Colon Targeting of 5-Fluorouracil Using Polyethylene Glycol Cross

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Colon Targeting of 5-Fluorouracil Using Polyethylene Glycol Cross-linked Chitosan Microspheres Enteric Coated with Cellulose Acetate Phthalate Kuntal Ganguly,† Tejraj M. Aminabhavi,† and Anandrao R. Kulkarni*,† †

SET’s College of Pharmacy, S.R. Nagar, Dharwad 580 002, India ABSTRACT: Polyethylene glycol cross-linked chitosan microspheres loaded with 5-fluorouracil (5-FU) have been prepared by emulsion cross-linking and enteric coated with cellulose acetate phthalate (CAP) to facilitate direct targeting of 5-FU to the colon. Spherical microspheres with surface ridges to moderate smooth surfaces loaded with different concentrations (i.e., 10, 20, and 30% w/w) of 5-FU have been evaluated for encapsulation efficiency, swelling and in vitro release characteristics in simulated gastro intestinal tract (GIT) conditions for both enteric coated and uncoated microspheres. In acidic pH, 8 to 17%, but in alkaline pH, approximately 88 to 97% of 5-FU was released with CAP coated microspheres. In the case of uncoated microspheres, 46 to 77% release of 5-FU occurred in acidic pH, but 93 to 97% was released in alkaline pH. The study demonstrates that chitosan coating with CAP and cross-linking with PEG is necessary for targeted delivery of 5-FU to the colon. The coated microspheres are found to be more suitable for colon targeting than the uncoated formulations as the former prolonged the 5-FU release from 6 to 12 h by protecting 5-FU in acidic environment of the stomach. Kinetics of drug release as investigated by empirical equations suggested Super Case II transport mechanism.

1. INTRODUCTION Chitosan (CS) and chemically modified chitosans, being the widely available naturally occurring biopolymers, have been extensively used in drug delivery applications,110 since these have proven to be nontoxic, biodegradable, nonallergic, and easily absorbable, and their properties can be easily tailor-made for specific applications.412 However, appropriate degree of deacetylation and molecular weight of CS is necessary for developing micro/nanoparticles.4 The ability of CS to control the release of drugs and solubility in aqueous acidic solution, thus alleviating toxic organic solvents during the formulation, having abundant free amino groups that are easily cross-linkable, in addition to its mucoadhesive character makes CS the most ideal candidate in drug delivery.1116 In the area of pharmaceutics, oral drug delivery has been the most promising and convenient route,17,18 but the delivery device for oral route should be capable of sustaining the drug in varying pH environments. Chitosan, particularly useful in oral delivery, is a cationic polysaccharide that is soluble or swellable in acidic pH (i.e., in stomach), while in neutral or alkaline pH, it deswells and precipitates. Innumerable authors have reported on the colon specificity of CS,1921 but its intestinal delivery i.e., especially to the colon is questionable22 because of the deswelling property of chitosan in alkaline media. However, the polyelectrolyte complex of CS (such as chitosan-pectin, chitosan-alginate, etc.) with watersoluble polyionic species, which are swellable in neutral pH, could be used in colon targetting.2329 In recent years, there is a growing interest in polyelectrolyte complexes of CS with carboxymethyl starch,26 chitosan-carboxymethyl konjac glucomannan27 and chitosanheparin for the delivery of albumin,28 heparin/ chitosan nanoparticle carriers prepared by polyelectrolyte complexation28 and chitosan-polyaspartate29 for the delivery of 5-FU. Folic acid-conjugated CS has also been used to formulate r 2011 American Chemical Society

nanoparticles for targeted delivery of 5-aminolevulinic acid (5-ALA) to the colon,30 wherein conjugation of folic acid imparts high affinity for colorectal cancer cells showing the overexpression of folate receptors. Chitosan and tamarind kernel powder interpolymer complex films have also been investigated for colon drug delivery.31 Realizing the above findings and based on the literature search, no studies have yet been made on developing CS microspheres that are enteric coated with cellulose acetate phthalate (CAP) and cross-linked with polyethylene glycol (PEG) for colon targeting of 5-FU. The chemical cross-linking of CS with a hydrophilic and low molecular weight PEG produces a swellable delivery system in both acidic and alkaline pH media. This can be achieved by cross-linking with PEG by facilitating the interaction between amino group of CS with the aldehydic group of formaldehyde (see Scheme 1) forming an intermediate Schiff’s base. However, crosslinking of CS with a conventional cross-linking agent such as glutaraldehyde (GA) has the disadvantage due to its toxic effects as well as nonswelling behavior in alkaline media.32 In this work, we have attempted to cross-link CS with PEG in the presence of formaldehyde to produce PEGylated chitosan (PEG-CS), which swells significantly in both stomach and intestine, in which PEG acts both as a swelling agent as well as a crosslinker.33 However, in colon targeting, the restriction of drug release in the stomach (acidic pH) is important, which can be achieved34 by the granular method of enteric coating with CAP. Drug release profiles of such delivery devices in simulated GIT conditions are investigated for plain CS, PEGylated CS as well as Received: July 27, 2011 Accepted: September 23, 2011 Revised: September 6, 2011 Published: September 23, 2011 11797

dx.doi.org/10.1021/ie201623d | Ind. Eng. Chem. Res. 2011, 50, 11797–11807

Industrial & Engineering Chemistry Research

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Scheme 1. Mechanism Showing the Cross-linking of CS with PEG

Figure 1. Flow diagram for the preparation of PEG-cross-linked CAPcoated and uncoated CS microspheres.

enteric coated CS and PEGylated CS microspheres. Furthermore, in vitro release kinetics parameters have been estimated through empirical equations that are widely used in the literature, to understand the nature of drug release mechanism.

2. MATERIALS AND METHODS 2.1. Materials. Polyethylene glycol with a molecular weight of 4000 Da, formaldehyde (37% w/w), formic acid (98100%), methanol, acetic acid, liquid paraffin, glutaraldehyde (GA), acetone, petroleum ether, and other chemicals used were all of analytical grade samples purchased from s.d fine chemicals, Mumbai, India. Chitosan of medium molecular weight (≈400 kDa) with ≈80% deacetylation was purchased from SigmaAldrich, USA. Cellulose acetate phthalate (CAP) was purchased from Yarrow Chem, Mumbai, India. 2.2. Preparation of Chitosan Microspheres. Microspheres of chitosan were prepared by an emulsification method followed by cross-linking with GA. In brief, CS solution (1% w/v) was prepared by dissolving CS in 1% v/v acetic acid solution. The solution was dispersed in light liquid paraffin oil containing Span20 (1% w/v) as an emulsifier and stirred using a stainless steel overhead stirrer between 2000 and 2500 rpm speed for 30 min. Further, GA was added and stirred for 3 h at 50°-55 °C. The cross-linked microspheres were centrifuged, washed several times with petroleum ether, followed by water and then with

acetone, dried, and stored in a desiccator. The 5-FU was loaded into these microspheres by adding an aqueous solution of 5FU (30% w/w) during emulsification step. Microspheres were enteric coated with CAP by a granule coating method.32 The uncoated 5-FU-loaded GA cross-linked CS microspheres were designated as C1, whereas those coated with CAP were denoted as CC1. 2.3. Preparation of PEG-cross-linked Chitosan Microspheres. PEG-cross-linked CS was prepared as per the method developed before.31 In brief, both CS as well as PEG were dissolved separately in formic acid (500 mg/10 mL); both these solutions were mixed by stirring on a magnetic stirrer for 15 min. Thereafter, formaldehyde (500 μL) was added and mixed thoroughly for 12 h to complete the cross-linking. The entire solution was poured into 100 mL of liquid paraffin oil containing 1% w/v Tween-80 and stirred for 4 h using an overhead stirrer at the rotation speed of 1500 rpm by maintaining the temperature at 45 °C. The microspheres were centrifuged and washed with petroleum ether followed by acetone and deionized water to remove the unreacted formaldehyde. For preparing 5-FU-loaded PEG cross-linked chitosan microspheres, a similar method was adopted by mixing weighed quantities of 5-FU i.e., 10%, 20%, and 30% w/w in the PEG-cross-linked CS polymer solution before emulsification. These are designated as F1, F2, and F3, respectively. The flow diagram for the preparation of PEG-cross-linked CAP-coated and uncoated CS microspheres is shown in Figure 1. 2.4. Characterization. 2.4.1. Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectra of the plain CS, physical mixture of PEG and CS, placebo microspheres of PEG-cross-linked CS and 5-FU-loaded PEG-cross-linked CS microspheres were all obtained on a MAGNA-IR 560 spectrophotometer (Nicolet Instrument Corp., Wisconsin, USA). Polymer samples were powdered in an agate mortar and pestled with KBr to make the pellets by compression using a hydraulic press. Spectra were scanned over the wavenumber range of 4000400 cm1 at ambient temperature. 2.4.2. Differential Scanning Calorimetry (DSC). DSC analysis was carried out on 5-FU, PEG-CS blend, placebo microspheres of PEG-CS and 5-FU-loaded PEG-CS microspheres using DSC Q20 V24.4 Build 116 TA Instruments, New Castle, Delaware, USA. 11798

dx.doi.org/10.1021/ie201623d |Ind. Eng. Chem. Res. 2011, 50, 11797–11807

Industrial & Engineering Chemistry Research Samples were heated at the rate of 10 °C/min under nitrogen atmosphere between 40° and 400 °C. 2.4.3. Thermogravimetric Analysis (TGA). Weight changes with respect to temperature of PEG-CS blend, placebo PEGCS microspheres, and 5-FU-loaded PEG-CS microspheres were analyzed using (SDT Q600 (V20.9 Build 20) instrument, TA Instruments-Waters, Schaumburg, Illinois, USA) thermal analyzer. Nitrogen was used as a carrier gas, and TGA analysis was carried out at the heating rate of 10 °C/min under inert nitrogen atmosphere. The sample size was ≈10 mg, and TGA curves were recorded in the temperature range of 40°-500 °C. Initial decomposition temperatures (Tonset) were determined directly from the thermograms. 2.4.4. X-ray Diffraction Analysis (XRD). XRD analysis was carried out to investigate the effect of microencapsulation on the crystallinity of drug as well as drug-loaded microspheres. XRD patterns of 5-FU crystals, placebo microspheres of PEG-CS and 5-FU-loaded PEG-CS microspheres were recorded using Philips X’PERT PRO X-ray diffractometer (Philips, Almelo, The Netherlands) with a PW3050/60 theta/theta goniometer and a PW3373/00 copper long fine focus X-ray tube with CuKα radiation. Results were collected in a continuous scan mode using a step size of 0.001° 2θ. Scanning was done up to 2θ of 80°, and these measurements were done at the Environmental Protection Agency, Cincinnati, OH, USA (courtesy of Dr. M. N. Nadagouda). 2.4.5. 13C Nuclear Magnetic Resonance Spectroscopy ( 13C NMR). Solid-state 13C NMR spectra of chitosan and PEG-crosslinked chitosan were recorded on a Bruker MSL-300 (Fremont, California, USA) NMR spectrometer, at ambient temperature, operating at a carbon frequency of 75.5 MHz. Samples were grounded in a glass mortar and pestle to get fine powder. Around 200 mg of samples were filled in zirconia rotors and spun at 34 kHz. About ∼1500 accumulations were made to get better spectra. The measurements were done by the courtesy of Dr. Manohar Badiger, Scientist, National Chemical Laboratory, Pune, India. 2.4.6. Scanning Electron Microscopy (SEM). Shape and surface morphology of the microspheres of both plain CS and PEGcross-linked CS were observed using SEM (Jeol, JSM-840A scanning electron microscope, Tokyo, Japan). Microspheres were fixed on the supports with carbon-glue and coated with a gold layer using a gold sputter coater (Jeol, JFC-1100E sputter coater, Tokyo, Japan) in a high vacuum evaporator (coating thickness 200 Å). Samples were observed by SEM under 20 kV energy. 2.4.7. Determination of Mean Particle Size. Particle size analysis of placebo and 5-FU-loaded PEG-CS microspheres was performed by optical microscopy using a compound microscope (BESTO, model 10A, Ambala, India). A small number of dry particles were spread onto a glass slide with a drop of acetone. After completely drying the acetone, glass slide was observed under a compound microscope at a magnification of 100X. Around 500 particles were selected for measurement using a precalibrated ocular micrometer. 2.5. Swelling Studies. Equilibrium swelling of placebo as well as 5-FU-loaded microspheres were determined by measuring the extent of swelling of the microspheres in pH 1.2 and pH 7.4 buffer media. To ensure complete equilibration, samples were allowed to swell for 24 h, and the excess surface-adhered liquid droplets were removed by blotting with soft tissue paper. The swollen microspheres were weighed to an accuracy of (1 mg on an electronic microbalance (Essae FB200, Tokyo, Japan). The

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microspheres were dried in an oven at 60 °C for 5 h until no change in weight gain of the dried samples was observed. Equilibrium swelling (%) was calculated as Ws Wd 100 ð1Þ %Swelling ¼ Wd where Ws is weight of the swollen microspheres, and Wd is weight of the dry microspheres. Experiments were performed in triplicate, but the average values with standard errors are reported. 2.6. Microsphere Production Yield. The yield of microsphere production was calculated by dividing the weight of the collected microspheres by the weight of all nonvolatile components used in the preparation of microspheres, which is expressed in % yield ! Wm %Yield ¼ 100 ð2Þ Wp where Wm is weight of the dried microspheres, and Wp is initial weight of the polymers. 2.7. Drug Loading (DL) and Encapsulation Efficiency (EE). About 10 mg of 5-FU-loaded microspheres were swollen in 50 mL of distilled water and crushed in a glass mortar and pestle. The resulting solution was diluted with 0.1 N HCl in a 100 mL volumetric flask, kept in a water bath, and maintained at 50 °C for 6 h. Aliquots were taken out and filtered, and the drug concentration was measured spectrophotometrically at the λmax of 266 nm (LABINDIA UV 3000+, Mumbai, India). Readings were taken in triplicate to calculate % DL and % EE using the following equations ! Mt %Drug loading ¼ 100 ð3Þ Mp where Mt is total weight of 5-FU extracted from the microspheres, and Mp is the weight of microspheres DL 100 ð4Þ %Encapsulation efficiency ¼ TL Here, DL is the actual drug loading, and TL is the theoretical loading. 2.8. Enteric Coating with CAP. A known weight of the microspheres was taken in a coating pan and sprayed with 2% w/v CAP solution in acetone using an atomizer of 100 mL capacity. The pan was preheated for contact drying. Once the temperature of the pan reached 42° to 46 °C, the coating solution was sprayed as a fine mist, which got dried just after when it came in contact with the microspheres. Coating was applied in many short and fast exposures until the weight of microspheres increased by about 1015%. The coated F1, F2, and F3 formulations are designated as CF1, CF2, and CF3, respectively. 2.9. In Vitro Release. Drug release from PEG-CS and CAPcoated PEG-CS microspheres loaded with different amounts of 5-FU was investigated in two different pH (1.2 and 7.4) media in order to mimic GIT conditions. The study was performed using USP XXIII dissolution test apparatus by the paddle method (Electrolab, Mumbai, India). Accurately weighed microspheres equivalent to 50 mg of 5-FU were suspended in 3 mL of 0.1 N HCl and placed in a dialysis membrane bag having a molecular weight cutoff between 12,000 and 14,000 Da. The bag was tied and immersed in 900 mL of dissolution medium maintained at 11799



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37 °C and stirred continuously at the paddle rotation speed of 100 rpm. During the first 2 h, 0.1 N HCl solution was taken as the dissolution medium, but after 2 h, this solution was replenished with phosphate buffer (pH 7.4). Aliquots of sample were withdrawn at the predetermined intervals of time and replaced with fresh solution to measure the absorbance at λmax of 266 nm on a UV spectrophotometer (LABINDIA UV 3000 +, Mumbai, India). The release of 5-FU was determined from the calibration curve. These studies were performed in triplicate for each sample, but the average values were considered in data analysis. 2.10. Model Fitting of In Vitro Release Data. Kinetics of drug release from all the coated formulations was investigated by fitting the in vitro release data using empirical equations viz., zeroorder, first order, Higuchi square root, Hixson-Crowell cube root, and the Korsemeyer-Peppas equations using the PCP Disso v3 software developed in Microsoft excel 97. Regression coefficients, r2, approaching to almost unity were considered to be the best fit model for the system with reference to a particular equation.

3. RESULTS AND DISCUSSION Microspheres of plain CS were prepared by emulsion crosslinking method using glutaraldehyde as a cross-linking agent, while the PEG-cross-linked CS microspheres were prepared by the method explained as per flow diagram given in Figure 1. The cross-linking of CS is necessary to prepare microspheres in order to retain their structural integrity in acidic pH condition. Since, glutaraldehyde is toxic to biological systems due to the pendant aldehydes in the final product, hence alternatively a nonaldehydic cross-linker such as PEG was used to prepare the cross-linked microspheres. Another reason to use PEG is that it helps to improve the swelling of CS in alkaline media by imparting pHindependent swelling properties. The cross-linking of CS with PEG was carried out as per the method prescribed before,33 and the mechanism is shown in Scheme 1. The free amino groups of CS when treated with formaldehyde would form an intermediate of the Schiff’s base (-NdCH2), which undergoes the addition reaction with hydroxyl group of PEG.33 3.1. Characterization. 3.1.1. Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectra of plain CS, PEG-CS blend, and

placebo microspheres of PEG-cross-linked CS shown in Figure 2 confirm the cross-linking reaction between CS and PEG. FTIR spectra of plain 5-FU and 5-FU-loaded PEG-CS microspheres were taken to rule out any incompatibility between 5-FU and PEG-CS. Notice that FTIR spectra of CS displays the characteristic peaks of CH stretching at 2923 and 2849 cm1, CdO stretching of the secondary amide at 1658 cm1, NH bending of amide II band at 1591 cm1, NH stretching of amide and ether bands at 1422 cm1, and bridge oxygen of cyclic ether (COC) stretching at 1158, 1062, 1023, and 889 cm1, respectively.19 The PEG-CS physical mixture shows two typical characteristic peaks of PEG at 1111 cm1 and 3428 cm1, respectively, for CO stretching and OH stretching vibrations of PEG moiety. The physical polymeric blend shows35 the characteristic peaks of PEG at 1281, 959, and 843 cm1. Hemiacetal and acetal reactions occur between alcohol and aldehydes in strong acidic conditions. Hemiacetals are very unstable in nature and the backward reaction is likely. Since, chitosan also contains hydroxy group, there is more possibility of the formation of acetal and hemiacetal during cross-linking with formaldehyde. However, the highly reactive free amino groups

Figure 2. FTIR of different polymers and formulations (a) chitosan, (b) PEG-CS blend, and (c) PEG-cross-linked CS.

on chitosan backbone readily undergo the addition of formaldehyde to form Schiff’s base. In the present research, substitution at the amino group was confirmed by a change in the intensity of amide II band observed at 1591 cm1. Usually acetal groups show a peak around 1150 cm1, but no such a peak was observed in PEG-cross-linked chitosan. In case of PEG-cross-linked CS, typical peaks of PEG and CS are present. The intensity of NH bending of amide II band of CS has increased significantly due to cross-linking. FTIR spectra (Figure 3) of placebo and 5-FU-loaded microspheres of PEG cross-linked CS were taken to investigate the incompatibility between drug (5-FU) and polymer (PEG-CS). The 5-FU shows a characteristic peak at 3134 cm1 due to NH stretching, whereas the peaks at 1731 cm1 and 1666 cm1 are due to CdO stretching. The peaks at 1248 and 814 cm1 are for CH in plane and CH out of plane deformation, respectively. In case of 5-FU-loaded PEG-cross-linked CS microspheres, characteristic peaks of 5-FU at 3073 cm1 (NH stretch), 1719 cm1 and 1657 cm1 (CdO stretch), 1246 cm1 (CH in plane deformation), and 813 cm1 (CH out of plane deformation) are present in addition to those of PEG-cross-linked CS. Overall, the FTIR results confirm the successful cross-linking between PEG and CS as well as the compatibility between 5-FU and crosslinked PEG-CS. 3.1.2. Differential Scanning Calorimetry (DSC). DSC studies were performed to understand the thermal behavior of plain CS, PEG-CS blend and the placebo microspheres of PEG-CS. These data are presented in Figure 4, while those of plain 5-FU and 5-FU-loaded PEG-CS microspheres shown in Figure 5 show an endotherm peak for CS at 96 °C, due to the loss of moisture, but the exotherm peak observed at 305 °C is due to the decomposition of CS. The PEG-CS blend shows a sharp endotherm at 58 °C, indicating the melting36 of PEG. An exothermic peak at 305 °C is observed due to the decomposition of CS as well as PEG in the blend. However, placebo microspheres show a peak 11800



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Figure 5. DSC curves of (a) 5-FU loaded PEG-cross-linked CS microspheres and (b) 5-FU.

Figure 3. FTIR of (a) 5-FU and (b) 5-FU-loaded PEG-cross-linked CS microspheres.

Figure 6. TGA curves of (a) chitosan, (b) blend of PEG and CS, (c) PEG-cross-linked CS, and (d) 5-FU-loaded PEG-cross-linked CS microspheres.

Figure 4. DSC curves of (a) chitosan, (b) PEG-CS blend, and (c) PEGcross-linked chitosan.

at 90.5 °C due to endothermic transition as a result of loss of moisture from the PEG-cross-linked CS matrix. A peak at 291.2 °C is due to exothermic transition of cross-linked matrix as a result of decomposition of CS as well as PEG. Notice that intensity of PEG-CS exotherm peak has decreased compared to the plain CS, confirming the reduction in rigid crystalline structure of CS after cross-linking with the soft PEG segment. In the case of 5-FU (see Figure 5), a sharp peak at 284.7 °C indicates its melting point, but 5-FU-loaded PEG-CS microspheres show the DSC patterns similar to that of placebo and no peaks of 5-FU are observed at 281 °C, indicating the amorphous nature of the polymer after encapsulation of 5-FU into PEG-cross-linked CS matrix. 3.1.3. Thermogravimetric Analysis. In order to evaluate thermal properties of the polymers and drug-loaded formulations, plain CS, PEG-CS blend, placebo PEG-CS, and 5-FU-loaded PEG-CS microspheres were characterized by TGA. TGA tracings displayed in Figure 6 for plain CS (Figure 6a) shows two

events of weight loss with respect to an increase in temperature; the first event occurred at 44 °C that continued up to 86 °C with a gradual loss of 10% weight, due to loss of moisture. The second event of weight loss occurred at 281 °C up to 311 °C with 43% weight loss corresponding to decomposition of CS. The TG curve for PEG-CS blend (Figure 6b) shows three significant weight loss events. The first one occurred at 65 °C that continued up to 114 °C with a weight loss of only 4.7% due to the loss of moisture and melting of PEG segment. The second event occurred at 281 °C up to 307 °C with a weight loss of 19.3% corresponding to CS decomposition that shifted slightly due to the presence of PEG. The third event occurred at 382 °C and continued up to 410 °C with a significant weight loss of 51%, due to the decomposition of PEG segment. It is observed that the initial thermal decomposition (Tonset) of CS took place at 281 °C, whereas Tonset in case of PEG-CS started at 257 °C. The PEGcross-linked chitosan (Figure 6c) showed nearly 90% weight loss around 400 °C, while CS alone has lost about 50% of its initial weight. The Tonset value is shifted for PEG-CS due to the addition of soft PEG segment, thereby reducing the rigidity of plain CS matrix. Figure 6(d) shows the TG curve of 5-FU-loaded PEG-CS microspheres, wherein four events of weight losses are observed. 11801



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Figure 7. XRD curves of (a) placebo PEG-cross-linked CS microspheres and (b) 5-FU and (c) 5-FU-loaded microspheres of PEG-crosslinked CS.

The first event occurred at 44 °C, which continued up to 66 °C with 9% weight loss. The second event started at 150 °C, which continued up to 202 °C with 14% loss in weight. This was followed by another event of weight loss, which occurred at 300 °C and continued up to 337 °C with 28% weight loss corresponding to TG curve of 5-FU. However, the last event of weight loss started at 393 °C that continued up to 423 °C with 36% weight loss. On the whole, TGA data indicate that PEG-cross-linked CS has shown that the degradation pattern i.e., the Tonset value, has shifted to lower temperature and follows four steps, indicating a reduction in the rigidity of CS matrix after cross-linking with PEG. However, incorporation of a drug into the PEG-crosslinked chitosan alter degradation due to the high melting point of the drug (i.e., 281 °C) resulted in a stabilized polymer matrix. 3.1.4. X-ray Diffraction Analysis. XRD analysis was carried out to investigate the drug’s polymorphism after encapsulation. XRD profiles of placebo PEG-CS microspheres, plain 5FU and 5-FUloaded PEG-CS microspheres are displayed in Figure 7 (a, b, and c, respectively). In case of placebo PEG-cross-linked CS microspheres, two sharp peaks are observed at 2θ of 10° and 20° that are characteristic of chitosan.37 The 5-FU-loaded microspheres of PEG-cross-linked CS show all the peaks at the same 2θ values as observed in case of placebo microspheres but with a much lower intensity. This together with the absence of peak at 2θ of 28°, which is the characteristic peak38 of 5-FU, indicates a molecular level dispersion of 5-FU in the polymer matrix. 3.1.5. 13C Nuclear Magnetic Resonance Spectroscopy ( 13C NMR). The cross-linking of chitosan with PEG was further confirmed by 13C NMR analysis shown in Figure 8 for chitosan and PEG-cross-linked CS that display all the characteristic chemical shifts of chitosan. The new chemical shifts observed between 42 and 81 ppm are due to CH2 groups of PEG.33 3.1.6. Morphology and Mean Particle Size of Microspheres. SEM images of chitosan, placebo PEG-cross-linked CS matrix, and 5-FU-loaded microspheres of PEG-cross-linked CS are shown in Figure 9. Chitosan microspheres are spherical with smooth surfaces, whereas those of PEG-cross-linked CS, though are almost spherical, have some wrinkles and ridged surfaces due to the cross-linking of PEG with CS. The pores observed on the surfaces of both placebo and 5-FU-loaded microspheres of PEGCS are due to the evaporation of solvent during particle hardening. On the other hand, 5-FU-loaded PEG-cross-linked CS and

Figure 8. Solid state 13C NMR spectra of chitosan (MVB1) and PEGcross-linked CS (MVB2).

Figure 9. SEM images of (A) placebo CS microspheres at 15000X magnification, (B) placebo PEG-cross-linked CS microspheres at magnification of 240X, and (C) 5-FU-loaded PEG-cross-linked CS microspheres at 300X magnification.

placebo PEG-cross-linked CS microspheres show almost identical morphologies. However, no drug crystals are seen on the surfaces of 5-FU-loaded PEG-cross-linked CS microspheres, which was also confirmed by DSC and XRD. SEM images of CAP coated PEG-CS microspheres are shown in Figure 10. After coating with CAP, the surface of the 11802



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microsphere became more smooth (Figure 10 A). However, there was formation of agglomerates consisting of 37 microspheres after the coating (Figure 10 B), and this may be due to the small size of the microspheres and tachifying nature of CAP. Results of mean particle size and equilibrium swelling in different pH media presented in Table 1 for placebo microspheres of CS, placebo PEG-cross-linked CS microspheres, uncoated 5-FU-loaded PEG-cross-linked CS microspheres, and enteric coated 5-FU-loaded PEG-cross-linked CS microspheres as determined by optical microscopy suggest that for placebo microspheres of CS, the mean particle size is 3.89 ( 0.68 μm, but for placebo PEG-cross-linked CS microspheres, the mean particle size is quite high i.e., 319 ( 32.5 μm, even though both were prepared by using the same stirring speed. On the other hand, CAP-coated placebo PEG-cross-linked CS microspheres have a mean particle size of 322 ( 18 μm, but 5-FU-loaded PEG-crosslinked CS microspheres (formulation F3) has a somewhat low mean particle size of 304 ( 12 μm. The CAP-coated PEG-CS microspheres loaded with 5-FU (formulation CF3) have the mean particle size of 316 ( 20 μm that ranged between those of 5-FU-loaded PEG-cross-linked CS and CAP-coated placebo PEG-cross-linked CS microspheres. Thus, the size of the microspheres depends on the nature of the delivery device. 3.2. Equilibrium Swelling. Swelling characteristics of the PEG-cross-linked CS matrix is important, since the release of 5-FU from such a matrix depends on polymer equilibrium swelling. The GA-cross-linked CS exhibits an equilibrium swelling of 92 ( 4% in pH 1.2 with a lower value of 43 ( 6% in pH 7.4. The % swelling of CAP-coated and GA-cross-linked CS microspheres exhibit 56 ( 10 and 19 ( 4% equilibrium swelling, respectively, in pH 1.2 and 7.4 media. In the case of uncoated PEG-crosslinked CS microspheres, the % equilibrium swelling is as high as 476 ( 15% and 370 ( 11%, respectively, in pH 1.2 and pH 7.4 buffer media. In any case, the matrix remained intact in both

acidic and alkaline media, suggesting a pH-independent swelling of PEG-cross-linked CS matrix. For CAP-coated PEG-cross-linked CS microspheres, equilibrium swelling of 49 ( 2% in pH 1.2 and 290 ( 12% in pH 7.4 suggests higher swelling in alkaline media, meaning higher release of 5-FU. For uncoated, CAP-coated, and 5-FU-loaded PEG-cross-linked CS microspheres, % equilibrium swelling was investigated to understand the effect of swelling on 5-FU release; the effect of enteric coating on drug release also plays a dominant role. For instance, 30% 5-FU-loaded PEG-cross-linked CS microspheres exhibit 429 ( 12 and 296 ( 10% equilibrium swelling, respectively, in pH 1.2 and 7.4 media, while the CAPcoated and 30% 5-FU-loaded PEG-cross-linked CS microspheres show only 47 ( 4% and 285 ( 13% equilibrium swelling in the same media. It can be noticed that the results of equilibrium swelling given in Table 1 are in agreement with earlier studies on PEG-cross-linked CS systems.33 Chitosan also is known to exhibit a pH and temperature dependent swelling characteristics due to the ionization of free amino groups,39 but in the case of PEG-cross-linked CS matrix, no free amino groups are available for interaction because after the incorporation of PEG segments, chitosan attains the ability to swell more irrespective of the pH of the media. This is indeed a favorable situation for 5-FU release in alkaline media, viz., colon. 3.3. Drug Loading (DL) and Encapsulation Efficiency (EE). The results of % drug loading and % drug encapsulation efficiency were calculated, which showed a dependence on drug loading. For three different formulations (F1, F2, and F3) with three different loadings (10, 20, and 30%), encapsulation efficiencies are 22.4 ( 3.5; 23.8 ( 3.2, and 30.4 ( 4%, respectively. These data suggest that as the theoretical drug loading increases, % entrapment efficiency also increases. However, the % yield for F1, F2, and F3 are 81 ( 1.2, 80 ( 1.3, and 81 ( 1.2%, respectively, which are almost the same without showing any dependence. 3.4. In Vitro Release. A colon-specific CR drug delivery system should prevent drug release in the stomach as well as in the small intestine. In the literature, a number of studies have been made on developing controlled release (CR) formulations of 5-FU-loaded microspheres using a variety of different kinds of polymers.4043 However, these reports, even though, have not directly dealt with the colon delivery of 5-FU, but mostly these have addressed the extended release of 5-FU over that of nascent drug, whose half-life is quite short (1020 min). Also, these could avoid the toxic effects of 5-FU. However, other studies on the use of chitosan as a delivery device for the CR of 5-FU have indicated some successful formulations to extend the in vitro release of 5-FU43 as well as for other drugs.44 Yassin et al.45 designed a new delivery system to target 5-FU directly to the colon much more effectively. This system was prepared by a compression coating technique using the granulated

Figure 10. SEM images of (A) CAP coated 5-FU-loaded PEG-crosslinked CS microspheres at 250X magnification and (B) agglomerates of CAP coated 5-FU-loaded PEG-cross-linked CS microspheres at 100X magnification.

Table 1. Results of Mean Particle Size and % Equilibrium Swelling of Different Formulations in pH 1.2 and 7.4 Media uncoated microspheres

CAP coated microspheres

% equilibrium swelling in pH formulation

1.2

% equilibrium swelling in pH

7.4

mean particle size (μm)

1.2

7.4

mean particle size (μm) 4.26 ( 1.1

placebo chitosan

92 ( 4

43 ( 6

3.89 ( 0.68

56 ( 10

19 ( 4

placebo PEG-CS

476 ( 15

370 ( 11

319 ( 32.5

49 ( 2

290 ( 12

322 ( 18

5-FU-loaded PEG-CS

429 ( 12

296 ( 10

304 ( 12

47 ( 4

285 ( 13

316 ( 20

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Scheme 2. Formation of Agglomerates after CAP Coating and Disaggregation As Well As Uncoating of PEG-CS Microspheres at Basic pH Solution

chitosan, which extended the release of 5-FU up to 6 h. The system was very effective in protecting against the effect of stomach acidic medium on chitosan and obviates the need for further enteric coating. In a recent study by Nayak et al.,46 CS microspheres were prepared by emulsification method using GA as the cross-linking agent, which were enteric coated with Eudragit to avoid the release of 5-FU in stomach and upper small intestine. In vitro drug release profile of the uncoated microspheres was typical like that of the conventional dosage form with 38 to 88% release at the end of 2 and 10 h, but the coated microspheres showed no drug release in 2 h in the simulated gastric media. In the present study, in vitro release of the enteric coated as well as uncoated 5-FU-loaded GA-cross-linked CS and PEGcross-linked CS microspheres have been performed in simulated gastric (pH 1.2) media for the initial 2 h, followed by the intestinal (pH 7.4) buffer media until longer time (up to 12 h). The extent of coating was controlled by measuring the weight gain, and preliminary experiments were conducted to optimize the quantity of 2% w/v CAP solution required to keep the microspheres intact in acidic media for 2 h. It was found that 10% weight gain was sufficient to keep the microspheres intact in the acidic media for about 2 h without hindering the drug release in the alkaline media. However, there was formation of agglomerates of PEG-CS microspheres after coating with CAP. Thus, CAP coating protects the microspheres in acidic pH, and as soon as these agglomerates are exposed to the basic pH, CAP coating get dissolved and disaggregation of microspheres occurs. The formation of agglomerates and CAP uncoating in basic pH is displayed in Scheme 2. The average % cumulative release vs time plots along with the standard deviations between three independent sets of readings for coated and uncoated GA-cross-linked CS microspheres (i.e., formulations C1 and CC1) are shown in Figure 11. Formulation C1 exhibits an overshoot effect by releasing 81.3% of 5-FU in 2 h in acidic pH, whereas only 98.6% release of 5-FU occurred in alkaline pH at the end of 12 h. However, a drastic decrease in % drug release was observed in case of CC1 formulation for which only 14.1% release occurred in acidic pH followed by 46% release in pH 7.4 at the end of 12 h. Figure 12 displays the % average cumulative release of 5-FU from the enteric coated and uncoated PEG-cross-linked CS microspheres prepared by taking three different 5-FU % loadings. All the uncoated formulations (F1, F2, and F3) showed a higher 5-FU release in acidic media than the enteric coated formulations. For instance, F1, F2, and F3 showed 46%, 67%, and 77% 5-FU release, respectively, in acidic pH in 2 h, followed by 93%, 97%, and 97% release in alkaline pH up to 12 h. Formulation F3

Figure 11. In vitro release of 5-FU from (a) uncoated CS and (b) CAPcoated GA-cross-linked CS microspheres at 37 °C in pH 1.2 for 2 h followed by pH 7.4 up to 12 h.

shows a higher release than formulations F1 and F2 due to higher % drug loading of F3 for the same degree of cross-linking in all three formulations. On the other hand, enteric coated formulations show entirely different release profiles than those of uncoated formulations. For instance, 5-FU release in acidic pH is around 10% for CF1, whereas it is around 8% and 17% for CF2 and CF3, respectively. The 5-FU release in alkaline pH is 88%, 89%, and 97% at the end of 12 h for formulations CF1, CF2, and CF3, respectively. Thus, comparatively a higher release is observed for a formulation with a higher % drug loading. However, the bulk of 5-FU-loaded in the enteric coated PEG-cross-linked CS microspheres is released in alkaline pH as shown in Figure 12, suggesting the targeting potentiality of the microspheres in alkaline environment of the intestine. Thus, the formulations of this study are potential targeted delivery systems for treating colon cancer using 5-FU, a well-known anticancer drug. Notice that there is not much difference in the release rates of 5-FU in the case of uncoated 5-FU-loaded GA-cross-linked chitosan (C1) and uncoated PEG-cross-linked CS formulations (F3) in pH 1.2 and pH 7.4 media. However, a significant difference in the release rates is observed in pH 7.4 media in the case of CAP-coated 5-FU-loaded GA-cross-linked CS microspheres (CC1) as well as CAP-coated PEG-cross-linked CS formulations (CF3). Only 46% of 5-FU is released with CC1, whereas 97% of release is observed for CF3. This is attributed to pH-independent swelling behavior of PEG-cross-linked CS, which exhibited varying degree of swelling under different pH conditions in contrast to GA-cross-linked CS that forms an insoluble matrix even after cross-linking, thereby showing a limited or nonswelling behavior in the alkaline pH media.

4. EMPIRICAL CORRELATIONS All the release data have been fitted to different empirical equations using the PCP Disso v 3 software to compute the regression coefficients, r2 given in Table 2. The r2 values for 5-FU-loaded CAP-coated PEG-cross-linked CS microspheres with respect to various empirical equations have been examined. Formulations CF1, CF2, and CF3 showed the regression coefficient values of 11804



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Figure 12. In vitro release of 5-FU from the coated and uncoated PEG-cross-linked CS microspheres (viz. F1, F2, F3, CF1, CF2, and CF3) at 37 °C in pH 1.2 for 2 h followed by pH 7.4 up to 12 h.

Table 2. Fitting Coefficients, r2 Estimated from Different Equations by the Least-Squares Method at the 95% Confidence Level Korsemeyer-Peppas

a

formulationsa

zero order r2

first order r2

Higuchi r2

Hixson-Crowell r2

N

r2

CF1

0.997

0.964

0.997

0.997

1.86

0.998

CF2 CF3

0.990 0.996

0.933 0.921

0.977 0.988

0.990 0.996

2.08 1.67

0.999 0.999

CF1, CF2, and CF3 are 5-FU-loaded CAP-coated PEG-CS microspheres with 10%, 20%, and 30% w/w drug loading.

0.998, 0.999, and 0.999, respectively, approaching almost close to unity for Korsemeyer-Peppas equation giving n > 1, indicating that the drug release follows Super Case II transport. However, almost a similar fitting was observed for zero order and HixsonCrowell equations; in both the latter cases, the fit was not good compared to Korsemeyer-Peppas equation. A poor fit compared to all the equations is observed for first order and Higuchi equations that seemed to be not suitable for the analysis of the systems of this study.

5. CONCLUSIONS PEG-cross-linked chitosan microspheres were prepared and enteric coated with cellulose acetate phthalate to regulate 5-FU loading in both the coated and uncoated PEG-cross-linked CS microspheres on their release profiles. The encapsulation efficiency of the microspheres varied from 22 to 30% and the absence of drug-polymer interactions between individual polymers, polymer blends and drug, both in placebo and 5-FU-loaded PEG-cross-linked CS microspheres have been confirmed by FTIR. DSC indicated molecular level dispersion of 5-FU in the formulations, which was also confirmed by XRD. TGA confirmed the thermal behavior of polymers after cross-linking, while 13 C NMR confirmed the cross-linking of CS with PEG.

The results of in vitro release profiles of 5-FU from the highest % 5-FU-loaded enteric coated and uncoated PEG-CS formulations (F3 and CF3) have been compared with those of GA-crosslinked CS microspheres (C1 and CC1). Higher CR values of 5-FU occurred in alkaline pH than in acidic pH, suggesting that CAP-coated PEG-cross-linked CS microspheres are able to bypass the gastric acidic environment, while at the same time, maintaining its slow release characteristics in intestinal conditions due to its pH-independent swelling behavior. The developed devices may be useful as the pH-independent swelling matrices and particularly well-suited for colon delivery of 5-FU. The release profiles when analyzed by empirical equations suggested the presence of Super Case II transport mechanism.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT The authors acknowledge the financial support from Council of Scientific and Industrial Research (CSIR) [grant no. 08/558(0001)/2010-EMR-I], New Delhi, India in providing a research fellowship to Mr. Kuntal Ganguly. Professor T. M. Aminabhavi 11805


Industrial & Engineering Chemistry Research acknowledges the CSIR [grant no. 21(0760)/09/EMR-II] for Emeritus Scientist.

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Colon Targeting of 5-Fluorouracil Using Polyethylene Glycol Cross

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