Iranian Polymer Journal / Volume 8 Number 4 (1999)

1626-1265199

Casein Cross-linked Polyacrylamide Hydrogels : Study ofSwelling and Drug Release Behaviour

S.K. BajpaiDepartment of Chemistry, Government Science College (Autonomous), Jabalpur (M .P .), India

'Received 21 June 1995: accepted 5 July 1999

ABSTRACT

The polymeric hydrogels composed of polyacrylamide and food protein caseinhave been synthesized for the purpose of studying their swelling and drugrelease behaviour. The two samples, differing in cross-linking ratio, showalmost Fickian swelling behaviour (n50 .50) except the less cross-linkedsample at pH 7 .0 for which swelling exponent n was 0 .62, thus showing non-Fickian swelling behaviour . The swelling parameters like diffusion exponent,penetration velocity, and diffusion coefficient have also been evaluated . Theswelling behaviour of hydrogels is greatly affected by variation in pH of theexternal medium . These gels show maximum swelling at pH 7 .0 and itdecreases in both acid and alkaline range . This behaviour has been explainedon the basis of osmotic swelling pressure theory, thus treating the hydrogel tobe of ionic nature due to presence of protein casein . The release of modeldrug bromocresol green (BCG) has been studied as a function of temperatureof the external medium, and it has been observed that the amount of drugreleased decreases beyond 40 ' C . The purpose of undertaking the presentwork is to explore the possibilities of using casein the natural protein, as adrug delivery device so that Ike other natural polymers (collagen, guargum,gum arebica etc.) casein may also be used without causing any toxic effect inthe human body system. These gels also undergo a number of reversibleswelling-deswelling cycles.

Key Words: hydrogels, equilibrium swelling capacity, cross-linking, casein, polyacrylamide

INTRODUCTION

researchers and technologists due to their widespreadapplications in contact lenses [i], wound dressings

Hydrogels are water-swellable, three-dimensional

[2], drug delivery systems [3, 4], controlled release ofpolymeric networks possessing both the cohesive

perfumes [5], adsorption of proteins [6] and metalproperties of solids and diffusive transport properties

ions [7] etc.of liquid. They have been increasingly attracted to

However, most of the research work on

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Casein Gyms-linked Pnlyaerylamide Hydrogels : Study a(Swelling and Aug Release

polymer gels is mainly focused on five syntheticpolymeric gels, thus neglecting the considerablevariety of networks in natural polymers such asnaturally occurring polysaccharides, proteins etc.Since the biological responses to polymer surfaces arecomplex, each polymer system should meet certainrequirements for biomedical applications.

Biocompatibility of the material is critical, andfor some applications biodegradability is desirable.The natural gels are ideal candidates for thesebiomedical applications . For example, they could beused to encapsulate the cultivate cells inside the gel,where the network will act as a semipermeablemembrane allowing only growth factors to enter to aidthe growth of the cells . The study of gels could beuseful in the development of novel synthetic polymernetworks that mimic natural gels [8], and a knowledgeof the response of natural gels to the changes in theenvironment could be invaluable varifying the trendsobserved in the experimental behaviour of syntheticpolymeric gels.

In recent past the researchers have focused theirattention on studies related with synthesis, swellingbehaviour and drug release analysis of naturallyoccurring polymeric hydrogels in the form ofnanoparticles, thin films, microspheres etc. Forexample, collagen [9], chitosan [10] guar gum [I1],agarose [12], gum arabica [13] have been successfully

Table 1 . Raw materials employed and their source.

CaseNo .

Name and description_

Source

1 Acrylamide (Mm) Robert Johnson '

2kW- Methylene bisacrylamide Central Drug(131S) House'

3 Casein powderLoba Chemie

4 Potassium persulphate (KPS)

Industries'Loba Chemie

5 Sodium metabisulphite (MSS)IndustriesS .D. FineChemicals '

6 Bromocresol green (BCG) Central DrugHousea . India

used as drug delivery devices for anticancer drugs,antibiotic drugs, colonic drugs, antimicrobial drugs,protein and peptide drug delivery respectively. In thisconnection, the author has already reported theswelling behaviour of hemoglobin cross-linkedpolyacrylamide hydrogels [14] and in continuation thepresent communication describes the analysis ofswelling behaviour of casein cross-linkedpolyacrylamide hydrogels . The equilibrium swellinghas been studied as a function of pH of the externalsolution . The model drug release behaviour ofhydrogels has also been analyzed.

EXPERIMENTAL

MaterialsThe raw materials used have been described in TableI. Acrylamide was recrystallized in methanol before

use and other material were used as received . Thedoubly distilled water was used throughout the work.

Synthesis of Cross-linked HydrogelsA definite amount of casein powder was dissolved inNaOH solution and then acrylamide (AAm) and N,N'-methylene bisacrylamide (BIS) were added in definiteamounts and mixed thoroughly, followed by additionof calculated quantities of potassium persulphate(KPS) and sodium metabisulphite (SMB) . The totalmixture was stirred well quickly to avoid lumping,poured into 10 x 75 mm test tubes and set asideundisturbed. The resulting smooth, semi-transparentcylindrical hydrogels were removed from the testtubes and then sliced into discs, washed with Tris-HCIbuffer, pH 7.0, followed by acetone and water anddried, in a dust free glass chamber at roomtemperature . In all, two samples of hydrogels wereprepared with different cross-linking ratio X(X = molBISlmol AAm). These samples will be denoted asCas-PAAm X (2 .1) and Cas-PAAm X (1 .7) wherenumber in the parentheses represent the cross-linkingratio in mol % (Table 2). Scheme I describes theformation of hydrogel in systematic way. In order toprepare the drug loaded gel, the calculated amount ofmodel drug bromocresol green (BCG) was added to

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Iranian Polymer Journal /Volume 8 Number 4 (1999)

Bajpai 5-K.

Casein

solution ofcasein

hydrogels . After the attainment of equilibrium theswelling capacity was calculated using the formula:

Grams of water per gram of gel = W W ( 1 )

Where W, is the weight of the equilibrated hydrogeland W is the initial weight of the dry hydrogel.

The penetration velocity (V) of buffer in eachpolymer was determined by weight gain method asreported elsewhere [15] . The penetration velocity wascalculated from the slope of the initial portion of thepenetrant uptake curve by using the equation:

1 dWgV =

2dA dt

dissolution in NaOH

:7.=ga.=:==ittsm

SMB + KPS AAm + BIS(2)

cross-linkedhydrogel

where V denotes penetration velocity, dW,/dt denotesthe slope of the weight gain versus time curve, ddenotes the density of water at 37 'C, and A denotesarea of one face of the disc.

The mass uptake of the swelling solution M, asa function of time t was analyzed according to the eqn(3) [16],

Schematic diagram showing the formation of Cas-PAAmXhydrogels.

M _ kt aM,, (3)

Scheme I

the reaction mixture before adding sodiummetabisulphite and potassium persulphate

. There is noparticular reason for selecting bromocresol green as amodel drug because the purpose of study is just tohave an understanding about the drug releasingcapacity of the proposed hydrogel, so that it could beused as a model drug-releasing device in near future.The semi-transparent greenish hydrogel discs wereobtained.

Dynamic Swelling StudiesHydrogels were swollen to equilibrium in water atphysiological temperature 37 'C . Equilibrium wasattained in 20 h, and the approach to equilibrium wasmonitored by measurement of mass of the swollen

Eqn (3) could be used to find out the Fickianand non-Fickian absorption of water by hydrogel.is the mass uptake of the solvent at equilibrium, k is aconstant and n is the exponent describing the Fickianor anomalous swelling mechanism.

On taking natural log of eqn (3):

InM =Ink+nlntM m

The values of n and k were calculated from theslope and intercept of the plot of In against In t,respectively.

The diffusion coefficient D of solvent wascalculated using the following equation [I7]:

0 = k(nr 2 ) a4

(5)

(4)

233Iranian Polymer Journal / Volume 8 Number 4 (1999)

Casein Cron-IiWcM Polyacrylamide Hydrogen :: Study of Swelling and Drug Release

Table 2 . Composition of Cas-PAAm hydrogels.

SI . System Composition (g)No . Cas AAm BIS BCG SMB KPS H2O

1 Cas-PAAmX(2.1) 4 .0 4 .0 0.19 0.00 0 .10 0 .10 402 Cas-PAAmX(1 .7) 3 .0 5 .0 0,19 0.00 0 .13 0 .13 403 Cas-PAAmX(2.1) 4 .0 4 .0 0.19 0.02 0 .10 0 .10 404 Cas PAAmX(1 .7)

(Drug loaded) 3 .0 5 .0 0.19 0.02 0 .13 0 .13 40

where, r is the radius of the gel disc.To study the swelling behaviour of the

hydrogels in medium of different pH, two pieces ofpreweighed hydrogels were placed in buffer solutionof required pH and allowed to equilibrate . Massmeasurements of the hydrogels were taken at differenttime intervals to monitor the attainment ofequilibrium, and swelling capacities (water sorbed perg of gel) were determined. In each case the final watercontent was determined for two pieces of hydrogels.Good agreement was found in the degree of swellingfor both pieces, each having the same history, theaverage of two determinations being used forcalculations.

In order to study the reversibility of theswelling process, the hydrogel samples were allowedto equilibrate in water and then placed in 2 M NaClsolution, which caused the gel to deswell . Thedeswelling was then followed by weighing the gel atvarious time intervals. The reversibility of swellingand deswelling was determined using the samesamples for consecutive swelling and deswellingexperiments . The degree of swelling has beenexpressed as the swelling ratio, the ratio of finalweight of the gel to initial weight of the gel.

In another set of experiments, completely driedhydrogel was allowed to equilibrate in water and afterattainment of the equilibrium it was dried again . Thedried hydrogel, was further allowed to swell in water,and then this process of drying and swelling wasrepeated a number of times.

In order to study the drug release analysis, thedrug-loaded hydrogels were placed in externalmedium at pH 7 .0 and the absorbance of the solutionwas measured (Systronix, India) at definite time

intervals. The amount of active ingradient released M,at a time t was determined using Beer-Lambert law.The total amount of drug incorporated in the disc wastaken as M,,.

RESULTS AND DISCUSSION

Dynamic Swelling StudiesA critical analysis of the swelling process reveals thatthere are two underlying molecular processes:penetration of the solvent molecules into the voidspaces in the network and subsequent stretching orrelaxation of the network segments . The fundamentalequation M,/M.,,=kt" defines the following threesituations:For a perfectly Fickian process where the rate ofsolvent penetration is the slowest and hence is the ratelimiting step, the value of n=0 .50.When the penetration velocity is far greater than thechain relaxation rate, the solvent uptake isproportional to time, i .e., n= 1 .0 (This is often calledrelaxation-controlled case-lll transport) [18].When both the diffusion and polymer relaxationcontrol the overall rate of water uptake, the diffusionmechanism is non-Fickian i .e ., 0 .5

5epai S .K.

Table 3. Swelling parameters of Cas-PAArnX hydrogels at different pH over a period of 20 h with a temperature=37 'C.

pH System Equilibrium swelling capacity Penetration velocity n k Diffusion coefficientH20 :gel (gig) V x 105 (curls) DK105 (cm2ls)2 .0 Cas-PAAmX(2 .1) 1 .10 0.98 0.11 0.18 1 .78

Cas-PAAmX(1 .7) 1 .86 1 .67 0.20 0.28 2 .864 .0 Cas-PAAmX(2 .1) 3.61 2.17 0.32 0.27 3 .04

Cas-PAAmX(1 .7) 4.92 3.06 0.42 0.40 4 .267 .0 Cas-PAAmX(2 .1) 6.19 5.02 0.56 0.41 4 .29

Cas-PAAmX(1 .7) 7.82 6.08 0.61 0.57 6.819 .0 Cas-PAAmX(2 .1) 5.20 3.15 0.49 0.38 4.01

Cas-PAAmX(1 .7) 6.15 4.87 0.55 0.47 5.01

thus following transport mechanism with a chainrelaxation contribution.

Effect of pHThe swelling behaviour of hydrogels is greatlyaffected by variation in pH of the external solution asdepicted in Figure I . The results so obtained are quitedifferent from those obtained in the case of gelatin-swelling was found to decrease with increase in pH of

9 .0D Cas-PAArnX (1 .7)

r r 1 r2 .0

4 .0

6 .0

8 .0

10.0pH

Figure 1 . Effect of pH on the equilibrium swelling capacity;temperature = 37 ' C .

polyacrylamide hydrogels [19], where the equilibriumthe external solution . It is clear from the Figure I thatthe equilibrium swelling capacity increases with risein pH of the external solution, attains maximum valueat pH 7 .0, and then starts decreasing, as the pH goes inthe alkaline range. It is also clear from the figure thatswelling is suppressed to a greater extent in the acidicrange as compared to the alkaline one.

In order to explain the observed experimentalfindings, two possible theories may be proposed. Firsttheory is based on the formation of a complexstructure in the polymer matrix through H-bondinginteractions between -COOH and -CONH2 groupsdue to hydrolysis of amide groups of polyacrylamidein the polymer matrix [20]

. This theory clearly impliesthat the variation in swelling behaviour of Cas-PAmXhydrogels must be due to possible hydrolysis ofpolyacrylamide and hence it should play a key role ingoverning pH dependent swelling behaviour.

Now, in order to verify the above theory,hydrogels of polyacrylamide, with the same cross-linking ratio, were prepared in absence of othercomponent casein . When these gels were put invarious buffer solutions of different pH, the swellingwas found to be almost the same for all pH values . Ifthe above proposed theory were correct then thehydrogels of cross-linked polyacrylamide should haveshown the similar pH dependent behaviour asobserved with Cas-PAAmX hydrogels . This clearlyverifies that no such complex structure formationtakes place within the polymer matrix and obviouslypolyacrylamide does not play any role in governingthe observed pH - effect. Therefore it must be the

7 .0

5.0

3 .0

1.0

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Casein Cross.linked Polyasrylamidc Hydrogen : Study of Swelling and Drug Rana.

other component of polymer matrix, namely casein,which should play the key role in the explanation ofobserved behaviour.

After failure of the above theory we nowproceed for the other theory, according to which theswelling behaviour of ionic hydrogels in diluteelectrolyte solution is mainly due to osmotic swellingpressure between the gel phase and external solution.At the moment, the above theory seems to be quite fitfor the present study because casein, one of thecomponents of the hydrogel, is a polyelectrolytecontaining charged and dissociable groups along themacromolecular chain.

The kinetic swelling behaviour of an ionichydrogel depends upon mass transfer limitations,Donnan equilibrium considerations, ion exchange andionic interactions [21] . When such an ionic hydrogelis immersed in a high dielectric constant medium,these ionic moieties will dissociate and create anoverall charge density along the chains, as well as ahigh concentration of mobile ions in the gel . Ascompared to the non-ionic gel behaviour, this ioniccharacter will introduce two "new player" forces inthe system: the osmotic pressure resulting fromdifference in ion concentrations between the swollengel and the external solution i .e., Ir is , (for"macroscopic" electroneutrality reasons, mobile ionsbelonging to the gel can not leave it and henceminimization of this osmotic pressure can only beachieved through dilution of the network chargedensity i .e ., swelling) and the net charge density alongthe chains will generate some electrostatic repulsion(i.e ., coulombic forces) between chain segments rze 1,.The resulting expansion of the network will contributeto the overall swelling behaviour [22] . It has beenshown by theoretical calculations and experimentalresults that

is typically similar than nia,.For a weakly charged polymer network in a

dilute electrolytic solution, the osmotic swellingpressure

is given as:

a 1e = RTE(C8 C;)

(6)

where Cg and C, are the molar concentrations ofcounter ions in the gel and solution phase

respectively, R is general gas constant and T is theabsolute temperature.

K-casein is an amphiphilic protein which hastwo identifiable regions : amino acids 1105containing predominantly hydrophobic residues and106169 containing hydrophilic charged amino acidresidues . It is the latter portion that contributestowards the hydrophilic nature of casein . In addition,oligosaccharide chains consisting of sialic acid,galactose and N-acetylgalactosamine are grafted ontothreonine at positions 131 and 135 in the amino acidsequence. This also contributes to the hydrophilicityof this portion of K-casein.

Since the synthesis of hydrogel involves thedissolution of casein in 0.1 N NaOH, the hydrogelcontains undiffusable COO- groups along themacromolecular chain and a high concentration ofmobile counter ions in the gel phase . When thishydrogel is placed in the doubly distilled water at pH7.0, obviously the difference Cg Ca becomes verylarge, thus resulting in a high osmotic swellingpressure nie,,, and ultimately high degree of swelling.The mutual repulsion of 000- groups along themacromolecular chain also promotes the swelling.

When the hydrogel is placed in the buffersolution of pH 4.0, the dissociation of COOH groupspresent along the macromolecular chain is suppressedand hence concentration of counter ions inside the gelphase i.e ., Cg decreases and hence Ce C, becomescomparatively small . Moreover, since the pH ofexternal solution is just below the isoelectric pH of thecasein (i .e ., 4 .6), NH2 groups present along thecasein molecules also get protonated to a little extent.In this way the mutual attraction between the twooppositely charges groups NH ; and 000- alsocauses the polymer segments to contract (n, ) . Bothof these factors cause a decrease in the degree ofswelling.

In the case of pH 2 .0 of external solution thedissociation of COOH groups is suppressed to such agreat extent that the difference C i Ci becomesextremely small . Therefore minimum swelling isobserved . Here it is worth mentioning that theswelling observed at this pH is mainly due to theelectrostatic repulsion between the similarly charge

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Bgpri S.K.

NH; groups along the segment. Moreover some

contribution of hydrophilic tendency of polyacryl-amide should also be taken into account.

In the alkaline range although the dissociationofCOOH is complete, thus providing a large numberof counter ions in the gel phase but at the same timethere exists a higher concentration of Na+ and OWions in the external solution

. As a result the differenceCg C, becomes small thus resulting in decrease inosmotic swelling pressure as well as extent ofswelling

. However the suppression in the degree ofswelling in alkaline medium is not to such a greatextent as found in the acidic medium. This may becontributed to the fact that in alkaline medium themutual repulsion among COO

- groups is morepredominant . The values of swelling exponent n atdifferent pH of the external solution (Table 3) alsosupport the proposed mechanism.

Reversibility of Hydrogel SwellingThe ability of hydrogel to undergo several cycles ofswelling and deswelling is shown in Figure 2

.,6. It canbe seen from Figure 2a that after the first cycle the geldid not achieve its original swollen state and in all thefollowing cycles it swelled back to its previousswollen state

. Since the virgin gel was used withoutany prior washing, some salt might have been presentwhich could have leached out upon deswelling, thusreducing the degree of successive swelling.

When the freshly prepared gel is placed in thedoubly distilled water at pH 7

.0, the swelling occursto maximum in accordance with the Donnan-membrane equilibrium

. Now when this swollen gel isplaced in 2 M NaCl solution, the concentration ofosmotically active ions in the external solutionbecomes high and hence this results into diffusion ofwater molecules from gel phase into outer solution,thus causing the gel to deswell

. Due to reversibilityand rapidity of swelling, the gel could be consideredas a mechanochemcial system in which chemicalionization energy could be transformed directly intomechanical energy [23].

It is also clear form the Figure 2b that whencompletely dried gel is placed in water it attainsalmost maximum swelling in every cycle

. This shows

0 .010 SI DS1 S2 DS2 S3DS3 S4 DS4

Cycles

(a)

8 .0

6.0

0

2 .0

0.0 .Di Si D2 S2 D3

Cycles(b)

Figure 2 . (a) : Reversibility of swelling, of Cas-PAAmX(l .7)hydrogels ; pH=7.0 ; temperature=37 'C ; 10 indicates initialcondition ; Si=swelling cycle equilibrium condition and DSi=deswelling cycle equilibrium condition . (b) : Reversibleswelling and drying of Cas-PAmX(I .7) hydrogels, pH=7.0;temperature=37 'C ; Di=dried state; Si=swollen state of gel.

that during the swelling process the gel does notundergo any irreversible structural change and hence

8 .0

2 .0

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Casein Crosslinked Nolyacrylamide Hydrogels : Study of Swelling and Drag Rckase

it swells back to maximum when placed in water.During swelling, the shape of the gel sample

followed a repeatable pattern. Initially a swelling front

moved inward separating the swollen surface layerand the unswollen inside core

. As a result, the sampleassumed a dumbbell shape

. Observation through anoptical microscope showed the presence of stresses.The surface of the gel was full of cracks whichdisappeared after a while, yet the dumbbell shape wasmaintained, although it gradually changed back tocylindrical shape . This behaviour was observed duringthe swelling of all samples including dried samples.Similar observations were reported by Tanaka et al.1241 as mechanical instabilities during the swelling ofpolyacrylamide gel beads

. It is concluded that the thinswollen layer would be in mechanical constraint dueto the free outer surface and fixed inner surface.Hence two opposing forces, one forcing the gel toswell and the other to remain unswollen would beresolved depending upon the osmotic pressuredeveloped.

Drug Release AnalysisThe model drug release behaviour of Cas-PAAmXhydrogels has been studied " as a function oftemperature of the external medium. When the drugloaded hydrogel is placed in the double distilled waterat pH 7 .0 at 37 'C, water diffuses into the polymermatrix owing to the swelling osmotic pressure . Thepenetrant molecules diffuse into the gel and displacethe drug particles which ultimately diffuses out of thedevice due to increased chain relaxation. It wasobserved that initial rate of drug release was higherwhich may be contributed to the fact that when gel isplaced in solvent, the outermost surface of thepolymer matrix immediately comes in contact with itand the solvent diffuses into the gel phase, followedby release of drug

. Similar observations have alsobeen reported elsewhere [25].

Figure 3 describes the effect of temperature ondrug releasing capacity of drug loaded hydrogel withthe cross-linking ratio 2.1 and 1 .7 mol % . Here theamount of drug released in a period of 20 h has beenplotted against the temperature of the externalsolution . It is clear from the figure that there is an

q Cam-PAAmX (1 .7)90

10

10

20

30

40

50

50Temperature (C)

Figure 3 . Amount of drug released in a period of 20 h as afunction of temperature of the external solution pH = 7

.0.

increase in the amount of drug delivered up to 40 'C,and then it decreases . Initially the observed decreasein the amount of drug released beyond 40 -C wasthought to be due to formation of some complexstructure between the amide groups and carboxylicgroups, which were produced due to possiblehydrolysis of polyacrylamide . However, when thesame experiment was conducted with the drug loadedhydrogel of cross-linked polyacrylamide alone, thetheory proved to be wrong, as there was no decreasein the amount of drug released beyond 40 *C. Thisclearly showed that no such complex structure formedinside the polymer matrix . In fact, an increase intemperature from 25 to 40 ' C shows a higher andfaster drug release due to the extensive swelling andchain relaxation

. An increase in temperature beyond40 'C shows a decrease in drug release due to decreasein solubility of casein.

Effect of Cross-linkingThe degree of cross-linking of a hydrogel networkplays a significant role in controlling its swellingbehaviour. In the present study, two hydrogel sampleswith the cross-linking ratio in mol % 2

.1 and 1 .7 havebeen used, of which sample Cas-PAAmX (1

.7)showed a greater swelling tendency than the other one(Figure I)

. This behaviour of hydrogels is very

70

30

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Baja. s .ic.

common and may be attributed to the fact that byincreasing molar percent of cross-linker to monomer(i .e ., lightly to highly cross-linked) the number ofefficient cross-links per unit volume increases withthe result that there is less free volume or roomavailable to accommodate solvent, and hence degreeof swelling decreases.

CONCLUSION

The swelling behaviour of casein cross-linkedpolyacrylamide hydrogels has been found to be pH-dependent and it favours the medium with neutral pH7 .0 . This effect could be explained well on the basis ofosmotic swelling pressure theory. These gels alsoundergo a number of reversible swelling-deswellingcycles while maintaining their structural integrity . ThepH-dependent and reversible swelling behaviourcould be very useful in the development of artificialmuscle or physiologically sensitive drug deliverysystems and extraction of biocomponents from dilutesolutions by a modified gel filtration technique.

The drug loaded gels release the drug over aperiod of 24 h and hence there is some possibility ofusing these gels in the case of short term drug deliveryapplications where an immediate or fast relief isneeded. The non-toxic nature of casein is also afavourable factor for its possible use as drug deliverydevice. In brief, the aim of the proposed study to lookfor the possibilities of using casein as a material fordrug delivery devices has been achieved to someextent.

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