Saudi Pharmaceutical Journal (2014) 22, 43–51

King Saud University

Saudi Pharmaceutical Journal www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE

Diisocyanate mediated polyether modified gelatin drug carrier for controlled release Vediappan Vijayakumar, Kaliappagounder Subramanian

*

Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam 638 401, Erode Dt, Tamil Nadu, India Received 14 October 2012; accepted 10 January 2013 Available online 26 January 2013

KEYWORDS Gelatin; Isophorone diisocyanate; Poly(ethylene glycol); Theophylline; In vitro drug release

Abstract Gelatin is an extensively studied biopolymer hydrogel drug carrier due to its biocompatibility, biodegradability and non-toxicity of its biodegraded products formed in vivo. But with the pristine gelatin it is difficult to achieve a controlled and desirable drug release characteristics due to its structural and thermal lability and high solubility in aqueous biofluids. Hence it is necessary to modify its solubility and structural stability in biofluids to achieve controlled release features with improved drug efficacy and broader carrier applications. In the present explorations an effort is made in this direction by cross linking gelatin to different extents using hitherto not studied isocyanate terminated poly(ether) as a macrocrosslinker prepared from poly(ethylene glycol) and isophorone diisocyanate in dimethyl sulfoxide. The crosslinked samples were analyzed for structure by Fourier transform-infrared spectroscopy, thermal behavior through thermogravimetric analysis and differential scanning calorimetry. The cross linked gelatins were biodegradable, insoluble and swellable in biofluids. They were evaluated as a carrier for in vitro drug delivery taking theophylline as a model drug used in asthma therapy. The crosslinking of gelatin decreased the drug release rate by 10–20% depending upon the extent of crosslinking. The modeled drug release characteristics revealed an anomalous transport mechanism. The release rates for ampicillin sodium, 5-fluorouracil and theophylline drugs in a typical crosslinked gelatin carrier were found to depend on the solubility and hydrophobicity of the drugs, and the pH of the fluid. The observed results indicated that this material can prove its mettle as a viable carrier matrix in drug delivery applications. ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.

1. Introduction

* Corresponding author. Tel.: +91 4295 226254, mobile: +91 9894372411; fax: +91 4295 223 775. E-mail address: [email protected] (K. Subramanian). Peer review under responsibility of King Saud University

Production and hosting by Elsevier

The use of natural polymers such as gelatin (Young et al., 2005), starch (Al-Karawi and Al-Daraji, 2010), chitosan (Sinha et al., 2004; Bertoldo et al., 2007; Subramanian and Vijayakumar, 2011), etc., as carriers in controlled drug delivery applications is gaining importance because of their inherent biocompatibility, biodegradability and biosafety (Young et al., 2005). But a common disadvantage of such natural polymers is their structural and thermal lability (Bigi et al., 2001), which require improvement for controlled and targeted

1319-0164 ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University. http://dx.doi.org/10.1016/j.jsps.2013.01.005

44

V. Vijayakumar, K. Subramanian O NH2

H3C

H NH

S

O-Na+

Theophylline

O

NH

CH

C

NH CH C CH 2 CH 2 CH 2 CH 2

CH 2 C O O

NH HC

O NH CH

O

N

NH CH2

C

C

NH

OH

CH

C

C

HC NH

H2C

CH2

C

NH

O

+

O

N

CH2

H2C

O

CH3

F 5-Fluorouracil

O

CH2

C NH C

HN

solubility in biofluids to have a controlled release characteristic with improved drug efficacy and broader application as a carrier for a wide range of drugs. Several chemical modifications such as crosslinking with formaldehyde (Digenis et al., 1994), glutaraldehyde (Narayani and Panduranga Rao, 1996), 1,4-diisocyanato butane, hexamethylene diisocyanate,

O

C

O

Chemical structures of drugs.

drug release applications. In this context, gelatin a well-known and widely used biopolymer drug carrier has been chosen for improving its drug release characteristics, because the high solubility, and poor mechanical and thermal stability of gelatin under physiological conditions may not facilitate a desirable sustained drug release. Hence it is necessary to modify its

O

H

CH3

O

Figure 1

O

N

O

N

N

O

CH3

O

Ampicillin sodium

H

CH3

N

O

N

N

H

O

CH

C

HC N

OH

O

C

NH2

C

OH

NH C O CH 3 CH

NH CH

O

OH

NH2

NH 2

-Asp-Lys -Ala-Gly-Pro-Arg-Gly-Glu-4Hyp-Gly-Pro-Tyr-

CH3

O C N

2

+

H3C H3C

CH2 OH

n

PEG-600

IPDI

CH3

O C N

HO CH2 CH2 O CH2

N C O

DMSO/ 35 °C / N2 / 24h O

O H3C

NH C O CH2 CH2 O CH2

H3C

CH2 O C

NH

CH3

n

ICTPE

H3C

H2N

2 HO

N C O

H3C DMSO/ 35 °C / N2 / 24h

COOH

Gelatin NH2

H3C

CH3 O

O H3C

NH C O CH2 CH2 O CH2

HN C

H3C

CH2

NH

CH2 O C

NH

CH3

n O

O H3C

O

O

O

C

Gelatin

NH

C NH

NH C

H3C

CH3

NH

H3C

C

O

NH C O CH2 CH2 O CH2

CH2 O C

NH

n H3C H3C

CH CH3 3

NH

O

H3C H3C

NH C O CH2 CH2 O CH2

CH2 O C

NH

C

+ O

CO 2

ICTPE Cross linked gelatin

NH

Figure 2

CH3

n H3C

NH

Gelatin O NH

O

O

O

C O NH

Crosslinking reactions of gelatin with ICPTE.

NH

H3C C NH

O

Diisocyanate mediated polyether modified gelatin drug carrier for controlled release Table 1

Composition of reaction ingredients for cross linked gelatin samples.

Cross linked gelatin

GEPI-5 GEPI-10 GEPI-20 GEPI-30

Table 2

45

Feed composition for prepolymer (ICTPE) (mole) PEG-600 (·103)

IPDI (·103)

0.4483 0.896 1.799 5.398

0.8966 1.793 3.599 5.398

Gelatin (g)

Insoluble crosslinked gelatin yield (g)

2 2 2 2

1.925 2.125 2.325 2.401

Intensity ratio of IR absorption peaks at 1705 and 3285 cm1 in crosslinked gelatins.

Polymer sample

h1705 cm1 (A)

h3285 cm1 (B)

A/B

GEPI-10 GEPI-10Da GEPI-20 GEPI-20Da GEPI-30 GEPI-30Da

1.3 1.5 0.9 1.1 0.9 1.2

1.8 2 2 1.7 2 1.8

0.72 0.75 0.45 0.64 0.45 0.66

A and B, Peak heights at 1705 and 3285 cm1 (Fig. 3), respectively. a Degraded cross linked gelatin in SIF at 37 C.

1,12-diisocyanto dodecane, 1,3-butadiene diepoxide, and 1,2,7,8-diepoxyoctane (Patil et al., 2000), grafting (Patil et al., 2000; Curcio et al., 2010, Va´zquez et al., 1995), etc. have been reported in the literature to bring down its solubility and rate of biodegradation and to improve the thermal and mechanical stability under in vivo conditions. Majority of these crosslinkers are reported to be toxic in nature either inherently or by its toxic degradation products formed in vivo. In the present study gelatin crosslinking was performed using the isocyanate terminated oligomeric poly(ethylene glycol) (PEG-600) prepared by reacting isophorone diisocyanate (IPDI) and PEG-600 to modify the gelatin properties because of the less toxicity of IPDI compared to other diisocyanate crosslinkers (Ferreira et al., 2007; Guelcher et al., 2005) and biocompatibility of PEG. Moreover, the PEG spacer may minimize the loss in the flexibility and swellability of the gelatin after crosslinking. The study also involved the evaluation of the crosslinked gelatin as a drug carrier through in vitro release studies in simulated biological fluids taking theophylline as a model drug. Structural effects of drugs on their release characteristics were also tested with a typical crosslinked gelatin carrier taking the drugs 5-fluorouracil, ampicillin sodium salt and theophylline as specific examples. 2. Experimental 2.1. Materials The gelatin (GE, intrinsic viscosity 0.135 dl/g in water at 30 C) used in the study was a gift sample from Sterling Biotech, Ooty, India. It was dried under vacuum at 60 C to remove moisture for seven days. Dimethyl sulfoxide (DMSO) and dichloromethane (SRL Mumbai) were dried overnight on anhydrous Na2SO4, distilled and used. Isophorone diisocyanate (IPDI, Sigma–Aldrich 99%), poly(ethylene glycol)-600 (PEG-600), disodium hydrogen orthophosphate and monosodium dihydrogen orthophosphate, (SD fine), hydrochloric

acid, sodium chloride, acetone and methanol (Rankem) were used as received. Simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 7.2) were prepared as per United States Pharmacopeia (USP) (Patel and Amiji, 1996). The drugs ampicillin sodium salt (AMP, antibiotic to treat gram +ve and gram ve, organisms), theophylline (THP, used in respiratory therapy) and 5-fluorouracil (5 FU, anti-metabolic drug, used in cancer chemotherapy) (Himedia) were used as received. The structures of these drugs are shown in Fig. 1. 2.2. Synthesis of isocyanate terminated polyether (ICTPE) Isocyanate terminated poly(ethylene glycol) was prepared by reacting PEG-600 and IPDI (mole ratio 1:2) at 35 C in DMSO solution in a 100 ml three necked RB flask provided with nitrogen inlet and magnetic stirrer, kept in a thermostated (35 C) water bath. In a typical reaction, 0.8966 · 103 mole of IPDI was dissolved in 5 ml of DMSO and added to 0.4483 · 103 mole of PEG-600 under vigorous stirring. The stirring was continued for 24 h to facilitate the completion of the isocyanate terminated poly(ether) formation. 2.3. Synthesis of ICTPE modified gelatin Dry gelatin (2 g) was taken in a three necked RB flask equipped with dry nitrogen inlet, mechanical stirrer and addition funnel and dissolved by adding 20 ml of dry DMSO at 35 C and stirred overnight under nitrogen atmosphere to complete the dissolution. To this solution, the above synthesized prepolymer ICTPE in DMSO was added drop wise over a period of 2 min. A sudden build-up in viscosity was observed followed by gelatin due to polyurea forming reaction. The reaction was continued for another hour to facilitate its completion. The yellowish transparent ICTPE cross linked gelatin gels with different degrees of cross linking levels designated as GEPI-5, GEPI-10, GEPI-20 and GEPI-30 (Table 1) were synthesized using different proportions of gelatin and ICTPE. The

46

V. Vijayakumar, K. Subramanian 7.0

(A) 7

Residual amino group(equ/g) (x 10 )

(i) (h)

Transmittance

(g) (f) (e)

(d)

6.5

6.0

5.5

5.0

4.5

4.0 5

(c)

10

15

20

25

30

Weight % of ICTPE

(b)

(B)

65

4000

3500

3000

2500

2000

1500

1000

% Residual Weight after 2 weeks

(a)

500

-1

Wavenumber(cm )

Figure 3 4 FT-IR spectra of, (a) PEG, (b) IPDI, (c) GE, (d) GEPI-5,(e) GEPI-10 (f) GEPI-30, (g) GEPI-10D, (h) GEPI-20D and (i) GEPI-30D in KBr matrix.

2.4. Characterization

55

a

50

b

45

40

35

30 0

5

10

15

20

25

30

% weight of ICTPE in feed 100

(C) 90

80

Weight(%)

crosslinked gelatin polymer gel thus obtained was removed, cut into small pieces and soaked in dichloromethane (100 ml) with mechanical stirring for 5 h to remove the hoarded DMSO and unreacted IPDI if any present in the gel. Then it was immersed in boiling distilled water for 3 h to remove unreacted gelatin and subsequently Soxhlet extracted using water/ethanol (1:1) mixture for 48 h and dried at 60 C under reduced pressure to constant weight. The dried polymer was kneaded in a mortar and pestle to make fine powder and sieved in 150 micron sieves (Jayant Test Sieves, India), vacuum dried at 60 C for 4 h and stored in air tight PP/HDPE storage vials until further usage. The structure of crosslinked gelatin was analyzed by FT-IR.

60

70

60

2.4.1. Extent of cross linking by ninhydrine method Since the number of free hydroxyl and carboxyl groups present in gelatin are very much less compared to the free amino groups, as a good approximation it may be assumed reasonably that majority of IPDI and gelatin reaction is due to the urea forming reaction between amino and isocyanate groups of gelatin and IPDI respectively. The extent of cross-linking in gelatin sample was determined by ninhydrine assay of free amino groups through absorbance measurement at 570 nm before and after cross-linking with IPDI, taking glycine as the reference standard (Yuan et al., 2007). Ninhydrine solution was prepared by mixing the solution A, containing 1.05 g of citric acid, 10 ml of 1.0 M NaOH and 0.04 g SnCl2Æ2H2O, diluted to 25 ml using deionized H2O with the solution B having 1 g ninhydrine dissolved in 25 ml 2-methoxy ethanol. The mixed solutions were stirred for 45 min and stored in an amber colored bottle.

SGF GEPI-5 GEPI-20

50

0

2

4

SIF GEPI-20 GEPI-5 6

8

10

12

Time(days)

Figure 4 Effect of weight% of ICTPE on residual amino group in cross linked gelatin (A) and% acetone insoluble fraction of aged cross linked gelatin (B) in SGF (a) and SIF (b), and biodegradation of GEPI-5 and GEPI-20 with time (C) in SGF and SIF.

2.4.2. Swelling studies Swelling studies were performed in simulated biofluids (SGF and SIF) at 37 C by keeping a known weight (100 mg) of cross linked copolymer compressed(5 ton, KBr press) pellet in a stainless steel (SS) cylindrical mesh (30 mm dia; 50 mm

Diisocyanate mediated polyether modified gelatin drug carrier for controlled release

paper. Then, the average degree of swelling (=(Wt–W0)/W0, where Wt and W0 are the weights of the polymer after and before swelling) for the triplicate experiments was plotted against time to get the swelling profile.

100

(A)

90

47

80

Weight (%)

70

(a) GE (b) GEPI-5 (c) GEPI-10 (d) GEPI-20

60 50

2.4.3. Degradation studies

40 30

a

20

b c d

10 0 100

200

300

400

500

600

700

800

900

Temperature (°C)

Degradability of the cross linked gelatin was tested in SGF and SIF by immersing 0.15 g of polymer in 20 ml each at 37 C in separate stoppered conical flasks and kept in a shaker (10 rpm) incubator for two weeks. Then the residual polymers in the biofluids at different time intervals were recovered through precipitation using ice cold acetone, dried and weighed. The residual polymer weights were then compared with the extent of cross linking in terms of ICTPE taken and number of amino groups reacted/or left behind after the reaction. The residual polymers recovered from degraded GEPI-10, GEPI-20 and GEPI-30 in biofluids were designated as GEPI-10 D, GEPI20D and GEPI-30 D, respectively.

(B) 2.4.4. Fourier infrared (FT-IR) spectrum FT-IR spectra of gelatin and modified gelatin were recorded on JASCO FT-IR-460 in KBr pellet for the spectral range 400–4000 cm1with 2 cm1 resolution by accumulating 48 scans.

23

(b) GEPI-5

22

(c)

21

(b) 20

(a)

50

200

(d)

19

(a) GE

100

(A)

GE(a) GEPI-5(b) GEPI-10(c) GEPI-30(d)

(c)GEPI-10

Heat flow endo up(mW)

ο

Derivative Weight (%/ C)

(d)GEPI-20

300 400 500 o Temperature( C)

600

700

100

150

200

Polymer

Onset degradation temperature (C)

GE GEPI-5 GEPI-10 GEPI-20

370 340 325 319

350

400

(B)

70

5FU(a) GEPI-10F(b) THP(c) GEPI-10T(d) AMP(e) GEPI-10A(f)

Heat flow endo up(mW)

60

Table 3 Onset degradation temperature data for gelatin and ICTPE crosslinked gelatins.

300 o

Figure 5 TGA (A) and DTG (B) traces of, (a) GE, (b) GEPI-5, (c) GEPI-10 and (d) GEPI-20.

height) immersed in 20 ml of SGF or SIF taken in a 25 ml beaker and allowed to swell. The weights of the swelled copolymers at predetermined time intervals were calculated after wiping the mesh containing the swelled polymer with a tissue

250

Temperature( C)

800

50

a

c

40

30

e

d

20

b f 0

50

100

150

200

250

300

350

400

ο

Temperature( C)

Figure 6 DSC thermograms of, (A) gelatin and crosslinked gelatins and (B) drugs and tablets.

48

V. Vijayakumar, K. Subramanian

2.4.5. X-ray diffraction (XRD)

3. Results and discussion

2.4.6. Thermo gravimetry (TG) TG/DTG studies were performed on TGA Q500 V20.10 Build 36 with a sample size of 1.5–3.5 mg under nitrogen atmosphere at a heating rate of 20 C/min for the temperature range from ambient to 800 C. 2.4.7. Differential scanning calorimetry (DSC) DSC thermograms of the cross linked polymers were recorded on Perkin Elmer Pyris 6DSC model in nitrogen atmosphere at a heating rate of 10 C/min. 2.4.8. In-vitro drug dissolution studies Compressed tablets of dia, 13 mm and thickness, 0.5 mm made by compressing (5 ton) a homogeneous mixture of drug(15 mg) and polymer(200 mg) were used for in vitro drug dissolution studies in an USP apparatus Type II (Veego Model VDA-6DR) in SGF and SIF at 37 C by embarking the tablet inside the SS basket immersed in a thermo stated and stirred (50 rpm) biological fluid. The tablets maintain their integrity and shape during swelling for a period of time >5 h. The amount of drug released was estimated UV-spectrophotometrically (Perkin Elmer Lamda 35 UV–VIS spectrophotometer) by withdrawing aliquots of sample from the drug release vessel at a time interval of 15 min up to 1 h followed by 30 min by measuring its absorbance values at 270 nm (Sloan et al., 1998). An average of three identical measurements were taken to determine the amount of drug released for a given set of experimental conditions. To maintain a constant volume of the experimental solution a volume equivalent of aliquot sample as incubated fresh fluids was added to the solution after each withdrawal.

3.1. Cross linking of gelatin with ICTPE Formation of ICTPE from IPDI and PEG-600 via the urethane reactions and the subsequent crosslinking of gelatin using ICTPE via the urea and urethane link forming reactions in DMSO are illustrated in Fig. 2. 3.2. FT-IR analysis FT-IR spectra of PEG, IPDI, gelatin and ICTPE cross linked gelatins (GEPI-5, GEPI-10 and GEPI-30) are shown in Fig. 3. The absorption peaks around 1652 and 1561 cm1 (Fig. 3(d–f)) were assigned to urea amide (CO–NH) band (I) and (II) respectively (Shen et al., 2009). The peak at 2259 cm1 (Fig. 3b) characteristic of isocyanate group (–NCO) was absent in Fig. 3 (d–f)) indicating the absence of unreacted –NCO groups in crosslinked gelatin. The peak at 1082 cm1 (Fig. 3d) was attributed to the C–O–C linkage of PEG segment. The absorption peak at 1705 cm1 was assigned to the >C‚O stretching of urethane group(Yilgor et al., 2000). The IR spectra also revealed that the intensity of the C–H

16

SGF(A)

14

Degree of swelling

X-ray diffractogram was recorded on Shimadzu XRD-6000 diffractometer with Cu Ka radiation operated at the voltage and current values of 40 kV and 30 mA respectively for the 2h values in the range 5–45at a scan speed of 5/min.

(a)

12

(b)

10 8

(c)

6

(d)

4 2 0 0

30

60

90

120

150

180

210

Time(min) 1200

a 1000

16

(a)GE (b)GEPI-5 (c)GEPI-10 (d)GEPI-20

d

SIF(B) 14

Degree of swelling

Intensity(CPS)

(a)

12

c 800

b

600

400

(b) 10 8 6

(c)

4

(d)

200 2

0

0

10

20

30

40

50

60

70

2θ (degree)

Figure 7 XRDs of gelatin (GE (a)) and cross linked gelatins GEIP-5 (b), GEIP-10 (c) and GEIP-20 (d).

0

15

30

45

60

75

90 105 120 135 150 165 180 195 210

Time(min)

Figure 8 Swelling profiles of GEPI-5 (a), GEPI-10 (b), GEPI-20 (c) and GEPI-30 (d) in SGF (A) and SIF (B) at 37 C.

Diisocyanate mediated polyether modified gelatin drug carrier for controlled release

49

100

100

(A)

90 80

% Drug released

80

% Drug released

70 60 50

SGF GEPI-5 GEPI-10 GEPI-20 GEPI-30

40 30 20

40

SGF THP AMP 5FU

20

10

SIF THP AMP 5FU

0 0

0 0

50

100

150

200

250

300

50

100

150

200

250

300

Time(min)

350

Time(min)

Figure 10 Release profiles of drugs THP, AMP and 5 FU in SGF and SIF at 37 C with GEPI-30 as carrier.

100

(B)

90

degraded decreased with increased percentage of crosslinker in the monomer feed. The degradation was also supported by the variation in IR absorption peak intensity ratio ðh1705 cm1 = h3285 cm1 Þ (Fig. 3(d) and (g), (e) and (h), (f) and (i) and Table 2) of peaks at 1705 cm1 (urethane carbonyl) and 3285 cm1 (amide N–H stretching) (Yilgor et al., 2000) after immersing in biofluids. If there was no degradation, the intensity ratio of the above peaks would have remained the same as that in the original sample.

80

% Drug released

60

70 60 50

SIF GEPI GEPI-5 GEPI-10 GEPI-20 GEPI-30

40 30 20 10

3.4. TGA/DTG

0 0

50

100

150

200

250

300

350

Time(min)

Figure 9 Release profiles of THP with carriers, (a) GEPI-5, (b) GEPI-10, (c) GEPI-20 and (d) GEPI-30 in SGF (A) and SIF (B) at 37 C.

stretching (2949 cm1) (Shen et al., 2009) increased with the increased concentration of ICTPE in the feed. 3.3. Degree of cross linking and degradability With the increased content of ICTPE in the crosslinking reaction mixture, the residual amino group in the cross linked polymer decreased (Fig. 4A) indicating participation of more amino groups in urea link formation. During ageing of the crosslinked polymer in SGF and SIF, the fluids become more viscous and this was more likely attributed to the dissolution of oligomers formed through biodegradation. Moreover, the acetone insoluble polymer fraction recovered from these fluids using the precipitant acetone at different immersion times decreased both in SGF and SIF supporting the biodegradation of the polymer. An increase in the weight of residual acetone insoluble polymer was observed with the extent of cross linking (Fig. 4B) indicating enhanced resistance toward biodegradation with increased crosslink density. The degradation profiles of GEPI-5 and GEPI-20 in SGF and SIF with respect to time displayed in. Fig. 4C revealed that the amount of the polymer

The TG/DTG traces of gelatin and cross linked gelatin shown in Fig. 5 indicated that the thermal stability of gelatin decreased after cross linking and this was more likely attributed to the lower thermal stability of urea linkage compared to peptide bond (Awad and Wilkie, 2010). Onset degradation temperature data for gelatin and ICTPE crosslinked gelatins are given in Table 3. The major degradation of gelatin starts at 220 C and ends at 370 C and the initial weight loss below150 C was attributed to the loss of moisture and other volatile impurities if any. The cross linked gelatin showed three step degradations around 270, 321 and 330 C, unlike gelatin which degraded by a single step route. The weight loss around 270–275 C was due to degradation initiated via urea linkage (Awad and Wilkie, 2010). The residual weights at 850 C for GEPI-5, GEPI-10 and GEPI-20 were 12%, 10% and 5%, respectively implying decreased residual weight with increased crosslinker level in the feed. This may be attributed to more number of degradation sites in the polymer due to introduction of more urea linkages. The weight loss in the temperature range 190–240 C was assigned to the degradation of hydrolytic by-products of IPDI (amines and polyureas) (Berthold et al., 1996). Comparison of the TG traces of gelatin and ICTPE crosslinked gelatin implied that the degradation step in the temperature range of 270– 450 C was due to the degradation of gelatin backbone. 3.5. DSC trace analysis The DSC traces of gelatin and ICTPE crosslinked gelatin (Fig. 6A) showed random glass transitions below the degradation

50 temperature due to crosslinking via urea and urethane links’ formation (Fig. 2). The endotherm around 100 C in all the samples was due to residual moisture loss. The origin of endotherm around 270 C in the ICTPE crosslinked gelatin may be more likely due to the endothermic degradation of hydrolyzed products of –NCO (Ofner and Bubnis, 1996) which was also revealed in the TG trace (Fig. 5) as a weight loss around 270 C. The inflexion point around 165 C in the cross linked gelatin (GEPI-10) may be attributed to glass transition as there was no weight loss around this temperature in TG (Fig. 5). The broad endotherm beyond 280 C in all the samples was ascribed to the massive endothermic degradation of polyurea and peptide links. Comparison of DSC thermograms of THP(c) and 5 FU (a) with those of their corresponding tablets in GEPI-10 matrix (GEPI-10F(b) &GEPI-10T(d) (Fig. 6B)indicted that the melting points of THP (273 C) and 5 FU (281 C, which overlapped with the broad endothermic matrix degradation temperature range of 250–320 C and hence was not visible as a sharp melting endotherm(Fig. 6B(b)) have been reduced significantly in the tablet and the melting endotherms were very broad unlike the sharp of melting endotherms of pure drugs. These observations tend to reveal that there may be a broad range of polymer matrix–drug physical interaction and the drugs are compatible with the matrix. AMP is unstable near its melting point (215 C) (Ampicillin Sodium Salt, 2012) and its DSC thermogram showed a broad exotherm around 235 C (Fig. 6B(e)). This exotherm was more prominent in the ampicillin tablet (Fig. 6B(f)) which may be due to the matrix effect. 3.6. XRD analysis XRD patterns of gelatin and ICTPE crosslinked gelatin (Fig. 7) revealed a broad peak at 2h value around 22 and this remains nearly the same both in virgin and cross linked gelatin. This implied the increased amorphous character with crosslinking. It is anticipated that the increased amorphous character may decrease the chain flexibility and hence may increase drug release rate if diffusion is the drug release mechanism. 3.7. Matrix swelling Increasing the concentration of ICTPE in the reaction recipe decreased the degree of swelling of ICTPE crosslinked gelatin both in SIF and SGF (Fig. 8) due to increased extent of cross linking. Since gelatin is highly soluble in water, the measurement of its correct degrees of swelling at different instants of time was difficult and hence its swelling profile is not displayed in Fig. 8. 3.8. In vitro drug release study The in vitro release profiles of theophylline in SGF and SIF are given in Fig. 9. The release rate with gelatin carrier was greater in SGF than in SIF perhaps due to enhanced swellability of carrier in SGF due to protonation of gelatin via its amino groups. During the initial 20 min of release, the quantum of drug released in SIF was nearly independent of extent of degree of cross linking due to poor swelling, and the cross linking effect on theophylline release rates was noticed only after 25 min. But in SGF the influence of degree of crosslinking

V. Vijayakumar, K. Subramanian on release rates was different right from the start of dissolution experiment, and this was more likely attributed to greater swellability of matrix in SGF than in SIF for the reason mentioned earlier. The release rate decreased with increased cross linking in spite of the increased amorphous character of the carrier (Fig. 7). If swelling-diffusion is the mechanism of drug release, the increased amorphous character may enhance release rate which was not observed. These facts corroborate that the decreased release rate with enhanced cross linking may be not only due to poor swelling but also due to decreased degradation rate. Hence, the mechanism of release may be interpreted in terms of competitive relaxational and diffusional mechanisms (Urdahl and Peppas, 1988) and carrier degradation. This was supported by the degradation of carrier in SGF and SIF (Fig. 4B). But for a release period of 5 h the matrix degradation may not be prominent. Hence the mechanism of release may be predominantly through segmental relaxation and diffusion. Even though increased cross linking decreased the swelling properties of the carrier significantly, the drug release rate was decreased to a lesser extent again substantiating the fact that diffusion may not be the exclusive mechanism of drug release. Typical release profiles of ampicillin sodium salt and 5 FU in SGF at 37 C were also made by the spectrophotometric estimations of the released drugs at 254 and 266 nm (Sunshine 1982; Huang et al., 2010), respectively with GEPI30 carrier and are displayed in Fig. 10 along with theophylline release curve. The higher release rates for ampicillin sodium salt and 5 FU compared to that of theophylline may be due to their high water solubility, and slow release of theophylline may be due to its more hydrophobicity compared to the other two drugs. 3.9. Drug release mechanism Drug release was modeled as per the following empirical kinetic equation (Curcio et al., 2010). Mt =M1 ¼ ktn where Mt/M1 is the fractional release at time t, k is rate constant and ‘n’ is the diffusional exponent. The exponent ‘n’ provides an indication of the release mechanism and generally ranges from 0.5 to 1. It was calculated from the slope of the logarithmic plot (i.e., ln(Mt/M1) vs. ln(t)) after linearizing the above kinetic expression. A value of n = 0.5 corresponds to a Fickian diffusion, whereas a value in the range 0.6–0.8 indicates anomalous transport as there exists an influence of swelling and/or erosion (Curcio et al., 2010). In the present study for the IPDI cross linked gelatin the experimental n-values were in the range of 0.6–0.8 revealing an anomalous transport mechanism (non-Fickian) for the drug release. The anomalous mechanism may also be attributed to the competing release through matrix degradation. However, its contribution may be very less for an initial release period of 5 h or less. 4. Conclusions Gelatin with polyether urea crosslinks was synthesized using the isocyanate terminated polyether prepared from IPDI and PEG-600, and its thermal stability, in vitro swellability, degradability and drug release characteristics as a carrier matrix were evaluated using theophylline as a model drug. Its

Diisocyanate mediated polyether modified gelatin drug carrier for controlled release in vitro swellability and drug release were decreased with increased extent of crosslinking. The release rate was more in SGF than in SIF. Comparison of typical release profiles for ampicillin sodium salt, theophylline and 5-fluorouracil in a typical carrier GEPI-30 indicated only marginal differences in release rates and this may be due to the minor variation in the hydrophobicity and solubility of drugs and their physical interaction with the matrix, and the lengthy chain of crosslinker, ICTPE. Modeling of the drug release indicated an anomalous transport mechanism. The cross linked gelatin is biodegradable in SIF and SGF and the biodegradability decreased with increased degree of cross linking. The degradability in SGF and SIF may also contribute for the drug release rate. But the release mechanism for the initial periods of 5 h or less seemed to be non-Fickian. The results suggested that the cross linked gelatin might be a promising vehicle for sustained release preparations in oral therapy. This material may also be used in tissue engineering as scaffold in medical field because of its biocompatibility and biodegradability. Acknowledgment The authors wish to thank and acknowledge the full financial support provided by theDepartment of Biotechnology (DBT), Government of India (Grant Reference No: BT/PR8768/BRB/ 10/533/2007) for this project. The authors also thank the management of Bannari Amman Institute of Technology (BIT), Sathyamangalam 638 401 for providing lab facilities to carry out this research project, Department of Chemistry and Sophisticated Analytical Instrumentation Facility (SAIF) of Indian Institute of Technology, Chennai, for recording TG traces, Karunya University for recording XRD and PSG college of Technology for recoding DSC. Mr. V. Vijayakumar also thanks DBT for awarding the fellowship. References Al-Karawi, A.J.M., Al-Daraji, A.H.R., 2010. Preparation and using of acrylamide grafted starch as polymer drug carrier. Carbohyd. Polym. 79, 769–774. Ampicillin Sodium Salt, 2012. Material Safety Data Sheet A9518 Sigma–Aldrich. http://www.sigmaaldrich.com/. Accessed 29.09.12. Awad, W.H., Wilkie, C.A., 2010. Investigation of the thermal degradation of polyurea: the effect of ammonium polyphosphate and expandable graphite. Polymer 51, 2277–2285. Berthold, A., Cremer, K., Kreuter, J., 1996. Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model for anti-inflammatory drugs. J. Control. Release 39, 17–25. Bertoldo, M., Bronco, S., Gragnoli, T., Ciardelli, F., 2007. Modification of gelatin by reaction with 1,6-diisocyanatohexane. Macromol. Biosci. 7, 328–338. Bigi, A., Cojazzi, G., Panzavolta, S., Rubini, K., Roveri, N., 2001. Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking. Biomaterials 22, 763–768. Curcio, M., Spizzirri, U.G., Iemma, F., Puoci, F., Cirillo, G., Parisi, O.I., Picci, N., 2010. Grafted thermo-responsive gelatin microspheres as delivery systems in triggered drug release. Eur. J. Pharm. Biopharm. 76, 48–55.

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