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 www.elsevier.com/locate/actabiomat

Preliminary evaluation of molecular imprinting of 5-fluorouracil within 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 2008 Available 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 a number of applications in the life sciences. Keeping in mind the therapeutic importance of 5-fluorouracil (5-FU) and the technological significance of molecular imprinting polymers, the present study is an attempt to synthesize 2-hydroxyethylmetacrylate- and acrylic acidbased 5-FU imprinted hydrogels. For the synthesis of these hydrogels, N,N0 -methylenebisacrylamide was used as a crosslinker, ammonium 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 study their recognition affinity, their swelling and the in vitro release dynamics of the drug. It was observed from this study that the recognition affinity of MIPs is increased when these are synthesized in a high concentration template solution. Ó 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 technique for the preparation of polymeric materials that are capable of molecular recognition for selective separation and chemical identification. To prepare molecularly imprinted polymers (MIPs), a functional monomer and a crosslinker are polymerized in the presence of a template molecule. The template is then extracted, leaving sites which are complementary in both shape and chemical functionality to those of the template. This polymer is capable of selectively absorbing the template species. Because of the stability, predesigned selectivity and easy preparation of MIPs, they were applied in a wide range of technologies for a wide range of purposes, such as catalysis [1], separation and purification [2,3], detection [4] and drug delivery [5]. Recently, there has been rapid growth in the area of drug discovery, facilitated by novel technologies, which *

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

has resulted in a more urgent focus on developing novel techniques to deliver these drugs more effectively and efficiently. This can be achieved by the use of polymeric matrix as a delivery system. Hydrogels have been used in the controlled delivery of drugs. Hydrogels are three-dimensional polymeric networks that swell quickly by imbibing a large amount of water or shrink in response to changes in their external environment. These changes can be induced by changing the surrounding pH, temperature, ionic strength and electrostimulus [6,7]. There is ongoing interest in identifying additional tools to modify the release profile of a drug from a polymer matrix, and molecular imprinting has been suggested as one of those tools [8]. Molecular imprinting technology has an enormous potential for creating satisfactory drug dosage forms. Although its application in this field is just at the incipient stage, the use of MIPs in the design of new drug delivery systems and devices useful in closely related fields, such as diagnostic sensors, is receiving increasing attention [9]. Examples of MIP-based drug delivery systems were found for the three main approaches developed to control the moment at

1742-7061/$ - see front matter Ó 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2008.03.017

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

which delivery should begin and/or the drug release rate and activation-modulated or feedback-regulated drug delivery. These systems were used for administering drugs by different routes, such as oral, ocular or transdermal [10–12]. The uses of MIPs in different drug delivery systems have been reported in the literature. Alvarez-Lorenzo and co-workers have developed norfloxacin delivery systems by imprinting it into soft contact lenses prepared from poly(hydroxyethylmethacrylate)-based hydrogels [5,13]. Hiratani et al. [14–16] have made ocular release of timolol possible from molecularly imprinted soft contact lenses. Affinity sites for an antibacterial drug, ampicillin, were created by Sreenivasan [12] on the surface of polyurethane using the technique of non-covalent molecular imprinting to study the interactions with two bacterial species, Escherichia coli and Staphylococcus aureus. The macromolecular recognition of biologically significant molecules, such as drugs, amino acids, steroids, nucleotide bases and carbohydrates, has also been carried out via molecular imprinting methods to observe the receptor–ligand association and dissociation constant [17]. Due to the high biocompatibility of MIPs and their ability to recognize the template, MIPs were considered as good means of delivering proteins as part of an implantable drug delivery system [18,19]. 5-Fluorouracil (5-FU) is an anticancer agent that is widely used in the clinical treatment of several solid cancers, such as breast, colorectal, liver and brain cancer. Because of its high rate of metabolism in the body, the maintenance of a high serum concentration improves its therapeutic activity, but this requires its continuous administration. However, concentration above a certain limit produces a severe toxic effect, and this must be avoided [20,21]. It has been reported in the literature that polypeptide- and polysaccharide-based drug delivery devices have improved the performance of 5-FU [22]. Keeping in mind the therapeutic importance of 5-FU and the technological significance of molecular imprinting polymers, the present study is an attempt to synthesize 2-hydroxyethylmetacrylate (HEMA) and acrylic acid (AAc) based 5-FU imprinted hydrogels. For the synthesis of these hydrogels, N,N0 -methylenebisacrylamide (N,N-MBAAm) was used as a crosslinker, ammonium persulfate (APS) as an initiator and N,N,N0 ,N0 -tetramethylethylenediamine (TEMED) as an accelerator. Both MIPs and non-imprinted polymers (NIPs) were synthesized at the optimum crosslinker concentration obtained from swelling studies and used to study their recognition affinity, their swelling and in vitro release dynamics of the drug. 2. Experimental 2.1. Materials and methods HEMA and AAc were obtained from Merck-Schuchardt, Germany; APS and N,N-MBAAm were obtained from S.D. Fine, Mumbai, India and were used as received. TEMED was obtained from the Sisco Research

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Lab. Pvt. Ltd. 5-FU was obtained from the Dabar India Ltd., India. 2.2. Synthesis of molecular imprinted hydrogels (poly(HEMA-cl-AAc)) The hydrogels were synthesized by chemically induced polymerization through the free radical mechanism. To determine the optimum crosslinker concentration required for the synthesis of MIPs, hydrogels were synthesized in triplicate with three different crosslinker concentrations (i.e. 1.297  102, 3.89  102 and 6.487  102 mol l1 N,N-MBAAm), along with 4.38  102 mol l1 APS, 7.68  101 mol l1 HEMA, 13.88  101 mol l1 AAc and 1.72  101 mol l1 TEMED in an aqueous solution without drug at 37 °C for 30 min. The hydrogels thus formed were washed with distilled water and dried at 37 °C in an oven. These were named poly(HEMA-clAAc) hydrogels. The optimum concentration of crosslinker obtained, on the basis of swelling of the hydrogels and structural integrity maintained by the gel after swelling, was 3.89  102 mol l1. At this crosslinker concentration the MIPs of two different drug concentrations (i.e. 50 and 25 lg ml1 5-FU) were prepared and named MIPs-50 and MIPs-25, respectively; polymers without drug were called non-imprinted polymers (NIPs), as mentioned above. Synthesis of molecular imprinted hydrogels was carried out with 4.38  102 mol l1 APS, 7.68  101 mol l1 HEMA, 13.88  101 mol l1 AAc, 3.89  102 mol l1 N,N-MBAAm and 1.72  101 mol l1 TEMED in the aqueous solution of a definite concentration of drug (5FU) at 37 °C temperature for 30 min. Synthesis of nonimprinted hydrogels was carried without drug under similar conditions. Both MIPs and NIPs were synthesized in triplicate and were subjected to swelling and drug release studies. The MIPs were synthesized with two different concentrations of the drug to observe the effect of the number of recognition sites in the imprinted polymers on the entrapment of drug and on the release pattern of the drug. After removal of the template (drug) from the MIPs, these were dried at 37 °C in an oven and reloaded again in 200 lg ml1 5-FU. Loading of NIPs was also carried out in a solution of the same concentration of the drug. The MIPs and NIPs obtained after this loading were again subjected to swelling and drug release studies. 2.3. Characterization Polymers were characterized by FTIR spectroscopy and swelling studies. FTIR spectra of polymers were recorded in KBr pellets on Nicolet 5700FTIR THERMO. Swelling of the polymers was carried out in distilled water by gravimetric method. A known weight of the polymers was taken and immersed in an excess of solvent for fixed time intervals at 37 °C and then the polymers were removed after 30 min, wiped with tissue paper to remove excess of solvent, and weighed immediately for 300 min. The gain in

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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 curves In this procedure, the absorbance of a number of standard solutions of the reference substance at concentrations encompassing the sample concentrations were measured on a UV–visible spectrophotometer (Cary 100 Bio, Varian) and a calibration graph was constructed. The concentration of the drug in the sample solution was read from the graph as the concentration corresponding to the absorbance of the solution. The calibration graph of 5-FU was made to determine the amount of drug release from the drug-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 during synthesis of the hydrogels by the procedure mentioned in Section 2.2. The hydrogels were prepared with two different drug concentrations and were then washed with water. The polymers were then dried at 37 °C to get the release device, i.e. MIPs. In vitro release studies of the drug were carried out by placing the dried and loaded sample into a specific volume of releasing medium at 37 °C. The amount of drug released was assayed spectrophotometrically after each 30 min. The absorbance of the solution was measured at 267.0 nm wavelength 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, the polymers were dried at 37 °C in an oven. Reloading of the drug into MIPs and loading of the drug into NIPs was carried out by the swelling equilibrium method. The hydrogels were allowed to swell in the drug solution of known concentration (200 lg ml1 5-FU) for 24 h at 37 °C and then dried to obtain the release device. 2.5. Mathematical modeling for drug release from polymer matrix In the hydrogels system, absorption of water from the environment changes the dimensions and physicochemical

properties of the system and thus the drug release kinetics. Although a number of reports deal with the mathematical modeling of drug release from swellable polymeric systems, no single model successfully predicts all the experimental observations. Since most complex models do not yield a convenient formula and require numerical solution techniques, generalized empirical equations were widely used to describe both the water uptake through the swellable glassy 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

ð1Þ

where k and n are constant. Normal Fickian diffusion is characterized 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 Fickian and Case II diffusion, which is usually called non-Fickian, or anomalous, diffusion. Ritger and Peppas showed that the above power law expression could be used for the evaluation of drug release from swellable systems [25,26]. In this case, Mt/M1 replaces Ms in the above equation to give Mt ¼ ktn M1

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 characteristic of the release mechanism. When the plot is drawn between ln Mt/M1 and ln t, the slope of the plot gives the value of n and the intercept gives k. This equation applies until 60% of the total amount of drug is released. It predicts that the fractional release of drug is exponentially related to the release time and adequately describes the release of drug from slabs, spheres, cylinders and discs from both swellable and non-swellable matrices. Fick’s first and second laws of diffusion adequately describe the most diffusion processes. For cylindrical hydrogels the integral diffusion is given by the simple equation:  0:5 Mt Dt ¼4 ð3Þ M1 p‘2 where D is the diffusion coefficient and ‘ is the thickness of the sample. In Eq. (3), the slope of the linear plot between Mt/M1 and t1/2 (the time required for 50% release of drug)

Table 1 Results of the diffusion exponent n, the gel characteristic constant k and various diffusion coefficients for the swelling kinetics of poly(HEMA-cl-AAc) hydrogels prepared with different concentrations of N,N-MBAAm [N,N-MBAAm] (102 mol l1)

Diffusion exponent, n

Gel characteristic constant, k (102)

1.30 3.89 6.49

0.6 0.5 0.4

1.35 2.91 3.94

Diffusion coefficients (cm2 min1) Initial, Di (104)

Average, DA (104)

Late time, DL (104)

7.24 13.09 6.42

13.20 18.17 14.89

1.22 2.15 1.24

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

yields the diffusion coefficient D. Therefore, the initial diffusion coefficient Di can be evaluated from the slope of the plot. The average diffusion coefficient DA may also be calculated for 50% of the total release by putting Mt/ M1 = 0.5 in Eq. (3), which finally yields Eq. (4). Late diffusion coefficients were calculated using the late time approximation as described by Peppas et al. given in Eq. (5) [25,26]: 0:049‘2 t1=2     Mt 8 ðp2 DtÞ ¼1 exp p2 M1 ‘2

DA ¼

ð4Þ ð5Þ

The slope of the plot between ln(1  Mt/M1) and t was used for the evaluation of DL. The values of diffusion coefficients for the swelling kinetics and release dynamics of the drug from the hydrogels were evaluated and the results are presented in Tables 1–3. 3. Results and discussion 3.1. Characterization Poly(HEMA-cl-AAc) hydrogels were characterized by FTIR and swelling studies. 3.1.1. Fourier transform infrared spectroscopy FTIR spectra of poly(HEMA-cl-AAc) was recorded and is presented in Fig. 1. The broad absorption band at 3430.9 cm1 is due to –OH stretching, indicating the strong association in this polymer. The infrared absorption bands at 1726.4 cm1 due to C@O stretching of the ester and at 1260.6 cm1 due to the C–O stretching of esters and at 1666 cm1 due to C@O stretching of the acid were observed in the poly(HEMA-cl-AAc). The CH2 asymmet-

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ric stretching vibration at 2926 cm1 and the symmetric CH2 absorption at 2856 cm1 along with the –CH deformation mode around 1457.5 cm1 were observed in the spectra. 3.1.2. Effect of crosslinker on swelling of poly(HEMA-clAAc) The structure of the imprinted cavities in the MIPs should be stable enough to maintain the conformation in the absence of the template and it should also be flexible enough to facilitate the attainment of a fast equilibrium between the release and re-uptake of the template in the cavity. This is particularly important if the device is used as a diagnostic sensor, as a trap of toxic substances in the gastrointestinal tract or to deliver a drug in a controlled and sustained manner in the colon. Therefore, in the present case, the polymers were prepared with three different concentrations of the crosslinker (that is 1.30  102, 3.89  102 and 6.49  102 mol l1). Polymers were synthesized in triplicate for each concentration of N,NMBAAm. In order to compromise between the rigidity and flexibility of the polymers, a swelling study was carried out for the polymers prepared with different concentrations of N,N-MBAAm. The amount of water taken up by the polymer matrix at 37 °C up to 300 min was studied after a fixed interval of 30 min and the results are shown in Fig. 2.1. It can be observed from Fig. 2.1 that the amount of water uptake by per gram of gel increases with time and decreases with increasing crosslinker concentration in the polymer. Further, it was observed from the equilibrium swelling after 24 h that maximum water uptakes of 3.87 ± 0.049, 1.53 ± 0.038 and 1.36 ± 0.076 g g1 gel were obtained for the polymers synthesized, respectively, with 1.30  102, 3.89  102 and 6.49  102 mol l1 N,NMBAAm (Fig. 2.2). This is probably due to the increased

Table 2 Results of the diffusion exponent n, the gel characteristic constant k and various diffusion coefficients for the swelling kinetics of the MIPs and NIPs of poly(HEMA-cl-AAc) in distilled water at 37 °C Different polymer matrices

Diffusion exponent, n

Gel characteristic constant, k (102)

MIPs-50 MIPs-25 NIP

0.42 0.44 0.47

3.13 3.11 3.18

Diffusion coefficients (cm2 min1) Initial, Di (104)

Average, DA (104)

Late time, DL (104)

3.16 3.78 5.34

10.60 11.56 14.08

0.77 0.82 1.09

Table 3 Results of the diffusion exponent n, the gel characteristic constant k and various diffusion coefficients for the release of 5-fluorouracil from the MIPs and NIPs of poly(HEMA-cl-AAc) in distilled water at 37 °C Drug release from polymers

Diffusion exponent, n

Gel characteristic constant, k (102)

MIPs-50 MIPs-25 NIP

0.268 0.235 0.143

9.98 11.09 15.96

Diffusion coefficients (cm2 min1) Initial, Di (104)

Average, DA (104)

Late time, DL (104)

2.53 1.72 0.46

13.91 12.26 10.60

0.767 0.698 0.286

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B. Singh, N. Chauhan / Acta Biomaterialia 4 (2008) 1244–1254 **HEMA AAc 96

80 78

802.1

520.8

629.5

1022.9

1160.6 1074.6

1726.4

82

1260.6

2855.2

84

2926.2

%Transmittance

86

1401.0

88

1655.0

2628.4

90

1561.7 1457.5

92

2364.4

3743.8

94

76

3430.9

74 72 70 68 66 4000

3500

3000

2500

2000

1500

1000

500

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

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

1.8

Amount of water uptake (g/ g of gel)

Amount of water uptake(g/g of gel) after 24 hrs.

2.0

1.6 1.4 1.2 1.0 0.8 0.6 0.4

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

4

2

0

0.2 0

30

60

90

120

150

180

210

240

270

300

Time (min)

Fig. 2.1. Swelling kinetics of the poly(HEMA-cl-AAc) hydrogels prepared with different [N,N-MBAAm] in distilled water at 37 °C. Reaction time = 30 min; reaction temperature = 37 °C; [HEMA] = 7.68  101 mol l1; [AAc] = 13.88  101 mol l1; [APS] = 0.438  101 mol l1; [TEMED] = 1.72  101 mol l1.

extent of crosslinking of polymeric chains in hydrogels that lead to a decrease in pore size and a decrease in the water uptake capacity of hydrogels. However, a higher total percentage uptake of water occurred with the polymers pre-

A

B

C

Fig. 2.2. Amount of water uptake for the poly(HEMA-cl-AAc) hydrogels prepared with different [N,N-MBAAm] after 24 h in distilled water at 37 °C.

pared with 3.89  102 mol l1 N,N-MBAAm (Fig. 2.3). The values of the diffusion exponent n and gel characteristic constant k were evaluated from the slope and intercept of the plot ln Mt/M1 vs. ln t (Fig. 2.4), and the results are presented in Table 1. It is clear from the table that values of n of both <0.5 and >0.5 were observed, which indicates that both Fickian- and non-Fickian-type diffusion mecha-

B. Singh, N. Chauhan / Acta Biomaterialia 4 (2008) 1244–1254 75 70

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

% Cumulative water uptake

65 60 55 50 45 40 35 30 25 20 15 10 0

30

60

90

120

150

180

210

240

270

300

Time (min)

Fig. 2.3. Percentage cumulative water uptake of the poly(HEMA-cl-AAc) hydrogels prepared with different [N,N-MBAAm] in distilled water at 37 °C.

-0.2

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

-0.4 -0.6

ln (Mt /Moo)

-0.8 -1.0 -1.2

1249

lg ml1 5-FU) were loaded into the polymers to observe the effect of the number of recognition sites in the imprinted polymers on the entrapment of drug and the release pattern of the drug. All the polymers were synthesized in triplicate and were used to study the release dynamics of the drug immediately after synthesis. The release pattern of the 5-FU is presented in Fig. 3.1. It can be observed from Fig. 3.1 that the release of 5-FU from the poly(HEMA-cl-AAc) hydrogels loaded with 50 lg ml1 of the drug was higher than from the hydrogels loaded with 25 lg ml1 5-FU in the first 300 min of the release. This observation is further supported by the fact that the release of 5-FU from MIPs-50 is also higher than that from MIPs25. The total amount of drug released from MIPs-50 and MIPs-25 were 280.66 ± 6.92 and 144.79 ± 5.04 lg (20 ml)1 g1 gel, respectively (Fig. 3.2). The higher release of 5-FU from MIPs-50 was due to the higher initial loading of the drug in the polymer during synthesis. The percentage of the total release was also observed to be higher in the case of MIPs-50 (Fig. 3.3). The value of the diffusion exponent n was observed to be 0.1 and 0.04, respectively, for MIPs-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 the MIPs slows the rate of release to below that expected from Fickian relationships [30,31] (Fig. 3.4). 3.3. Reloading of the drug or recognition affinity of MIPs

-1.4 -1.6 -1.8 -2.0 -2.2 3.0

3.5

4.0

4.5

5.0

5.5

6.0

Reloading was carried out in both MIPs and loading was carried in NIPs in triplicate to observe the recognition capacity of the hydrogels for the template. Molecular imprinting is a technique producing synthetic materials containing highly specific receptor sites that have an affin-

Fig. 2.4. Plot for the evaluation of the diffusion exponent n and the gel characteristic constant k for the swelling of the poly(HEMA-cl-AAc) hydrogels prepared with different [N,N-MBAAm] in distilled water at 37 °C.

nisms occurred for the diffusion of water molecules in the polymer prepared with different crosslinker concentrations. The values of the diffusion coefficient are presented in the Table 1. It is clear from the table that the values obtained for the average diffusion coefficient (DA) were higher than the initial and late diffusion coefficients (Di and DL, respectively). From the above discussion, the optimum concentration of N,N-MBAAm (3.89  102 mol l1) was obtained for the further synthesis of polymers with drug and 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 Section 2.2. Two different concentrations of drug (50 and 25

Amount of drug release (ug/20mL/g of gel)

ln t 220 200 180 MIP loaded with (50 ug/mL) of 5-FU MIP loaded with (25 ug/mL) of 5-FU

160 140 120 100 80 0

50

100

150

200

250

300

Time (min.)

Fig. 3.1. Release profile of 5-fluorouracil from MIPs of poly(HEMA-clAAc) hydrogels loaded with different drug concentrations in distilled water at 37 °C. Reaction time = 30 min; reaction temperature = 37 °C; [HEMA] = 7.68  101 mol l1; [AAc] = 13.88  101 mol l1; [APS] = 0.438  101 mol l1; [N,N-MBAAm] = 3.89  102 mol l1; [TEMED] = 1.72  101 mol l1.

B. Singh, N. Chauhan / Acta Biomaterialia 4 (2008) 1244–1254 -0.20

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

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

-0.25

300

-0.30 -0.35

250

ln (Mt /Moo)

Amount of drug release (ug/20 mL/g of gel) after 24 hrs.

1250

200

-0.40 -0.45 -0.50

150

-0.55 100

-0.60 -0.65

50

3.0

3.5

4.0

4.5

5.0

5.5

6.0

ln t 0

A

B

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.

Amount of drug entraped (ug/g of gel) after reloading

80

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

% Cumulative release

75

70

65

60

55

0

50

100

150

200

Fig. 3.4. Plot for the evaluation of the diffusion exponent n and the gel characteristic constant k of 5-fluorouracil from MIPs of poly(HEMA-clAAc) hydrogels loaded with different drug concentration in distilled water at 37 °C.

250

300

Time (min.)

Fig. 3.3. Percentage cumulative release of 5-fluorouracil from MIPs of poly(HEMA-cl-AAc) hydrogels loaded with different drug concentration in distilled water at 37 °C.

ity for a target molecule, and MIPs can mimic the recognition and binding capabilities of the template molecule. In the present case it was observed that the recognition affinity of the MIPs for 5-FU was higher than that of NIPs when reloading of drug was carried out by the swelling equilibrium method whereby both the MIPs (MIPs-50 and MIPs-25) and NIPs were kept in a 200 lg ml1 solution of 5-FU for 24 h at 37 °C and then dried to obtain the further release device. The results are present in the Fig. 4. It can be seen from the figure that the MIPs which were initially loaded with 50 lg ml1 5-FU entrapped a larger amount of the drug (1328.13 ± 55.36 lg g1 gel) than the MIPs loaded with 25 lg ml1 5-FU (1102.15 ± 12.32

1800 1600

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)

1400 1200 1000 800 600 400 200 0

MIP-1

MIP-2

NIP

Fig. 4. Total amount of drug entrapped by reloaded MIPs and NIPs of poly(HEMA-cl-AAc) after 24 h in distilled water at 37 °C. Stock concentration = 200 lg ml1.

lg g1 gel) and NIPs (716.01 ± 6.56 lg g1 gel). This is because the number of recognition sites (that is template sites) was higher in the MIPs initially loaded with the higher concentration of the drug. There were no recognition sites available in the NIPs, and hence these showed a lower 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 were dried at room temperature (25 °C) and then used to study the swelling of the poly(HEMA-cl-AAc) hydrogels and release dynamics of the drug from these hydrogels.

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55 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)

1.6

% Cumulative uptake after reloading

Amount of water uptake (g/g of gel) after reloading

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

1.4 1.2 1.0 0.8 0.6 0.4 0.2

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)

50 45 40 35 30 25 20 15 10

0

50

100

150

200

250

300

0

30

60

90

120

Time (min.)

3.4.1. Swelling kinetics The swelling of the MIPs and NIPs is presented in Fig. 5.1. More swelling of the MIPs was observed than of the NIPs. It is also clear from the Fig. 5.1 that the MIPs that were initially loaded with 50 lg ml1 5-FU (MIPs50) showed more swelling than those initially loaded with 25 lg ml1 5-FU (MIPs-25) and the NIPs. Total amount of water taken up by MIPs-50, MIPs-25 and NIPs was 3.73 ± 0.046, 3.11 ± 0.015 and 2.26 ± 0.036 g g1 gel, respectively (Fig. 5.2). Fifty percent of the total swelling occurred in 666.12, 462.25 and 359.10 min, respectively, for MIPs-50, MIPs-25 and NIPs (Fig. 5.3). It shows that the rate of swelling is higher in MIPs as compare to NIPs. The values of diffusion exponent and gel characteristics constant k were evaluated from the slope and intercept of the plot ln Mt/M1 vs. ln t (Fig. 5.4) and results are presented in Table 2. It is clear from the table that the value

Amount of water uptake (g/g of gel) after 24 hrs.

180

210

240

270

300

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)

2

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

-0.6

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)

-0.8 -1.0

ln (Mt /Moo)

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

4

150

Time(min)

-1.2 -1.4 -1.6 -1.8 -2.0 3.0

3.5

4.0

4.5

5.0

5.5

6.0

ln t

Fig. 5.4. Plot for the evaluation of the diffusion exponent n and the gel characteristic constant k for the swelling of reloaded MIPs and NIPs of 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 and 0.47, respectively, which indicates that a Fickian-type diffusion mechanism has occurred in all three polymers. The diffusion coefficients obtained are shown in Table 2. It is clear from the table that the values obtained for the average diffusion coefficient (DA) were higher than both the initial and late diffusion coefficients (Di and DL, respectively). This shows that during the early and the late stages of swelling the diffusion of water molecules into the hydrogels was slow. From this discussion, it is concluded that the imprinted gels swelled at faster rates than the nonimprinted ones.

0

MIP-1

MIP-2

NIP

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

3.4.2. Release dynamics of the drug The 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

B. Singh, N. Chauhan / Acta Biomaterialia 4 (2008) 1244–1254 55 480

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)

440

% Cumulative release after reloading

Amount of drug release (ug/20 mL per g gel) after reloading

1252

400 360 320 280 240 200 160

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)

50

45

40

35

30

25

20

120

0 0

50

100

150

200

250

50

100

Time (min.)

Fig. 6.1. Release profile of 5-fluorouracil from reloaded MIPs and NIPs of poly(HEMA-cl-AAc) hydrogels in distilled water at 37 °C. Reloading concentration of 5-FU = 200 lg ml1.

150

200

250

300

Time (min.)

300

Fig. 6.3. Percentage cumulative release of 5-fluorouracil from reloaded MIPs and NIPs of poly(HEMA-cl-AAc) hydrogels in distilled water at 37 °C. Reloading concentration of 5-FU = 200 lg ml1.

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)

-0.8 -0.9

800

600

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)

-0.7

ln (Mt /Moo )

Amount of drug release (ug/20 mL/g of gel) after 24 hrs.

-0.6

-1.0 -1.1 -1.2

400

-1.3 -1.4 -1.5

200

3.0

3.5

4.0

4.5

5.0

5.5

6.0

ln t 0

Fig. 6.2. Drug release pattern of 5-fluorouracil from reloaded samples of MIPs and NIPs of poly(HEMA-cl-AAc) hydrogels after 24 h in distilled water at 37 °C. Reloading concentration = 200 lg ml1.

Fig. 6.1 that the amount of drug released from the MIPs-50 was higher than from the NIPs, because grater entrapment of drug occurred in these polymers than in the NIPs. The drug in the MIPs was released in a controlled manner. The total amounts of gel released from MIPs-50, MIPs25 and NIPs were 875.33 ± 4.76, 731.46 ± 9.92 and 420.98 ± 3.89 lg (20 ml)1 g1, respectively (Fig. 6.2). The 50% total release was higher in the case of the MIPs (Fig. 6.3). The values of the diffusion exponent n and the gel characteristic constant k for the swelling of polymers in distilled water were evaluated from the slope and intercept of the plot ln Mt/M1 vs. ln t (Fig. 6.4) and the results

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

are presented in Table 3. It is clear from the table that a value for n of <0.5 was obtained in each release case, which indicates that a non-Fickian-type diffusion mechanism is responsible 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 of the monomers 1-b-allyloxycarbonyloxymethyl-5-fluorouracil and 1,3-bis(b-allyloxycarbonyloxymethyl)-5-fluorouracil separately with N-vinylpyrrolidinone (NVP) to form linear copolymers and crosslinked polymer networks, respectively. It was observed that the hydrolytic scission of the carbonate groups resulted in release of 5-FU. The time-dependent fractional release of the 5-FU was seen to be fitted by a power relationship with exponents between 0.10 and 0.25. These values are significantly lower than

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

the value of 0.5 expected for Fickian release or the values of between 0.5 and 1.0 that are usually observed when the release is moderated by interaction of the polymer with the diffusate. The data thus indicate that the release is controlled not only by Fickian diffusion and interaction with the polymer chain, but also, as expected, the rate of degradation of the covalent linkages to the poly(NVP) chains plays a major role. In another observation it was observed that the total fraction of 5-FU released over the time scale of the measurement is also a function of the initial loading. This result suggests that the rate of fractional release is lower for the hydrogel containing the lower concentration of 5-FU. This is a potentially useful observation since the hydrogel water content at all times during the degradation increases as the loading decreases, and in some medical applications this may be advantageous [30,31]. The values of the diffusion coefficient for the release of drug from these polymers are presented in Table 3. It can be seen from the table that the values obtained for the average diffusion coefficient (DA) were higher than both the initial and the late diffusion coefficients (Di and DL, respectively). This means that in both the early and late stages of drug release the rate of diffusion of drug from the polymer matrix was slow. This observation is also supported by the results obtained for the swelling of the MIPs and NIPs. 4. Conclusion It is concluded from the forgone discussion that concentration of the crosslinker during synthesis can play a very important role in deciding the flexibility and rigidity of MIPs. It is also concluded from the drug entrapment study that the concentration of the drug molecule determines the drug recognition capacity of MIPs. The swelling of MIPs increases with the increasing template concentration in the MIPs, which also helps in the release of the drug in a controlled manner. Because of enhanced binding capacity of the MIPs, these can be further exploited for the use in separation, purification detection and drug delivery technologies for biomolecules, and can be used for the developing the biomimic devices. References [1] Sellergren B, Karmalkar RN, Shea KJ. Enantioselective ester hydrolysis catalyzed by imprinted polymers. J Org Chem 2000;65(13):4009–27. [2] Haginaka J, Kagawa C. Chiral resolution of derivatized amino acids using uniformly sized molecularly imprinted polymers in hydroorganic mobile phases. Anal Bioanal Chem 2004;378(8):1907–12. [3] Baggiani C, Giraudi G, Vanni A. A molecular imprinted polymer with recognition properties towards the carcinogenic mycotoxin ochratoxin A. Bioseparation 2001;10(6):389–94. [4] Dickert FL, Hayden O, Bindeus R, Mann KJ, Blaas D, Waigmann E. Bioimprinted QCM sensors for virus detection-screening of plant sap. Anal Bioanal Chem 2004;378(8):1929–34. [5] Alvarez-Lorenzo C, Concheiro A. Molecularly imprinted polymers for drug delivery. J Chromatogr B: Anal Technol Biomed Life Sci 2004;804(1):231–45.

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