (Pam-g-MS)
For microwave assisted synthesis of polyacrylamide graftedmaize starch, briefly, 2 g of maize starch was slowly dispersed in100 mL of distilled water with a constant stirring in 1 L borosil-icate glass beaker placed on a magnetic stirrer. To this, desiredquantities of acrylamide (0.12 mol) and ammonium persulphate(0.002 mol) were added and mixed well. The reaction mixturethus obtained was placed on turntable of microwave (20 L Bajajmicrowave oven, model: 2801ETB) oven and irradiated at 720 Wof microwave power for selected time intervals, while keeping allother factors constant (Table 1). After the completion of intendedperiod of irradiation, the obtained viscous or gel like mass wascooled to room temperature and left undisturbed overnight tofacilitate the completion of grafting process. Later, the saturatedsolution of hydroquinone was poured into reaction vessel to ter-minate the reaction. Synthesized graft copolymer was precipitatedfrom the reaction mixture by adding excess of acetone and washedseveral times with methanol:water (80:20) to remove the un-reacted monomers. Finally, the obtained mass of graft copolymerwas dried overnight under vacuum (60 mm of Hg) at 40 ◦C, pulver-ized, sieved (#60) and stored in a desiccator.
To remove the competing homopolymer formed (possibly)during the synthesis, the purification process was carried out.For purification, the solvent extraction method using mixture offormamide and acetic acid (1:1), as reported elsewhere [15] wasemployed. The purified graft copolymer (Pam-g-MS) thus obtainedwas dried under vacuum (60 mm of Hg) and final mass wasrecorded to calculate the following:
% grafting
= mass of graft copolymer − mass of polysaccharidemass of polysaccharide
∗ 100 (1)
% grafting efficiency
= mass of graft copolymermass of monomer + mass of polysaccharide
∗ 100 (2)
of Bio
2m
Mafrwc(Fvs
2
2
sg(F4
2
ctc
2
tvNmar
2
phe&
N
wgNac
2
doco(hbao
C.M. Setty et al. / International Journal
.2.2. Synthesis of partially hydrolyzed polyacrylamide graftedaize starch (HPam-g-MS)
For alkaline hydrolysis, 2 g of purified graft copolymer (Pam-g-S) was dissolved in 100 mL of sodium hydroxide solution (0.9 N)
nd stirred at 50 ◦C on a thermostatically controlled water bathor 1 h. On completion of reaction time, the solution was cooled tooom temperature and hydrolyzed graft copolymer (HPam-g-MS)as precipitated by adding excess of methanol. The precipitate was
ollected by filtration and washed repeatedly with methanol:water90:10) to remove the adhered alkali and to dewater it completely.inally, the hydrolyzed graft copolymer was dried overnight underacuum (60 mm of Hg and at 40 ◦C), pulverized, sieved (60#); andtored in desiccator until used.
.3. Characterization of graft copolymer
.3.1. Fourier transform infrared spectroscopy (FTIR)For FTIR analysis, the pellets of dried sample of plain maize
tarch (MS), purified form of best grade of graft copolymer (Pam--MS1) and hydrolyzed form of best grade of graft copolymerHPam-g-MS1) with potassium bromide were scanned with aTIR spectrophotometer (Shimadzu 8400S) between 400 cm−1 and000 cm−1.
.3.2. Elemental analysisElemental analysis of MS, Pam-g-MS1 and HPam-g-MS1 was
arried out using Elemental CHNS analyzer (Elementar; Vario EL III)o determine the percentage content of four elements viz. nitrogen,arbon, hydrogen and sulphur.
.3.3. Intrinsic viscosity measurementIntrinsic viscosity measurements of all four grades of syn-
hesized graft copolymers were carried out using Ubbelodheiscometer at 25 ± 0.5 ◦C. The viscosities were measured in 0.1 MaNO3 solution. The time of flow for solutions of copolymer waseasured at four different polymer concentrations (0.1, 0.05, 0.025
nd 0.0125 g/dL) and intrinsic viscosity was calculated as per theeported method [16].
.3.4. Estimation of carboxylic functional groupThe extent of conversion of amide groups ( CONH2) of grafted
olyacrylamide chains into carboxylic groups ( COOH) by alkalineydrolysis was determined by titrimetric method (neutralizationquivalent) and calculated using an equation reported by Tripathi
Singh [17].
.E. = X ∗ 1000Y ∗ Z
(3)
here N.E. is neutralization equivalent, X is weight of sample inrams, Y is volume of NaOH consumed and Z is normality of NaOH..E. value corresponds to the number of basicity of a polybasic acidnd the greater number of carboxyl groups (basicity) on polysac-haride backbone will lower its N.E. value.
.3.5. Acute oral toxicity studyAcute oral toxicity study of hydrolyzed graft copolymer was
esigned and conducted as per Organization of Economic Co-peration and Development (OECD) guideline for the test ofhemicals, section 4; test number 425, taking necessary approvalf study protocol from institutional animal ethics committeeIAEC approval number: SDPC-AFC/2013/153). Selected batch of
ydrolyzed graft copolymer (HPam-g-MS1) was mixed with dou-le distilled water (as vehicle) to make a semi viscous solution anddministered to single female Wister rat by oral route at dose levelf 2000 mg/kg body weight (in the dose volume of 20 mL/kg body
logical Macromolecules 70 (2014) 1–9 3
weight) and observed carefully for first 4 h. Next day, after the sur-vival of first animal four more female rats were fed in the sameway, and all five animals were observed carefully twice daily for14 days to find the sign of toxicity and mortality if any. Serum bio-chemical studies were performed at definite intervals till 14 days.On 15th day, all animals were euthanized and required tests wereperformed as per the guideline [18].
2.4. Preparation of ferric ion cross-linked drug loaded microbeads
An accurately weighed quantity of a model drug aceclofenac(10%, w/w of polymer) was slowly dispersed into aqueous solu-tion of hydrolyzed graft copolymer (4%, w/v) using the magneticstirrer to prepare a homogenous suspension. The prepared bubblefree suspension was then carefully dropped into stagnant solu-tion of ferric ion (8%, w/v) using 25 mL glass hypodermic syringe(needle gauge 22). Viscous microgels thus obtained were left tocure for a suitable time (60 min) to produce firm yellowish browncolored microbeads. Developed microbeads were collected care-fully by decantation and washed sufficiently with distilled waterto remove the un-reacted ferric ions. Finally, the microbeads weredried at 50 ◦C for 48 h in vacuum oven (60 mm of Hg) and stored indesiccator until used.
2.5. Evaluation of drug loaded microbeads
2.5.1. Drug polymer compatibility study by FTIR analysisFTIR analysis was carried out to ascertain the chemical com-
patibility between synthesized graft copolymer and the drug. Ithelps to avoid the problems in later stages of formulation devel-opment and shelf life estimation. The scans of dried sample of puredrug aceclofenac, drug free blank microbeads and selected batch ofdrug loaded microbeads (F1) were recorded by potassium bromidepellet method using a FTIR spectrophotometer (Shimadzu 8400S)between 400 cm−1 and 4000 cm−1.
2.5.2. Drug entrapment efficiencyFor calculating the drug entrapment efficiency (%), accurately
weighed 50 mg of microbeads were incubated overnight in 100 mLof pH7.4 phosphate buffer maintained at 37 ± 0.5 ◦C, to facilitatethe complete swelling of microbeads. Later, this suspension wassonicated for 30 min to rupture the microbeads and release theentrapped drug completely into the buffer solution. The polymericdebris were removed by filtration (through 0.4 micron membranefilter) and the clear filtrate was analyzed for drug using UV–visiblespectrophotometer (Shimadzu-1700, Japan) at 274 nm, taking asolution of drug free blank microbeads prepared under identicalcondition as reference. The percentage drug entrapment efficiency(DEE, %) was calculated using the equation:
Drug entrapment efficiency (%)
= Experimentally calculated drug contentTheoretical drug content
∗ 100 (4)
2.5.3. Measurement of size of microbeadsThe size of different batches of microbeads was measured using
trinocular microscope (Lyzer, Model: LT-13A) attached with digitalcamera and Motic Images Plus software. The average diameter ofthe 100 beads per batch was calculated [11].
2.5.4. Swelling behavior of microbeads
To examine the pH responsive swelling behavior of prepared
microbead formulations, equilibrium mass % uptake and pulsatileswelling study were carried out using two different pH media (pH1.2 acid buffer and pH 7.4 phosphate buffer) by gravimetric method
4 of Bio
ac
Q
wm
2
ovldm5(vma
2
radiatsuoMnis
3
3
fitwwcumamicotpdpimh
ibgi
C.M. Setty et al. / International Journal
s reported earlier [10]. The equilibrium mass% uptake (Q) wasalculated using an equation:
= Ms − Md
Md∗ 100 (5)
here Ms is mass of swollen microbeads and Md is mass of dryicrobeads.
.5.5. Scanning electron microscopy (SEM).To study the overall, surface and cross-sectional morphologies
f developed microbeads, SEM analysis was performed. The pre-iously dried samples of both intact and half cut portion of drugoaded microbeads (F1) were mounted on a copper stub usingouble sided adhesive tape and sputtered coated with gold toake them conducting. The scanning was performed using JSM-
610 (JEOL, Kyoto, Japan) at 15 kV under required magnifications500 �m and 10 �m) and at room temperature. For cross-sectionaliew, soon after the completion of cross linking time, the weticrobeads were collected, washed, cut into two halves with blade
nd dried along with intact microbeads as mentioned before.
.5.6. In vitro drug release studyIn vitro drug release study from developed microbeads was car-
ied out at 37.0 ± 0.5 ◦C using USP Type-II paddle type dissolutionpparatus (Electrolab, Model: TDT-08L) rotated at 100 rpm. For therug release, accurately weighed 100 mg of microbeads was stud-
ed in 900 mL of both simulated gastric fluid of pH 1.2 (for initial 2 h)nd simulated intestinal fluid of pH 7.4 (for next 6 h) to simulatehe normal G.I transit conditions. At predetermined time intervals,ample aliquots were withdrawn and replaced with the same vol-me of fresh buffer. Solutions collected were analyzed for contentf dissolved drug, using UV–visible spectrophotometer (Shimadzu,odel: UV-1800) at the wavelength maxima of 274 nm. Simulta-
eously, the dissolution of blank microbeads was also carried outn similar conditions, to collect the solution for reference cell. Eachample was tested and analyzed in triplicate.
. Results and discussion
.1. Synthesis and characterization of graft copolymer
In the present study, the formation of HPam-g-MS was con-rmed by FTIR, intrinsic viscosity study and elemental analysis andhe content of COOH groups introduced by alkaline hydrolysisere estimated by titrimetry. In addition, acute oral toxicity studyas carried out to examine the biocompatibility of developed graft
opolymer. Microwave assisted free radical grafting technique wassed to synthesize the different grades of polyacrylamide graftedaize starch (Pam-g-MS), herein by keeping all other factors (such
s microwave power, concentration of starch, concentration ofonomer and concentration of initiator) constant, only microwave
rradiation time was sequentially modulated to control the per-entage grafting of copolymer (shown in Table 1,). As the timef microwave irradiation can be accurately controlled by digitalime controller of microwave oven, the degree of grafting thus gotrecisely controlled as a function of exposure time. Furthermore,uring the synthesis, the power of microwave was fixed at lowower output (720 W) to achieve the high degree of reproducibil-
ty and to decrease the formation of competing homopolymer. Theost desirable batch or best grade was selected on the basis of
ighest percentage grafting and intrinsic viscosity.From the results (Table 1) it was observed that percentage graft-
ng decreased as the time was increased. This phenomenon coulde explained by the fact that, during microwave irradiation, polarroups (such as OH group of both water and starch) tend to rotaten alignment with the electric field component of the microwave,
logical Macromolecules 70 (2014) 1–9
which rapidly reverses its direction. As polar groups of polysac-charide fail to align (due to bulkiness) themselves as quickly asthe direction of the electric field of microwave, friction is createdand this friction causes localized heating [19]. Localized heat playsa critical role in grafting process. If an optimum quantity of heatis generated, it works jointly (in synergism) with chemical freeradical initiator to generate free radical sites on both acrylamidemonomer (by microwave) and polar OH group of polysaccha-ride (by APS) to give higher grafting efficiency. On the contrary,if an excess of heat is generated, it leads to the occurrence oftwo major events: (a) decomposition of polymeric backbone and(b) increase in homopolymer fraction. Both these conditions candecrease percentage grafting (copolymerization). Hence, it is clearfrom the results (Table 1) that increase in exposure time has actu-ally increased the localized heating which in turn decreased thecopolymerization.
At irradiation time lesser than 60 s, (data not shown) a negligi-ble amount of grafting took place. This could be due to generationof suboptimal quantity of heat, which was insufficient to generatefree radical sites for copolymerization reaction. On the contrary,at exposure time more than 120 s (data not shown), the degree ofgrafting decreased significantly, probably due to the over exposureof microwave irradiation, causing rapid breakdown of polymericbackbone.
The main objective behind the partial alkaline hydrolysis ofsynthesized graft copolymers was to introduce an ionic chargeupon them. The ionic charge was essential for two reasons: (a)to carry out an ionic cross-linking reaction for the preparationof microbeads and b) to bring the pH sensitive characteristics onmicrobeads necessary for site specific drug delivery. During alkalinehydrolysis the amide groups ( CONH2) of grafted polyacrylamidechains were converted into ionic carboxylic groups ( COOH). How-ever, Khalil et al. [4] reported that at higher concentrations ofalkali and at higher temperature, the ether group of starch break-downs and this may lead to depolymerization. Therefore, to avoidsuch chemical changes in the polymer except the conversion ofamide groups into ionic carboxylic groups, saponification reactionwas carried out at 50 ◦C in 0.9 N NaOH for 1 h. From the results(Table 1), it was observed that the value of neutralization equivalentincreased (i.e. content of carboxyl group decreased) proportion-ally with decrease in percentage grafting. This phenomenon can beattributed to the availability (quantity) of amide groups for con-version on the backbone of copolymer with change in percentagegrafting.
To confirm both the grafting of polyacrylamide chains on thebackbone of maize starch (MS) and the partial hydrolysis of amidegroups into carboxyl groups, FTIR spectroscopy was performed(Fig. 1). The FTIR spectrum of MS showed broad peaks at 3600 cm−1
due to stretching vibration of O H group and a smaller peak at2927.12 cm−1 attributed to the C H stretching vibrations of methy-lene group. The observed small peak at 929.72 cm−1 is due toC O C stretching vibrations.
In the spectrum of Pam-g-MS the peaks at 3634.01 cm−1,3399.45 cm−1 and 3206.08 cm−1 are observed, which can beassigned to the overlap of N H stretching band of amide groups ofacrylamide and O H stretching band of hydroxyl groups of starch.Small peaks at 2930.15 cm−1 and 929.23 cm−1 can be attributedto C H stretching vibrations of methylene group and C O Cstretching vibrations respectively. Further the absorption bandsat 1722.49 cm−1, 1674.27 cm−1 and 1508.38 cm−1 can be assignedto C O and N H stretching of amide-I and amide-II respectively.A sharp peak at 1323.14 cm−1 can be attributed to C N stretch-
ing. Appearance of these new peaks in Pam-g-MS compared to MSconfirms the grafting process.
In case of HPam-g-MS, decrease in intensity of sharp peaks ofN H stretching band at 3399 cm−1 and 3206 cm−1 indicate the
C.M. Setty et al. / International Journal of Biological Macromolecules 70 (2014) 1–9 5
artial conversion of amide groups into carboxylic groups. Peak at674.34 cm−1 can be assigned to C O of amide-I. But emergence ofew and intense peaks at 1564.32 cm−1 and1408.08 cm−1 can bettributed to COO− groups and this result confirms the conversionf amide groups into carboxylic groups after the alkaline hydrolysis.
The results of elemental analysis are shown in Table 2. In theample of MS apart from carbon and hydrogen a negligible amountf sulphur was present. The presence of sulphur can be attributed tonsolicited impurity present in the sample. However, the presencef substantial quantity of nitrogen in the sample of best grade ofraft copolymer Pam-g-MS1 (which was completely absent in theample of MS) can be accounted to the grafting of polyacrylamidehains onto starch backbone. In sample of hydrolyzed form of bestrade of graft copolymer (HPam-g-MS1), a threefold decrease inhe content of nitrogen is noted and there was not much varia-ion in percent carbon and hydrogen, this confirms the conversionf amide groups into carboxyl groups after the alkaline hydrolysiseaction.
From the results (Table 1) of intrinsic viscosity study, it wasbserved that intrinsic viscosity of all grades of hydrolyzed graft
opolymer was more than plain starch. This observation is quiteasy to understand because the intrinsic viscosity of any polymers a function of its molecular weight. When the percentage grafting
-MS), and hydrolyzed polyacrylamide grafted maize starch (HPam-g-MS).
of starch has increased the molecular weight of polymer has alsoincreased (as more number of polyacrylamide chains got grafted onbackbone of starch) and this in turn increased the intrinsic viscosity.This result further justify the Mark–Houwink–Sakurada equation(intrinsic viscosity [�] = KMa, where ‘K’ and ‘a’ are constants forgiven polymer–solvent system and ‘M’ is molecular weight). Similartrend of viscosity behavior was observed in earlier studies carriedout for graft copolymers [8,20]. However, in our study an inversecorrelation was observed between the irradiation time and intrin-sic viscosity, i.e. as the irradiation time was increased the values ofintrinsic viscosity decreased.
During the 14 days acute oral toxicity study, all animals survivedand had shown no sign of toxicity. On completion of study time,animals were fasted overnight and both urine and blood sampleswere collected to perform the biochemical and hematological testsrespectively. Later on 15th day, all animals were sacrificed by over-dosing of ketamine hydrochloride injection (60 mg/kg body weight,I.P. route) to perform the necropsy and histopathological studies.The results (data not shown) showed that all animals were healthyand had no sign of toxicity, even at the dose level of 2000 mg/kgbody weight. As per the OECD guideline 425, any chemical whoseLD50 is greater than 2000 mg/kg fall under the “Category 5” ofGlobally Harmonized System (GHS) and considered to be orallynon-toxic. This finding suggests that HPam-g-MS is non-toxic andcan be used safely for further clinical investigations.
3.2. Development of graft copolymer based drug loadedmicrobeads
Four batches (formulations) of pH responsive microbeads(Table 3) were developed from four different grades of synthesizedHPam-g-MS using simple, economic and eco-friendly ionic gelation
6 C.M. Setty et al. / International Journal of Biological Macromolecules 70 (2014) 1–9
Table 3Evaluation parameters of graft copolymer based drug loaded microbeads.
Formulation code Polymer grade Drug entrapmentefficiency (±SD); n = 3
a Partial disintegration of microbeads was observed at the end of study time.
tfsabotmaiaa
3
3
tatN2gpem
sbcc
c(bd
3
mcrbbtrgcoci
b Complete disintegration of microbeads was observed at the end of study time.
echnique. An aqueous solution of ferric chloride (Fe3+) was usedor ionic cross-linking of microbeads. During the preparation, asoon as the viscous solution of HPam-g-MS comes in contact with
solution containing trivalent Fe3+ cations, a cross-linking processegins. A rapid substitution between monovalent Na+ ions presentn a polymeric carboxylic site, with trivalent Fe3+ ions in a solutionakes place. These substituted Fe3+ ions then interact with two
ore Na+ ions containing carboxylic site on chains of copolymernd forms a viscous, ionically cross-linked, three dimensionalnsoluble spherical network, which gradually solidifies with timend leads to spherical, spongy, uniform in size (3–4 mm when wet)nd brownish in color microbeads [11].
.3. Evaluation of graft copolymer based drug loaded microbeads
.3.1. Drug polymer compatibility study using FTIRThe results of FTIR analysis is shown in Fig. 2. The FTIR spec-
rum of pure drug aceclofenac (Fig. 2A), shows the prominent peakt 3319 cm−1 along with a small broad peak attached, it may be dueo O H hydrogen bonding. Peak at 3060 cm−1 may be attributed to
H aromatic stretching. Several peaks near 2900 cm−1 including937 cm−1 may be due to C H stretching of CH2 groups. Carbonylroup vibration was observed at 1770 cm−1 and 1723 cm−1. Theeaks at 1572 cm−1, 1506 cm−1 and 1451 cm−1 indicate the pres-nce of C C ring stretching and peaks at 668 cm−1 and 616 cm−1
ay be due to C Cl stretching.In the spectrum of blank microbeads (Fig. 2B), COO− group
tretching and vibration bands were shifted to lower wave num-ers (from 1674.34 to 1653 cm−1 and 1564 to 1541 cm−1), whichould be due to the complex formation between ferric ions and thearboxyl group of polymer.
In the spectrum of selected formulation F1 (Fig. 2C), allharacteristic peaks of pure drug and polymer are presentwithout any significant variations) between the wave num-er 1500 cm−1–3400 cm−1. This result discards the possibility ofrug–polymer interaction in the formulation.
.3.2. Measurement of size of microbeadsFrom the results (Table 3), it was observed that the size of
icrobeads was inversely correlated to percentage grafting ofopolymer. Therefore, with decrease in percentage grafting cor-esponding increase in the size of microbeads was observed. Theead size of formulation F1 was found to be smallest while theead size of formulation F4 was the largest. As seen already fromhe results (Table 1) of neutralization equivalent, that a direct cor-elation exists between percentage grafting and content of carboxylroups. Therefore, it is assumed that during the cross-linking pro-
ess, comparatively, the polymeric solution with higher amountf carboxyl content would have gone under rapid ionic exchange,ausing rapid shrinkage and densely packed matrix structure, yield-ng a smaller bead sizes.
3.3.3. Drug entrapment efficiencyThe drug entrapment efficiency (DEE, %) was found between 80
and 86% in all the formulations. From the results (Table 3), it wasobserved that DEE of microbeads decreased progressively from for-mulation F1 to F4. It indicates a direct correlation between DEE (%)and percentage grafting. Most likely, as the percentage grafting ofcopolymer decreased, the porosity in matrix of microbeads wouldhave increased. This increased porosity might have caused morequantity of drug molecules to leak into the solution of cross-linkingagent, causing poor DEE (%).
3.3.4. Swelling studyStimuli responsive polymeric materials are known to respond
to the triggering agents present in the surrounding environment.Therefore, in order to investigate the effect of surrounding pH(as trigger) on overall swelling behavior of developed microbeads,equilibrium mass % uptake (Q) was studied in both simulatedgastric buffer (pH 1.2) and simulated intestinal buffer (pH 7.4)solutions maintained at 37 ± 0.5 ◦C for the period of 12 h by massmeasurement method. From the results (Table 3) it has beenobserved that by increasing the pH from acidic (pH 1.2) to alkaline(pH 7.4), a considerable increase in water uptake has occurred inall the microbeads formulations. This pH induced swelling behav-ior can be accounted to the uncross-linked COOH groups presentin the matrix of microbeads. At alkaline pH, the carboxyl groupsmay get dissociated into its ionic ( COO−) form, creating the highosmotic pressure inside the microbeads. To balance this osmoticpressure a large quantity of water ingress into the matrix, result-ing in higher percentage swellings. Whereas in acidic medium,the carboxyl groups remain un-dissociated and create compact orimpervious micro-arrangement of polymeric chains, making it dif-ficult for water molecules to enter into microbeads and limits theirswelling.
The swelling of microbeads in both the media also dependsupon the extent of cross-linking density in matrix. In case oflow cross-linking density, a loose network structure with agreater hydrodynamic free volume is created; this accommodateslarge volume of solvent molecules, causing massive swelling ofmicrobeads. Hence in formulation F1, where the cross-linking den-sity is expected to be the highest among all, the lowest equilibriummass% uptake is observed in both the medium.
During the swelling study it was noted that formulation F3and F4 degraded partially in acidic medium (Table 3). Two fac-tors can be accounted for this result: (a) due to the occurrenceof proton–iron exchange phenomenon, the leaching of ferric ionsis generally more in acidic condition [21] and (b) the strength ofionic interaction between iron and polymeric network dependsupon both the availability of complexing groups and its accessi-
bility [22]. From the results of neutralization equivalent (Table 1),it was already seen that total content of carboxyl functional groupsin formulation F3 and F4 is less (as they were prepared from thegraft copolymer of low percentage grafting). Hence, the density of
C.M. Setty et al. / International Journal of Biological Macromolecules 70 (2014) 1–9 7
nac, (
ptaso
mfitplt1afam
wsp
F
Fig. 2. Compatibility study: FTIR spectra of (A) aceclofe
olymeric carboxyl group-Fe3+ ion complex would also be less inhem. Both these conditions (low density of iron–polymer complexnd proton–acid exchange together) have possibly led to more ero-ion of the polymeric matrix leading to decomposition or erosionf the microbeads.
While performing an equilibrium swelling study in alkalineedium, two important observations (Table 3) were made. The
rst, the value of equilibrium swelling of formulation F1 was lowerhan formulation F2. It can be attributed to slow rate of solventenetration in former (due to more densely packed matrix) than
ater. And the second, a partial degradation of formulation F3 andhe complete degradation of formulation F4 is observed during2 h study period. The degradation of these microbeads could bettributed to low cross-linking density of their matrices, which thenailed to retain its structural integrity against the high pressure cre-ted by rapid ingress of water into them, resulting into breaking oficrobeads with time.Since microbeads swelled differently in different pH conditions,
e considered it worthwhile to investigate their pH dependentwelling reversibility or pulsatile swelling behavior. The results ofulsatile swelling study are presented in Fig. 3. From the results,
ig. 3. Pulsatile swelling study of drug loaded microbeads in alternate pH change.
B) blank microbeads, and (C) drug loaded microbeads.
it is observed that developed microbeads are pH sensitive and therate of swelling is slower than the rate of de-swelling. FormulationF4 was excluded from the study owing to its instability in both themedium. The reason behind the pulsatile swelling can be under-stood by the fact that during the de-swelling process (in acidic pH),the diffusing H+ ions neutralizes the negatively charged carboxylic( COO−) groups present on the microbeads. It creates a neutralor de-swelled layer of polymer at the surface of the microbeadsfirst, surrounding the ionized and swollen core. A continuous mov-ing front (from outwards to inwards) separating the non-ionic shellfrom an ionized core will develop. It facilitates the diffusion of moreamounts of H+ ions into the core, where they can be rapidly cap-tured by the ionized COO− groups and thus a rapid de-swellingis observed. Whereas, in case of swelling process (at alkaline pH),the matrix was initially in its nonionic ( COOH) form, and wasthen converted to ionized ( COO−) state. Therefore, as soon as theionized shell was formed around the nonionized core, diffusion ofH+ ions become slow and more difficult (due to charge repulsion)resulting in slow rate of swelling [23].
3.3.5. Scanning electron microscopyThe SEM image (Fig. 4), of selected formulation F1 shows that
developed microbeads were uniform in size and spherical in shapewith a small crater like view on the top. The crater formation is aresult of escape of entrapped surface air (at the interface), duringthe cross-linking process. The overall surface study of microbeadsshows the uniform surface morphology with few surface folding.The folding of surface can be attributed to the contraction (dueto the removal of water) of cross-linked matrix during the dryingprocess. And finally, in cross-sectional view a densely packed corewith a finely knitted structural arrangement is observed. It confirmsthe uniform cross-linking of polymeric network.
3.3.6. In vitro drug release studyThe main objective behind the study was to develop the
microbeads capable of releasing its content specifically in responseto varying pH conditions of the GI tract. Therefore, the in vitro drugrelease study was carried out in two set of pH i.e., in simulated gas-tric fluid of pH 1.2 for initial 2 h then in simulated intestinal fluid of
8 C.M. Setty et al. / International Journal of Biological Macromolecules 70 (2014) 1–9
Fs
psomcotcFmfb
ig. 4. SEM micrographs of (a) microbeads, (b) surface morphology, and (c) cross-ectional view.
H 7.4 for next 6 h. It facilitates the simulation of normal. GI tran-it conditions. From the drug release data (Fig. 5), three significantbservations were made: (a) the release of drug from all the for-ulations was relatively quicker in alkaline medium (pH 7.4) as
ompared to acidic medium (pH 1.2). It confirms that the devel-ped microbeads are pH responsive, and are capable of releasingheir content more specifically at alkaline pH; (b) the percentageumulative drug release increased progressively from formulation
1 to F4. It indicates that a direct correlation exists between theatrix composition (percent grafting) and overall drug release
rom the formulations. Further, both of these observations (a and) are found to be in good agreement with the results obtained
Fig. 5. Drug release profile from developed microbeads in buffer media pH 1.2 andpH 7.4.
from equilibrium study and pulsatile swelling study; and (c) thetriphasic pattern of drug release is observed in all the formulations,i.e. initially in acidic medium (pH 1.2), a very low or negligibleamount of drug is released but as the pH was raised to alkaline(pH 7.4), a burst in release of drug is observed, which graduallystabilized with time, giving a slow and steady release pattern thenafter. As reported in our previous study [11], this type of releasepattern could be attributed to the property of both the drug andpolymeric matrix configuration. Initially in acidic medium (pH 1.2),due to the unionization of carboxyl groups of polymeric matrix, themicrobeads remain in their shrunken (un-swollen) state. And thedrug due to its limited solubility at this pH [14], may also remaininsoluble. Together, it might have suppressed the release rate ofthe enclosed drug from the microbeads. But as the pH of the dis-solution medium is increased (pH 7.4), a rapid solubility of drugpresent on the surface of microbeads along with the dissociation ofcarboxyl groups (pKa ≈ 4.6) would have occurred, and contributedto burst release. However in the later phase of dissolution study,the swelling of polymeric chains attained the equilibrium and itincreased the overall diffusional path length traveled by the drugmolecules, causing slow and uniform drug release.
From the results of T50% and T80% (time for 50% and 80% of theenclosed drug to be released respectively) release data (Table 3), it isseen that overall drug release rate from the microbeads was directlyproportional to the cross-linking density (iron–graft copolymercomplex). Therefore, with increase in percentage grafting, a pro-portionate decrease in percent release of enclosed drug is observed.Formulation F1, prepared from the graft copolymer of highest per-centage grafting (highly cross-linked matrix) had the slowest andminimum drug release in both the media. On the contrary, in for-mulation F4 prepared with lowest percentage grafting, offered theloosely cross-linked matrix arrangements, and showed the mostrapid (T50% is 137.53 min) and maximum drug release. In formu-lation F2 and F3 intermediate release patterns between the twoformulations (F1 and F4) were observed. From the results of T%, theoverall release rate of the enclosed drug decreased in the followingorder: F4 > F3 > F2 > F1.
4. Conclusion
In the present study we explored the potential of synthesizedHPam-g-MS as promising carrier material in the design and devel-opment of pH sensitive microbeads for site specific sustaineddelivery of therapeutic agents. During synthesis, the percentage
of Bio
gtiuoropuhpsitmspd
C
A
SMcS(
[
[[
[
[
[
[
[[
[[
http://dx.doi.org/10.1002/masy.200950313.
C.M. Setty et al. / International Journal
rafting was successfully controlled by adjusting the net irradia-ion time of microwave. The effect of percentage grafting on bothn vitro drug release and overall microbeads characteristics is eval-ated. Acute oral toxicity study in rodents ensured the biosafetyf developed copolymer for further clinical investigations. Fromesults it was observed that increase in percentage grafting notnly decreases the drug release but also improves the overallharmacotechnical parameters of microbeads formulations. Sim-lated intestinal (alkaline) pH triggered the higher swelling andigher release of drug from the microbeads as compared to acidicH, which is very crucial in the design and development of site-pecific drug delivery. Finally the study opens a door for furthernvestigation on optimization of processing factors, to controlhe performance of HPam-g-MS based pH responsive microbeads,
ore precisely, to meet the desired therapeutic need. In future, theynthesized graft copolymer of starch could also be explored asromising polymeric material for designing the other novel drugelivery system.
onflict of interest
No conflict of interest.
cknowledgements
Authors express sincere gratitude to Rameshwardasji Birlamarak Kosh, Medical Research Center, Bombay Hospital Avenue,
umbai [grant number: Fel. 2011-2012/2] (India) for their finan-
ial assistance, to carry out this research work and authorities ofhree Dhanvantary Pharmaceutical Analysis and Research CenterSDPARC), Kim for providing the necessary analytical facilities.
[[
[
logical Macromolecules 70 (2014) 1–9 9
References
[1] G. Sen, S. Mishra, U. Jha, S. Pal, Int. J. Biol. Macromol. 47 (2010) 164–170.[2] C. Freire, F. Podczeck, F. Veiga, J. Sousa, Eur. J. Pharm. Biopharm. 72 (3) (2009)
587–594.[3] A.L. Serrero, S.P. Trombotto, P. Cassagnau, Y. Bayon, P. Gravagna, S. Montanari,
L. David, Biomacromolecules 11 (6) (2009) 1534–1543.[4] M.I. Khalil, S. Farag, A.S. El Fattah, J. Appl. Polym. Sci. 57 (1995) 335–342.[5] A.J.M. Al-Karawi, A.H.R. Al-Daraji, Carbohydr. Polym. 79 (2010) 769–774.[6] S. Mishra, A. Mukul, G. Sen, U. Jha, Int. J. Biol. Macromol. 48 (2011) 106–111.[7] C. Banerjee, P. Gupta, S. Mishra, G. Sen, S. Shukla, R. Bandopadhyaya, Int. J. Biol.
Macromol. 51 (2012) 456–461.[8] G. Sen, S. Ghosh, U. Jha, S. Pal, J. Clin. Eng. 171 (2011) 495–501.[9] M. Eutamene, A. Benbakhti, M. Khodja, A. Jade, Starch 61 (2) (2009) 81–91.10] K.S. Soppimath, A.R. Kulkarni, T.M. Aminabhavi, J. Contr. Release 75 (2001)
331–345.11] C.M. Setty, A.S. Deshmukh, A.M. Badiger, Int. J. Biol. Macromol. 67 (2014) 28–36.12] K. Parfitt, Analgesic anti-inflammatory and antipyretics, in: J.E.F. Reynolds (Ed.),
Martindale, The Complete Drug Reference, Massachusetts, 1999, pp. 2–12.13] A.E. Kay, A. Alldred, Rheumatoid arthritis and osteoarthritis, in: R. Walker,
C. Edward (Eds.), Clinical Pharmacy and Therapeutics, Churchill Livingstone,London, 2003, pp. 791–807.
14] S. Mutalik, A. Naha, A.N. Usha, A.K. Ranjith, P. Musmade, K. Manoj, P. Anju, S.Prasanna, Arch. Pharmacal. Res. 30 (2) (2007) 222–234.
15] G.F. Fanta, Synthesis of graft and block copolymers of starch, in: R.J. Ceresa (Ed.),Block and graft copolymerization, Wiley & Sons, New York, 1973, p. 11.
16] E.A. Collins, J. Bares, F.W. Billmeyer, Experiments in polymer science, Wiley &Sons, New York, 1973, pp. 394–399.
17] T. Tripathy, R.P. Singh, Eur. Polym. J. 36 (2000) 1471–1476.18] OECD, Test No.: 425, Acute oral Toxicity: Up-and-Down Procedure, OECD
Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, 2008,doi10.1787/9789264071049-en.
19] S.A. Galema, Microwave chemistry, Chem. Soc. Rev. 26 (1997) 233–238.20] G. Sen, S. Pal, Macromol. Symp. 277 (2009) 100–111,
21] D.S. Marlin, P.K. Mascharak, Chem. Soc. Rev. 29 (2000) 69–74.22] H.S. AlKhatib, M.O. Taha, K.M. Aiedeh, Y. Bustanji, B. Sweileh, Eur. Polym. J. 42
(2006) 2464–2474.23] F.E.I. Jianqi, Z. Zhang, L. Zhong, L. Gu, J. Appl. Polym. Sci. 85 (2002) 2423–2430.
