Preliminary evaluation of molecular imprinting of 5-fluorouracil within hydrogels for use as drug delivery systems

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Acta Biomaterialia 4 (2008) 1244–1254

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Preliminary evaluation of molecular imprinting of 5-fluorouracilwithin hydrogels for use as drug delivery systems

Baljit Singh *, Nirmala Chauhan

Department of Chemistry, Himachal Pradesh University, Shimla 171 005, India

Received 5 November 2007; received in revised form 2 March 2008; accepted 28 March 2008Available online 8 April 2008

Abstract

Molecular imprinting is a new and rapidly evolving technique used to create synthetic receptors and it possesses great potential in anumber of applications in the life sciences. Keeping in mind the therapeutic importance of 5-fluorouracil (5-FU) and the technologicalsignificance of molecular imprinting polymers, the present study is an attempt to synthesize 2-hydroxyethylmetacrylate- and acrylic acid-based 5-FU imprinted hydrogels. For the synthesis of these hydrogels, N,N0-methylenebisacrylamide was used as a crosslinker, ammo-nium persulfate as an initiator and N,N,N0,N0-tetramethylethylenediamine as an accelerator. Both molecular imprinted polymers (MIPs)and non-imprinted polymers were synthesized at the optimum crosslinker concentration obtained from swelling studies and used to studytheir recognition affinity, their swelling and the in vitro release dynamics of the drug. It was observed from this study that the recognitionaffinity of MIPs is increased when these are synthesized in a high concentration template solution.� 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Drug delivery devices; Hydrogels; Molecular imprinted polymers; Release dynamics

1. Introduction

Molecular imprinting is a rapidly developing techniquefor the preparation of polymeric materials that are capableof molecular recognition for selective separation and chem-ical identification. To prepare molecularly imprinted poly-mers (MIPs), a functional monomer and a crosslinker arepolymerized in the presence of a template molecule. Thetemplate is then extracted, leaving sites which are comple-mentary in both shape and chemical functionality to thoseof the template. This polymer is capable of selectivelyabsorbing the template species. Because of the stability,predesigned selectivity and easy preparation of MIPs, theywere applied in a wide range of technologies for a widerange of purposes, such as catalysis [1], separation andpurification [2,3], detection [4] and drug delivery [5].

Recently, there has been rapid growth in the area ofdrug discovery, facilitated by novel technologies, which

1742-7061/$ - see front matter � 2008 Acta Materialia Inc. Published by Else

doi:10.1016/j.actbio.2008.03.017

* Corresponding author. Tel.: +91 1772830944; fax: +91 1772633014.E-mail address: [email protected] (B. Singh).

has resulted in a more urgent focus on developing noveltechniques to deliver these drugs more effectively and effi-ciently. This can be achieved by the use of polymeric matrixas a delivery system. Hydrogels have been used in the con-trolled delivery of drugs. Hydrogels are three-dimensionalpolymeric networks that swell quickly by imbibing a largeamount of water or shrink in response to changes in theirexternal environment. These changes can be induced bychanging the surrounding pH, temperature, ionic strengthand electrostimulus [6,7]. There is ongoing interest in iden-tifying additional tools to modify the release profile of adrug from a polymer matrix, and molecular imprintinghas been suggested as one of those tools [8]. Molecularimprinting technology has an enormous potential for creat-ing satisfactory drug dosage forms. Although its applica-tion in this field is just at the incipient stage, the use ofMIPs in the design of new drug delivery systems anddevices useful in closely related fields, such as diagnosticsensors, is receiving increasing attention [9]. Examples ofMIP-based drug delivery systems were found for the threemain approaches developed to control the moment at

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B. Singh, N. Chauhan / Acta Biomaterialia 4 (2008) 1244–1254 1245

which delivery should begin and/or the drug release rateand activation-modulated or feedback-regulated drugdelivery. These systems were used for administering drugsby different routes, such as oral, ocular or transdermal[10–12]. The uses of MIPs in different drug delivery systemshave been reported in the literature. Alvarez-Lorenzo andco-workers have developed norfloxacin delivery systemsby imprinting it into soft contact lenses prepared frompoly(hydroxyethylmethacrylate)-based hydrogels [5,13].Hiratani et al. [14–16] have made ocular release of timololpossible from molecularly imprinted soft contact lenses.Affinity sites for an antibacterial drug, ampicillin, were cre-ated by Sreenivasan [12] on the surface of polyurethaneusing the technique of non-covalent molecular imprintingto study the interactions with two bacterial species, Esche-

richia coli and Staphylococcus aureus. The macromolecularrecognition of biologically significant molecules, such asdrugs, amino acids, steroids, nucleotide bases and carbohy-drates, has also been carried out via molecular imprintingmethods to observe the receptor–ligand association anddissociation constant [17]. Due to the high biocompatibilityof MIPs and their ability to recognize the template, MIPswere considered as good means of delivering proteins aspart of an implantable drug delivery system [18,19].

5-Fluorouracil (5-FU) is an anticancer agent that iswidely used in the clinical treatment of several solid can-cers, such as breast, colorectal, liver and brain cancer.Because of its high rate of metabolism in the body, themaintenance of a high serum concentration improves itstherapeutic activity, but this requires its continuous admin-istration. However, concentration above a certain limitproduces a severe toxic effect, and this must be avoided[20,21]. It has been reported in the literature that polypep-tide- and polysaccharide-based drug delivery devices haveimproved the performance of 5-FU [22]. Keeping in mindthe therapeutic importance of 5-FU and the technologicalsignificance of molecular imprinting polymers, the presentstudy is an attempt to synthesize 2-hydroxyethylmetacry-late (HEMA) and acrylic acid (AAc) based 5-FU imprintedhydrogels. For the synthesis of these hydrogels, N,N0-meth-ylenebisacrylamide (N,N-MBAAm) was used as a cross-linker, ammonium persulfate (APS) as an initiator andN,N,N0,N0-tetramethylethylenediamine (TEMED) as anaccelerator. Both MIPs and non-imprinted polymers(NIPs) were synthesized at the optimum crosslinker con-centration obtained from swelling studies and used to studytheir recognition affinity, their swelling and in vitro releasedynamics of the drug.

2. Experimental

2.1. Materials and methods

HEMA and AAc were obtained from Merck-Schuc-hardt, Germany; APS and N,N-MBAAm were obtainedfrom S.D. Fine, Mumbai, India and were used asreceived. TEMED was obtained from the Sisco Research

Lab. Pvt. Ltd. 5-FU was obtained from the Dabar IndiaLtd., India.

2.2. Synthesis of molecular imprinted hydrogels

(poly(HEMA-cl-AAc))

The hydrogels were synthesized by chemically inducedpolymerization through the free radical mechanism. Todetermine the optimum crosslinker concentration requiredfor the synthesis of MIPs, hydrogels were synthesized intriplicate with three different crosslinker concentrations(i.e. 1.297 � 10�2, 3.89 � 10�2 and 6.487 � 10�2 mol l�1

N,N-MBAAm), along with 4.38 � 10�2 mol l�1 APS,7.68 � 10�1 mol l�1 HEMA, 13.88 � 10�1 mol l�1 AAcand 1.72 � 10�1 mol l�1 TEMED in an aqueous solutionwithout drug at 37 �C for 30 min. The hydrogels thusformed were washed with distilled water and dried at37 �C in an oven. These were named poly(HEMA-cl-AAc) hydrogels. The optimum concentration of crosslinkerobtained, on the basis of swelling of the hydrogels andstructural integrity maintained by the gel after swelling,was 3.89 � 10�2 mol l�1. At this crosslinker concentrationthe MIPs of two different drug concentrations (i.e. 50 and25 lg ml�1 5-FU) were prepared and named MIPs-50 andMIPs-25, respectively; polymers without drug were callednon-imprinted polymers (NIPs), as mentioned above.

Synthesis of molecular imprinted hydrogels was carriedout with 4.38 � 10�2 mol l�1 APS, 7.68 � 10�1 mol l�1

HEMA, 13.88 � 10�1 mol l�1 AAc, 3.89 � 10�2 mol l�1

N,N-MBAAm and 1.72 � 10�1 mol l�1 TEMED in theaqueous solution of a definite concentration of drug (5-FU) at 37 �C temperature for 30 min. Synthesis of non-imprinted hydrogels was carried without drug under simi-lar conditions. Both MIPs and NIPs were synthesized intriplicate and were subjected to swelling and drug releasestudies. The MIPs were synthesized with two different con-centrations of the drug to observe the effect of the numberof recognition sites in the imprinted polymers on theentrapment of drug and on the release pattern of the drug.After removal of the template (drug) from the MIPs, thesewere dried at 37 �C in an oven and reloaded again in200 lg ml�1 5-FU. Loading of NIPs was also carried outin a solution of the same concentration of the drug. TheMIPs and NIPs obtained after this loading were again sub-jected to swelling and drug release studies.

2.3. Characterization

Polymers were characterized by FTIR spectroscopy andswelling studies. FTIR spectra of polymers were recordedin KBr pellets on Nicolet 5700FTIR THERMO. Swellingof the polymers was carried out in distilled water by gravi-metric method. A known weight of the polymers was takenand immersed in an excess of solvent for fixed time inter-vals at 37 �C and then the polymers were removed after30 min, wiped with tissue paper to remove excess of sol-vent, and weighed immediately for 300 min. The gain in

1246 B. Singh, N. Chauhan / Acta Biomaterialia 4 (2008) 1244–1254

weight at different time intervals could thus be obtained.The equilibrium swelling was taken after 24 h.

2.4. Release dynamics of drug from poly(HEMA-cl-AAc)

2.4.1. Preparation calibration curvesIn this procedure, the absorbance of a number of stan-

dard solutions of the reference substance at concentrationsencompassing the sample concentrations were measured ona UV–visible spectrophotometer (Cary 100 Bio, Varian)and a calibration graph was constructed. The concentra-tion of the drug in the sample solution was read from thegraph as the concentration corresponding to the absor-bance of the solution. The calibration graph of 5-FU wasmade to determine the amount of drug release from thedrug-loaded MIPs at wavelength 267.0 nm.

2.4.2. Drug loading/release to/from the MIPs

The loading of a drug into MIPs was carried out duringsynthesis of the hydrogels by the procedure mentioned inSection 2.2. The hydrogels were prepared with two differentdrug concentrations and were then washed with water. Thepolymers were then dried at 37 �C to get the release device,i.e. MIPs.

In vitro release studies of the drug were carried out byplacing the dried and loaded sample into a specific volumeof releasing medium at 37 �C. The amount of drug releasedwas assayed spectrophotometrically after each 30 min. Theabsorbance of the solution was measured at 267.0 nmwavelength in each case.

2.4.3. Drug reloading to the MIPs or recognition affinity of

the MIPs and NIPs

After removal of drug (the template) from the MIPs, thepolymers were dried at 37 �C in an oven. Reloading of thedrug into MIPs and loading of the drug into NIPs was car-ried out by the swelling equilibrium method. The hydrogelswere allowed to swell in the drug solution of known con-centration (200 lg ml�1 5-FU) for 24 h at 37 �C and thendried to obtain the release device.

2.5. Mathematical modeling for drug release from polymer

matrix

In the hydrogels system, absorption of water from theenvironment changes the dimensions and physicochemical

Table 1Results of the diffusion exponent n, the gel characteristic constant k and variohydrogels prepared with different concentrations of N,N-MBAAm

[N,N-MBAAm](�102 mol l�1)

Diffusion exponent,n

Gel characteristic const(�102)

1.30 0.6 1.353.89 0.5 2.916.49 0.4 3.94

properties of the system and thus the drug release kinetics.Although a number of reports deal with the mathematicalmodeling of drug release from swellable polymeric systems,no single model successfully predicts all the experimentalobservations. Since most complex models do not yield aconvenient formula and require numerical solution tech-niques, generalized empirical equations were widely usedto describe both the water uptake through the swellableglassy polymers and the drug release from these devices[23–29]. In the case of water uptake, the weight gain, Ms,is described by the following empirical equations:

M s ¼ ktn ð1Þwhere k and n are constant. Normal Fickian diffusion ischaracterized by n = 0.5 and Case II diffusion by n = 1.0.A value of n between 0.5 and 1.0 indicates a mixture of Fic-kian and Case II diffusion, which is usually called non-Fic-kian, or anomalous, diffusion. Ritger and Peppas showedthat the above power law expression could be used forthe evaluation of drug release from swellable systems[25,26]. In this case, Mt/M1 replaces Ms in the above equa-tion to give

Mt

M1¼ ktn ð2Þ

where Mt/M1 is the fractional release of drug at time t (Mt

and M1 are the drug released at time t and at equilibrium,respectively), k is the constant characteristic of the drug–polymer system and n is the diffusion exponent characteris-tic of the release mechanism. When the plot is drawn be-tween lnMt/M1 and ln t, the slope of the plot gives thevalue of n and the intercept gives k. This equation appliesuntil 60% of the total amount of drug is released. It pre-dicts that the fractional release of drug is exponentially re-lated to the release time and adequately describes therelease of drug from slabs, spheres, cylinders and discsfrom both swellable and non-swellable matrices. Fick’s firstand second laws of diffusion adequately describe the mostdiffusion processes. For cylindrical hydrogels the integraldiffusion is given by the simple equation:

Mt

M1¼ 4

Dt

p‘2

� �0:5

ð3Þ

where D is the diffusion coefficient and ‘ is the thickness ofthe sample. In Eq. (3), the slope of the linear plot betweenMt/M1 and t1/2 (the time required for 50% release of drug)

us diffusion coefficients for the swelling kinetics of poly(HEMA-cl-AAc)

ant, k Diffusion coefficients (cm2 min�1)

Initial, Di

(�104)Average, DA

(�104)Late time, DL

(�104)

7.24 13.20 1.2213.09 18.17 2.156.42 14.89 1.24


yields the diffusion coefficient D. Therefore, the initial dif-fusion coefficient Di can be evaluated from the slope ofthe plot. The average diffusion coefficient DA may also becalculated for 50% of the total release by putting Mt/M1 = 0.5 in Eq. (3), which finally yields Eq. (4). Late dif-fusion coefficients were calculated using the late timeapproximation as described by Peppas et al. given in Eq.(5) [25,26]:

DA ¼0:049‘2

t1=2ð4Þ

Mt

M1¼ 1� 8

p2

� �exp

ð�p2DtÞ‘2

� �ð5Þ

The slope of the plot between ln(1 �Mt/M1) and t wasused for the evaluation of DL. The values of diffusion coef-ficients for the swelling kinetics and release dynamics of thedrug from the hydrogels were evaluated and the results arepresented in Tables 1–3.

3. Results and discussion

3.1. Characterization

Poly(HEMA-cl-AAc) hydrogels were characterized byFTIR and swelling studies.

3.1.1. Fourier transform infrared spectroscopy

FTIR spectra of poly(HEMA-cl-AAc) was recorded andis presented in Fig. 1. The broad absorption band at3430.9 cm�1 is due to –OH stretching, indicating the strongassociation in this polymer. The infrared absorption bandsat 1726.4 cm�1 due to C@O stretching of the ester and at1260.6 cm�1 due to the C–O stretching of esters and at1666 cm�1 due to C@O stretching of the acid wereobserved in the poly(HEMA-cl-AAc). The CH2 asymmet-

Table 3Results of the diffusion exponent n, the gel characteristic constant k and variouNIPs of poly(HEMA-cl-AAc) in distilled water at 37 �C

Drug release frompolymers


Gel characteristic constan(�102)

MIPs-50 0.268 9.98MIPs-25 0.235 11.09NIP 0.143 15.96

Table 2Results of the diffusion exponent n, the gel characteristic constant k and variopoly(HEMA-cl-AAc) in distilled water at 37 �C

Different polymermatrices


Gel characteristic constan(�102)

MIPs-50 0.42 3.13MIPs-25 0.44 3.11NIP 0.47 3.18

ric stretching vibration at 2926 cm�1 and the symmetricCH2 absorption at 2856 cm�1 along with the –CH defor-mation mode around 1457.5 cm�1 were observed in thespectra.

3.1.2. Effect of crosslinker on swelling of poly(HEMA-cl-AAc)

The structure of the imprinted cavities in the MIPsshould be stable enough to maintain the conformation inthe absence of the template and it should also be flexibleenough to facilitate the attainment of a fast equilibriumbetween the release and re-uptake of the template in thecavity. This is particularly important if the device is usedas a diagnostic sensor, as a trap of toxic substances inthe gastrointestinal tract or to deliver a drug in a controlledand sustained manner in the colon. Therefore, in the pres-ent case, the polymers were prepared with three differentconcentrations of the crosslinker (that is 1.30 � 10�2,3.89 � 10�2 and 6.49 � 10�2 mol l�1). Polymers were syn-thesized in triplicate for each concentration of N,N-MBAAm. In order to compromise between the rigidityand flexibility of the polymers, a swelling study was carriedout for the polymers prepared with different concentrationsof N,N-MBAAm. The amount of water taken up by thepolymer matrix at 37 �C up to 300 min was studied aftera fixed interval of 30 min and the results are shown inFig. 2.1. It can be observed from Fig. 2.1 that the amountof water uptake by per gram of gel increases with time anddecreases with increasing crosslinker concentration in thepolymer. Further, it was observed from the equilibriumswelling after 24 h that maximum water uptakes of3.87 ± 0.049, 1.53 ± 0.038 and 1.36 ± 0.076 g g�1 gel wereobtained for the polymers synthesized, respectively, with1.30 � 10�2, 3.89 � 10�2 and 6.49 � 10�2 mol l�1 N,N-MBAAm (Fig. 2.2). This is probably due to the increased

s diffusion coefficients for the release of 5-fluorouracil from the MIPs and

t, k Diffusion coefficients (cm2 min�1)

Initial, Di

(�104)Average, DA

(�104)Late time, DL

(�104)

2.53 13.91 0.7671.72 12.26 0.6980.46 10.60 0.286

us diffusion coefficients for the swelling kinetics of the MIPs and NIPs of

t, k Diffusion coefficients (cm2 min�1)

Initial, Di (�104) Average, DA

(�104)Late time, DL (�104)

3.16 10.60 0.773.78 11.56 0.825.34 14.08 1.09

520.

8629.

5

802.

1

1022

.910

74.6

1160

.6

1260

.6

1401

.014

57.515

61.7

1655

.017

26.4

2364

.4

2628

.4

2855

.229

26.2

3430

.9

3743

.8

**HEMA AAc

66

68

70

72

74

76

78

80

82

84

86

88

90

92

94

96

%Tr

ansm

ittan

ce

500 1000 1500 2000 2500 3000 3500

Wavenumbers (cm-1)4000

Fig. 1. FTIR spectra of poly(HEMA-cl-AAc).

0 30 60 90 120 150 180 210 240 270 300

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Am

ount

of w

ater

upt

ake

(g/ g

of g

el)

Time (min)

[N,N-MBAAm]- 1.30×10-2mol / L 3.89×10-2mol / L 6.49×10-2mol / L

Fig. 2.1. Swelling kinetics of the poly(HEMA-cl-AAc) hydrogels preparedwith different [N,N-MBAAm] in distilled water at 37 �C. Reactiontime = 30 min; reaction temperature = 37 �C; [HEMA] = 7.68 � 10�1

mol l�1; [AAc] = 13.88 � 10�1 mol l�1; [APS] = 0.438 � 10�1 mol l�1;[TEMED] = 1.72 � 10�1 mol l�1.

A B C

0

2

4

Am

ount

of w

ater

upt

ake(

g/g

of g

el)

afte

r 24

hrs

.

[N,N-MBAAm]=A- 1.30× 10-2 mol / LB- 3.89×10-2mol / LC- 6.49×10-2mol / L

Fig. 2.2. Amount of water uptake for the poly(HEMA-cl-AAc) hydrogelsprepared with different [N,N-MBAAm] after 24 h in distilled water at37 �C.


extent of crosslinking of polymeric chains in hydrogels thatlead to a decrease in pore size and a decrease in the wateruptake capacity of hydrogels. However, a higher total per-centage uptake of water occurred with the polymers pre-

pared with 3.89 � 10�2 mol l�1 N,N-MBAAm (Fig. 2.3).The values of the diffusion exponent n and gel characteris-tic constant k were evaluated from the slope and interceptof the plot lnMt/M1 vs. ln t (Fig. 2.4), and the results arepresented in Table 1. It is clear from the table that values ofn of both <0.5 and >0.5 were observed, which indicatesthat both Fickian- and non-Fickian-type diffusion mecha-

0 30 60 90 120 150 180 210 240 270 300

10

15

20

25

30

35

40

45

50

55

60

65

70

75

% C

umul

ativ

e w

ater

upt

ake

Time (min)

[N,N-MBAAm]- 1.30× 10-2 mol / L 3.89×10-2mol / L 6.49×10-2mol / L

Fig. 2.3. Percentage cumulative water uptake of the poly(HEMA-cl-AAc)hydrogels prepared with different [N,N-MBAAm] in distilled water at37 �C.

0 50 100 150 200 250 30080

100

120

140

160

180

200

220

Am

ount

of d

rug

rele

ase

(ug/

20m

L/g

of g

el)

Time (min.)

MIP loaded with (50 ug/mL) of 5-FU MIP loaded with (25 ug/mL) of 5-FU

Fig. 3.1. Release profile of 5-fluorouracil from MIPs of poly(HEMA-cl-AAc) hydrogels loaded with different drug concentrations in distilled waterat 37 �C. Reaction time = 30 min; reaction temperature = 37 �C;[HEMA] = 7.68 � 10�1 mol l�1; [AAc] = 13.88 � 10�1 mol l�1; [APS] =0.438� 10�1 mol l�1; [N,N-MBAAm] = 3.89� 10�2 mol l�1; [TEMED] =1.72 � 10�1 mol l�1.

3.0 3.5 4.0 4.5 5.0 5.5 6.0

-2.2

-2.0

-1.8

-1.6

-1.4

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

ln (

Mt/M

oo)

ln t

[N,N-MBAAm]- 1.30× 10-2 mol / L 3.89×10-2mol / L 6.49×10-2mol / L

Fig. 2.4. Plot for the evaluation of the diffusion exponent n and the gelcharacteristic constant k for the swelling of the poly(HEMA-cl-AAc)hydrogels prepared with different [N,N-MBAAm] in distilled water at37 �C.


nisms occurred for the diffusion of water molecules in thepolymer prepared with different crosslinker concentrations.The values of the diffusion coefficient are presented in theTable 1. It is clear from the table that the values obtainedfor the average diffusion coefficient (DA) were higher thanthe initial and late diffusion coefficients (Di and DL, respec-tively). From the above discussion, the optimum concen-tration of N,N-MBAAm (3.89 � 10�2 mol l�1) wasobtained for the further synthesis of polymers with drugand imprinting of the same in the polymers.

3.2. Loading of 5-FU in poly(HEMA-cl-AAc) and release

thereafter

MIP hydrogels were fabricated as mentioned in Section2.2. Two different concentrations of drug (50 and 25

lg ml�1 5-FU) were loaded into the polymers to observethe effect of the number of recognition sites in theimprinted polymers on the entrapment of drug and therelease pattern of the drug. All the polymers were synthe-sized in triplicate and were used to study the releasedynamics of the drug immediately after synthesis. Therelease pattern of the 5-FU is presented in Fig. 3.1. It canbe observed from Fig. 3.1 that the release of 5-FU fromthe poly(HEMA-cl-AAc) hydrogels loaded with 50 lg ml�1

of the drug was higher than from the hydrogels loaded with25 lg ml�1 5-FU in the first 300 min of the release. Thisobservation is further supported by the fact that the releaseof 5-FU from MIPs-50 is also higher than that from MIPs-25. The total amount of drug released from MIPs-50 andMIPs-25 were 280.66 ± 6.92 and 144.79 ± 5.04 lg (20ml)�1 g�1 gel, respectively (Fig. 3.2). The higher releaseof 5-FU from MIPs-50 was due to the higher initial loadingof the drug in the polymer during synthesis. The percentageof the total release was also observed to be higher in thecase of MIPs-50 (Fig. 3.3). The value of the diffusion expo-nent n was observed to be 0.1 and 0.04, respectively, forMIPs-50 and MIPs-25. As these values are less than 0.5,no specific type of mechanism can be assigned in this case.The data clearly indicate, however, that the binding to theMIPs slows the rate of release to below that expected fromFickian relationships [30,31] (Fig. 3.4).

3.3. Reloading of the drug or recognition affinity of MIPs

Reloading was carried out in both MIPs and loadingwas carried in NIPs in triplicate to observe the recognitioncapacity of the hydrogels for the template. Molecularimprinting is a technique producing synthetic materialscontaining highly specific receptor sites that have an affin-

A B0

50

100

150

200

250

300

350

Am

ount

of d

rug

rele

ase

(ug/

20 m

L/g

of g

el)

afte

r 24

hrs

.

A-MIP loaded with (50 ug/mL) of 5-FUB-MIP loaded with (25 ug/mL) of 5-FU

Fig. 3.2. Drug release pattern of 5-fluorouracil from MIPs of poly(HEMA-cl-AAc) hydrogels after 24 h in distilled water at 37 �C.

0 50 100 150 200 250 300

55

60

65

70

75

80

% C

umul

ativ

e re

leas

e

Time (min.)

MIP loadedwith (50 ug/mL) of 5-FU MIP loadedwith (25 ug/mL) of 5-FU

Fig. 3.3. Percentage cumulative release of 5-fluorouracil from MIPs ofpoly(HEMA-cl-AAc) hydrogels loaded with different drug concentrationin distilled water at 37 �C.

3.0 3.5 4.0 4.5 5.0 5.5 6.0-0.65

-0.60

-0.55

-0.50

-0.45

-0.40

-0.35

-0.30

-0.25

-0.20

ln (

Mt/M

oo)

ln t

MIP loaded with (50 ug/mL) of 5-FU MIP loaded with (25 ug/mL) of 5-FU

Fig. 3.4. Plot for the evaluation of the diffusion exponent n and the gelcharacteristic constant k of 5-fluorouracil from MIPs of poly(HEMA-cl-AAc) hydrogels loaded with different drug concentration in distilled waterat 37 �C.

MIP-1 MIP-2 NIP0

200

400

600

800

1000

1200

1400

1600

1800

Am

ount

of d

rug

entr

aped

(ug

/g o

f gel

) af

ter

relo

adin

g MIP-1=MIP initial loaded with (50 ug/mL) of 5-FUMIP-2=MIP initial loaded with (25 ug/mL) of 5-FUNIP= Non imprinted polymer (Control)

Fig. 4. Total amount of drug entrapped by reloaded MIPs and NIPs ofpoly(HEMA-cl-AAc) after 24 h in distilled water at 37 �C. Stockconcentration = 200 lg ml�1.


ity for a target molecule, and MIPs can mimic the recogni-tion and binding capabilities of the template molecule. Inthe present case it was observed that the recognition affinityof the MIPs for 5-FU was higher than that of NIPs whenreloading of drug was carried out by the swelling equilib-rium method whereby both the MIPs (MIPs-50 andMIPs-25) and NIPs were kept in a 200 lg ml�1 solutionof 5-FU for 24 h at 37 �C and then dried to obtain the fur-ther release device. The results are present in the Fig. 4. Itcan be seen from the figure that the MIPs which were ini-tially loaded with 50 lg ml�1 5-FU entrapped a largeramount of the drug (1328.13 ± 55.36 lg g�1 gel) than theMIPs loaded with 25 lg ml�1 5-FU (1102.15 ± 12.32

lg g�1 gel) and NIPs (716.01 ± 6.56 lg g�1 gel). This isbecause the number of recognition sites (that is templatesites) was higher in the MIPs initially loaded with thehigher concentration of the drug. There were no recogni-tion sites available in the NIPs, and hence these showed alower entrapment of drug per gram of gel.

3.4. Swelling and release dynamics of the drug from the

MIPs and NIPs after reloading

After reloading of the drug, the MIPs and NIPs weredried at room temperature (25 �C) and then used to studythe swelling of the poly(HEMA-cl-AAc) hydrogels andrelease dynamics of the drug from these hydrogels.

0 50 100 150 200 250 3000.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Time (min.)

Am

ount

of w

ater

upt

ake

(g/g

of g

el)

afte

r re

load

ing

MIP-1=MIP initial loaded with (50 ug/mL) of 5-FUMIP-2=MIP initial loaded with (25 ug/mL) of 5-FU NIP= Non imprinted polymer (Control)

Fig. 5.1. Swelling kinetics of reloaded MIPs and NIPs of poly(HEMA-cl-AAc) hydrogels in distilled water at 37 �C.

0 30 60 90 120 150 180 210 240 270 30010

15

20

25

30

35

40

45

50

55

Time(min)

% C

umul

ativ

e up

take

afte

r re

load

ing


Fig. 5.3. Percentage cumulative uptake of reloaded MIPs and NIPs ofpoly(HEMA-cl-AAc) hydrogels in distilled water at 37 �C.

3.0 3.5 4.0 4.5 5.0 5.5 6.0

-2.0

-1.8

-1.6

-1.4

-1.2

-1.0

-0.8

-0.6ln

(M

t/Moo

)

ln t


Fig. 5.4. Plot for the evaluation of the diffusion exponent n and the gelcharacteristic constant k for the swelling of reloaded MIPs and NIPs of


3.4.1. Swelling kinetics

The swelling of the MIPs and NIPs is presented inFig. 5.1. More swelling of the MIPs was observed than ofthe NIPs. It is also clear from the Fig. 5.1 that the MIPsthat were initially loaded with 50 lg ml�1 5-FU (MIPs-50) showed more swelling than those initially loaded with25 lg ml�1 5-FU (MIPs-25) and the NIPs. Total amountof water taken up by MIPs-50, MIPs-25 and NIPs was3.73 ± 0.046, 3.11 ± 0.015 and 2.26 ± 0.036 g g�1 gel,respectively (Fig. 5.2). Fifty percent of the total swellingoccurred in 666.12, 462.25 and 359.10 min, respectively,for MIPs-50, MIPs-25 and NIPs (Fig. 5.3). It shows thatthe rate of swelling is higher in MIPs as compare to NIPs.The values of diffusion exponent and gel characteristicsconstant k were evaluated from the slope and intercept ofthe plot lnMt/M1 vs. ln t (Fig. 5.4) and results are pre-sented in Table 2. It is clear from the table that the value

MIP-1 MIP-2 NIP0

2

4

MIP-1=MIP initial loaded with (50 ug/mL) of 5-FUMIP-2=MIP initial loaded with (25 ug/mL) of 5-FUNIP= Non imprinted polymer (Control)

Am

ount

of w

ater

upt

ake

(g/g

of g

el)

afte

r 24

hrs

.

Fig. 5.2. Swelling pattern of reloaded MIPs and NIPs of poly(HEMA-cl-AAc) hydrogels after 24 h in distilled water at 37 �C.

poly(HEMA-cl-AAc) hydrogels in distilled water at 37 �C.

of n for MIPs-50, MIPs-25 and NIPs are 0.42, 0.44 and0.47, respectively, which indicates that a Fickian-type diffu-sion mechanism has occurred in all three polymers. The dif-fusion coefficients obtained are shown in Table 2. It is clearfrom the table that the values obtained for the average dif-fusion coefficient (DA) were higher than both the initial andlate diffusion coefficients (Di and DL, respectively). Thisshows that during the early and the late stages of swellingthe diffusion of water molecules into the hydrogels wasslow. From this discussion, it is concluded that theimprinted gels swelled at faster rates than the non-imprinted ones.

3.4.2. Release dynamics of the drugThe profile of the release of 5-FU per gram of the MIPs

and NIPs is presented in Fig. 6.1. It can be seen from

0 50 100 150 200 250 300

120

160

200

240

280

320

360

400

440

480

Time (min.)Am

ount

of d

rug

rele

ase

(ug/

20 m

L pe

r g

gel)

afte

r re

load

ing


Fig. 6.1. Release profile of 5-fluorouracil from reloaded MIPs and NIPsof poly(HEMA-cl-AAc) hydrogels in distilled water at 37 �C. Reloadingconcentration of 5-FU = 200 lg ml�1.

0

200

400

600

800

1000 MIP-1=MIP initial loaded with (50 ug/mL) of 5-FU MIP-2=MIP initial loaded with (25 ug/mL) of 5-FU NIP= Non imprinted polymer (Control)

Am

ount

of d

rug

rele

ase

(ug

/20

mL/

g of

gel

) af

ter

24 h

rs.

Fig. 6.2. Drug release pattern of 5-fluorouracil from reloaded samples ofMIPs and NIPs of poly(HEMA-cl-AAc) hydrogels after 24 h in distilledwater at 37 �C. Reloading concentration = 200 lg ml�1.

0 50 100 150 200 250 30020

25

30

35

40

45

50

55

% C

umul

ativ

e re

leas

e af

ter

relo

adin

g

Time (min.)


Fig. 6.3. Percentage cumulative release of 5-fluorouracil from reloadedMIPs and NIPs of poly(HEMA-cl-AAc) hydrogels in distilled water at37 �C. Reloading concentration of 5-FU = 200 lg ml�1.

3.0 3.5 4.0 4.5 5.0 5.5 6.0-1.5

-1.4

-1.3

-1.2

-1.1

-1.0

-0.9

-0.8

-0.7

-0.6

ln (

Mt/M

oo)

ln t


Fig. 6.4. Plot for the evaluation of the diffusion exponent n and the gelcharacteristic constant k of 5-fluorouracil from reloaded MIPs and NIPsof poly(HEMA-cl-AAc) hydrogels in distilled water at 37 �C.


Fig. 6.1 that the amount of drug released from the MIPs-50was higher than from the NIPs, because grater entrapmentof drug occurred in these polymers than in the NIPs. Thedrug in the MIPs was released in a controlled manner.The total amounts of gel released from MIPs-50, MIPs-25 and NIPs were 875.33 ± 4.76, 731.46 ± 9.92 and420.98 ± 3.89 lg (20 ml)�1 g�1, respectively (Fig. 6.2). The50% total release was higher in the case of the MIPs(Fig. 6.3). The values of the diffusion exponent n and thegel characteristic constant k for the swelling of polymersin distilled water were evaluated from the slope and inter-cept of the plot lnMt/M1 vs. ln t (Fig. 6.4) and the results

are presented in Table 3. It is clear from the table that avalue for n of <0.5 was obtained in each release case, whichindicates that a non-Fickian-type diffusion mechanism isresponsible for the diffusion of the drug from the polymers.A similar observation was reported by Liu and co-workers[30,31] in hydrogels prepared by the copolymerization ofthe monomers 1-b-allyloxycarbonyloxymethyl-5-fluoroura-cil and 1,3-bis(b-allyloxycarbonyloxymethyl)-5-fluoroura-cil separately with N-vinylpyrrolidinone (NVP) to formlinear copolymers and crosslinked polymer networks,respectively. It was observed that the hydrolytic scissionof the carbonate groups resulted in release of 5-FU. Thetime-dependent fractional release of the 5-FU was seen tobe fitted by a power relationship with exponents between0.10 and 0.25. These values are significantly lower than


the value of 0.5 expected for Fickian release or the valuesof between 0.5 and 1.0 that are usually observed whenthe release is moderated by interaction of the polymer withthe diffusate. The data thus indicate that the release is con-trolled not only by Fickian diffusion and interaction withthe polymer chain, but also, as expected, the rate of degra-dation of the covalent linkages to the poly(NVP) chainsplays a major role. In another observation it was observedthat the total fraction of 5-FU released over the time scaleof the measurement is also a function of the initial loading.This result suggests that the rate of fractional release islower for the hydrogel containing the lower concentrationof 5-FU. This is a potentially useful observation since thehydrogel water content at all times during the degradationincreases as the loading decreases, and in some medicalapplications this may be advantageous [30,31]. The valuesof the diffusion coefficient for the release of drug from thesepolymers are presented in Table 3. It can be seen from thetable that the values obtained for the average diffusioncoefficient (DA) were higher than both the initial and thelate diffusion coefficients (Di and DL, respectively). Thismeans that in both the early and late stages of drug releasethe rate of diffusion of drug from the polymer matrix wasslow. This observation is also supported by the resultsobtained for the swelling of the MIPs and NIPs.

4. Conclusion

It is concluded from the forgone discussion that concen-tration of the crosslinker during synthesis can play a veryimportant role in deciding the flexibility and rigidity ofMIPs. It is also concluded from the drug entrapment studythat the concentration of the drug molecule determines thedrug recognition capacity of MIPs. The swelling of MIPsincreases with the increasing template concentration inthe MIPs, which also helps in the release of the drug in acontrolled manner. Because of enhanced binding capacityof the MIPs, these can be further exploited for the use inseparation, purification detection and drug delivery tech-nologies for biomolecules, and can be used for the develop-ing the biomimic devices.

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Documents

Preliminary evaluation of molecular imprinting of 5-fluorouracil within hydrogels for use as drug delivery systems