and Cellulose Acetate Phthalate for Colon Delivery of 5-Fluorouracil

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Blend Microspheres of Poly(3-hydroxybutyrate) and Cellulose Acetate Phthalate for Colon Delivery of 5-Fluorouracil Kiran Chaturvedi, Anandrao R. Kulkarni,* and Tejraj M. Aminabhavi* SET’s College of Pharmacy, S.R. Nagar, Dharwad, 580 002 India ABSTRACT: Blend microspheres of poly(3-hydroxybutyrate) (PHB) and cellulose acetate phthalate (CAP) have been prepared in compositions of 2:1, 1:1, and 1:3 (w/w) by the solvent evaporation technique. These pH-sensitive polymers were utilized to investigate the colon delivery of 5-fluorouracil (5-FU), an anticancer drug. The surface morphology of the microspheres was studied by scanning electron microscopy (SEM), which confirmed their spherical nature with sizes ranging from 29 to 67 μm, as well as pore structures before and after the dissolution experiments. Fourier transform infrared (FTIR) spectroscopy was used to confirm the polymer blend compatibility and to confirm the absence of drugpolymer interactions. Differential scanning calorimetry (DSC) was employed to study the crystalline nature and molecular-level dispersion of 5-FU in the matrixes after encapsulation. In vitro release experiments were performed at 37 °C in simulated buffer medium of the stomach (pH 1.2) for 2 h, followed by simulated intestinal medium (pH 7.4). It was found that the release of 5-FU from blend microspheres followed pH-dependent release as compared to that of plain PHB microspheres. From the in vitro release studies, it was found that blend microspheres are more efficient at delivering 5-FU to the colon than plain PHB microspheres. Furthermore, release data were fitted to empirical equations to understand the nature of the drug release profiles.

1. INTRODUCTION Biodegradable polymers are of great interest in biomedical research and are currently receiving growing scientific attention because of their inherent properties such as biocompatibility, predictability of degradation kinetics, and ease of fabrication.1 However, biopolymers of both synthetic and natural origin, such as polylactides,2 poly(lactide-co-glycolide) (PLGA) copolymers,3 albumin,4 chitosan,5,6 and alginates,7 have been used to develop controlled-release (CR) formulations for a wide variety of drugs. The concept of novel drug delivery systems (NDDS) aims at achieving systemically improved therapeutic success for drugs including those that have limited use because of toxicity, uneven distribution pattern, stability, and formulation difficulties. Highly toxic anticancer drugs can be targeted using NDDS, thereby avoiding the possible toxicity issues due to drug distribution in the whole body, thus offering an improved therapeutic index and patient compliance. Therefore, NDDS are quite promising means to provide new directions for approved drugs to extend patent protection for older drugs produced by pharmaceutical companies. Among the various biodegradable polymers used in NDDS, polyhydroxyalkanoates (PHAs) have received special attention because they can be produced in micrometer and nanometer size particles for the CR of drugs.8 A class of biodegradable polyesters that are synthesized by a wide range of microorganisms have been used either alone or in combination with other polymers for several biomedical applications such as sutures, repair patches, repair devices, slings, cardiovascular patches, orthopedic pins, adhesion barriers, stents, guided tissue repair/regeneration devices, articular cartilage repair devices, nerve guides, tendon repair devices, bone marrow scaffolds, and wound dressings.911 These polymers are well-suited for use as biomaterials because of the similarity between their mechanical properties and those of polypropylene and PLGA.12 Therefore, such polymers have r 2011 American Chemical Society

drawn renewed attention from the scientific community for the development of NDDS.13 One of the most widely studied and well-characterized homopolymers of the PHA family is poly(3-hydroxybutyrate) (PHB), which has been used in a wide range of biomedical applications.9,14 It is a natural, linear, and homochiral thermoplastic biopolyester produced by microorganisms as intercellular fat deposits in response to limited nutrient availability. It is optically active and biodegradable, having a high crystallinity and hydrophobicity.13,15 The fact that monomeric components of PHB are present in the blood of healthy adults reinforces its compatibility with the human body system.16 PHB has a lower water barrier property than polypropylene, but has a good resistance to solubility in water; it is biocompatible and highly biodegradable. The degradation of PHB depends on the microbial activity of the environment and on the surface area of the sample.17 The degradation of PHB leads to the production of 3-hydroxybutyric acid, which is one of the three endogenous ketones produced by a process known as ketogenesis.11 PHB degrades more slowly than PLGA, but its degradation rate can be tailored by blending it with other natural polymers such as amylose, dextran, dextrin, and sodium alginate that can alter the hydrolytic degradation rate of PHAs.18 For a polymer to be employed successfully in colon targeting, it is necessary to induce a pH-sensitive character. In this regard, cellulose acetate phthalate (CAP), a cellulose ester derivative, has been used as an enteric polymer.19 CAP contains anhydroglucose units, of which about one-half of hydroxyl groups are acetylated and about one-fourth are esterified with one of the two acidic Received: May 25, 2011 Accepted: August 8, 2011 Revised: August 2, 2011 Published: August 08, 2011 10414

dx.doi.org/10.1021/ie2011005 | Ind. Eng. Chem. Res. 2011, 50, 10414–10423

Industrial & Engineering Chemistry Research groups of phthalic acid.20 CAP dissolves in buffer medium at pH > 5.5, is inexpensive, and is considered to be nontoxic as it is free from any adverse effects. The polymer has been widely investigated in aqueous and nonaqueous21 media to microencapsulate drugs by the coacervation phase-separation technique. Recently, CAP was used to prepare enteric microspheres for targeted oral delivery of theophylline.22 To the best of our knowledge, not much work has been done on blends of CAP with other polymers and, in particular, with PHB. In this research, an attempt was made to prepare microspheres from blends of CAP and PHB in different ratios with a view toward inducing pH sensitivity in PHB while, at the same time, retaining its CR properties, thus enabling colon targeted delivery. 5-Fluorouracil (5-FU), a pyrimidine antimetabolite, is one of the most widely used cancer chemotherapeutic agents in clinical practice. It is commonly used for treating cancers of the colon, stomach, breast, and pancreas.23 5-FU is sparingly soluble in water but slightly soluble in alcohol, and it has a broad spectrum of activity against solid tumors. However, its short biological halflife of 1020 min, due to rapid metabolism; incomplete and nonuniform oral absorption, due to metabolism by dihydropyrimidine dehydrogenase;24 toxic side effects on bone marrow and the gastrointestinal tract (GIT); and nonselective action against healthy cells limits its usage in clinical practice. Therefore, 5-FUbased NDDS with targeting capability would not only reduce the systemic side effects, but also provide an effective and safe therapy for colon cancer with a reduced dose and duration of therapy.25 Several methods have been reported for site-specific delivery of drugs to the colon. These are classified into four types,26 namely, temporal control of delivery,27 pH-based,28 pressurebased,29 and enzyme-based30 systems. In the present study, pHsensitive PHB-based microspheres were prepared by blending with CAP during microencapsulation. The sustained-release property of PHB is tailored to restrict the release of 5-FU in the stomach, but to enhance the release rates at alkaline pH (i.e., in the colon). This system takes advantage of the pH-sensitive nature of CAP, which does not dissolve at acidic pH, but easily dissolves at alkaline pH, thus preventing the unwanted effects by specifically delivering the drug to the colon. The system would thus serve the dual purpose, namely, CR of 5-FU through the use of PHB and inhibition of drug release in the acidic environment of the stomach. Drug release profiles from the blend microspheres are compared with those of 5-FU-loaded nascent PHB microspheres to study the effects of blending of CAP on the CR of 5-FU. In addition, the possible mechanism of 5-FU release from PHB/CAP blend microspheres is explained on the basis of theoretical hypotheses, as well as actual observations. Furthermore, release profiles have been analyzed using empirical equations.

2. MATERIALS AND METHODS Poly(3-hydroxybutyrate) (PHB) and 5-fluorouracil (5-FU) were purchased from Sigma-Aldrich, St. Louis, MO. Dichloromethane (DCM), isopropyl alcohol (IPA), acetone, chloroform, and hydrochloric acid of analytical reagent grade were purchased from S.D. Fine Chemicals, Mumbai, India. Polyvinyl alcohol (PVA) was purchased from Rolex Chemicals, Mumbai, India; cellulose acetate phthalate (CAP) was purchased from Yarrow Chem Products, Mumbai, India; potassium dihydrogen orthophosphate, purified, was purchased from HiMedia Laboratories

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Figure 1. Schematic representation of the method of preparation of 5-FU-loaded PHB/CAP (1:1) blend microspheres.

Pvt. Ltd., Mumbai, India; and sodium hydroxide pellets were purchased from RFCL Ltd., New Delhi, India. 2.1. Preparation of 5-FU-Loaded PHB Microspheres. A precisely weighed (60 mg) quantity of PHB was dissolved in 3 mL of DCM with gentle heating. The loss of solvent during the dissolution of PHB in solvent was compensated by further addition of DCM. An exactly weighed quantity of previously micronized 5-FU [30%, 40% and 50% (w/w) PHB] was dispersed in PHB solution on a magnetic stirrer in a screw-cap container. The entire dispersion was then added dropwise into 240 mL of DI water containing 1% (w/v) PVA as the protective colloid and stirred on a magnetic stirrer until complete evaporation of the solvent. Microspheres were filtered using Whatman No. 1 filter paper, washed 34 times with distilled water to remove the adhered PVA particles from the surface, and dried completely in an incubator maintained at a constant temperature of 40 °C. 2.2. Preparation of 5-FU-Loaded pH-Sensitive Microspheres of PHB. The pH-sensitive microspheres were prepared from the PHB and CAP blend as per the scheme shown in Figure 1. Briefly, 50 mg of PHB was dissolved in 3 mL of DCM and 50 mg CAP was dissolved separately in 3 mL mixture of IPA and DCM (ratio 1:2). Then, both the polymer solutions were mixed to which a specified volume of 5-FU solution was emulsified using Span 80. After the formation of a stable emulsion, it was added dropwise into 25 mL of 1% w/v PVA solution and stirred to completely evaporate the solvent. Few drops of 0.1N HCl solution were added to make the PVA solution slightly acidic to prevent the dissolution of CAP. After the complete evaporation of solvent, the dispersion medium was centrifuged and microspheres were collected, washed 34 times with distilled water to remove the adhered PVA particles and allowed to dry completely in an incubator at 40 °C. Thereafter, the microspheres were stored in a desiccator at ambient temperature. 2.3. Production Yield. The yield of the product was calculated by dividing the weight of the collected microspheres by the weight of all nonvolatile components used to prepare the microspheres and 10415

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Industrial & Engineering Chemistry Research is expressed as a percentage yield ! Wm 100 percent yield ¼ Wp

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using the equations drug loading ð%Þ ¼

ð1Þ

where Wm is the weight of dried microspheres and Wp is the initial weight of the polymers. 2.4. Scanning Electron Microscopy (SEM). The surface morphology of the microspheres before and after dissolution experiments was observed by scanning electron microscopy (SEM; JSM-7000, JEOL Ltd., Tokyo, Japan) after samples had been coated with gold using a sputter coater (JFC-1600, JEOL Ltd.), and actual photographs were recorded at different magnifications. The mean diameter of the microspheres was estimated from the SEM micrographs. 2.5. Fourier Transform Infrared (FTIR) Spectroscopy. FTIR spectra of PHB, CAP, their blend microspheres, 5-FU, 5-FUloaded PHB microspheres, and 5-FU-loaded blend microspheres were recorded on a MAGNA-IR 560 FTIR spectrometer (Nicolet Instrument Corp., Madison, Wi). Drug and polymer samples were powdered separately in an agate mortar and pestle with KBr. Pellets were made by compression using a hydraulic press under a pressure of 600 kg/cm2 to scan the spectra in the wavelength range of 4000400 cm1 at ambient temperature. 2.6. Differential Scanning Calorimetry (DSC). DSC scans of accurately weighed samples of PHB, CAP, their blend microspheres, 5-FU, 5-FU-loaded PHB microspheres, and 5-FUloaded blend microspheres were performed using a differential scanning calorimeter (DSC Q20, V24.4 Build 116, TA Instruments, New Castle, DE) under an inert atmosphere of nitrogen. Sealed aluminum cups were used in the experiments for all samples, and scanning was done by first cooling the sample below ambient to 50 °C at a cooling rate of 10 °C/min and then heating it to 350 °C at the same rate. 2.7. Thermogravimetric Analysis (TGA). TGA scans were obtained using the samples of about 10 mg in platinum crucibles, under nitrogen atmosphere, in the temperature range of 40500 °C at a heating rate of 5 °C/min in an SDT Q600 instrument (V20.9 Build 20; TA Instruments-Waters, Schaumburg, IL). The initial decomposition temperatures (Tonset) were determined directly from the thermograms. 2.8. X-ray Diffraction (XRD). XRD analysis was carried out to determine the effect of microencapsulation on the crystallinity of the drug. XRD patterns of unloaded and 5-FU-loaded PHB/ CAP blend microspheres were recorded using a Philips X’PERT PRO X-ray diffractometer with a PW3050/60 theta/ theta goniometer and a PW3373/00 copper long fine focus X-ray tube with Cu KR radiation. Results were collected in continuous scan mode using a step size of 0.001° 2θ, and scanning was done to 2θ = 80°. 2.9. Drug Loading (DL) and Encapsulation Efficiency (EE). Accurately weighed quantities of 5-FU-loaded blend microspheres (10 mg) were dissolved in 2 mL of chloroform in a glass vial. To this was added a suitable volume of phosphate buffer (pH 7.4), and the mixture was transferred to a 100 mL volumetric flask to extract 5-FU. Complete recovery of 5-FU and evaporation of chloroform were achieved by heating the solution at 70 °C in a water bath. The solution was filtered and scanned using a UV spectrophotometer (LabIndia UV 3000+, Mumbai, India) at a maximum fixed wavelength of 266 nm. Readings were taken in triplicate to compute percentage DL and EE values

Mt Mp

! 100

ð2Þ

Where Mt is the total amount of 5-FU extracted from the microspheres and Mp is the weight of the microspheres DL encapsulation efficiency ð%Þ ¼ 100 ð3Þ TL where DL is the actual percentage drug loading and TL is the theoretical percentage loading. A similar method was followed for 5-FU-loaded PHB microspheres to calculate the percentage drug loading and encapsulation efficiency. 2.10. In Vitro Drug Release. In vitro drug release from the 5-FU-loaded microspheres was determined in two different buffer solutions of pH 1.2 and 7.4 to mimic the actual GIT conditions using a dissolution apparatus (USP XXIII dissolution test apparatus, LabIndia, DS 8000, Mumbai, India). Accurately weighed microspheres equivalent to 50 mg of 5-FU were suspended in 3 mL of 0.1 N HCl solution and placed in a dialysis membrane bag with a molecular weight cutoff between 12000 and 14000 Da. The bag was tied and immersed in 900 mL of dissolution medium maintained at 37 °C and stirred continuously at 100 rpm. HCl (0.1 N) was used as a dissolution medium for the first 2 h, after which the buffer was replenished with phosphate buffer (pH 7.4). Aliquots of sample were withdrawn at predetermined intervals of time and replaced with fresh buffer to measure the absorbance at 266 nm using a UV spectrophotometer (LabIndia UV 3000+, Mumbai, India). Readings were taken in triplicate, and the release of 5-FU was determined from the calibration curve. 2.11. Model Fitting of in Vitro Release Data. Kinetics of drug release from all of the formulations were calculated by fitting the in vitro release data using various mathematical equations, namely, zeroth-order, first-order, Higuchi square root, Hixson Crowell cube root, and KorsemeyerPeppas equations using the PCP Disso v3 software developed in Microsoft Excel 97. Regression coefficients, r2, approaching unity were considered to provide the best-fit models for the system under consideration.

3. RESULTS AND DISCUSSION In the present study, pH-sensitive microspheres of PHB loaded with 5-FU were prepared by the solvent evaporation method according to the scheme shown in Figure 1. PHB is soluble in DCM upon gentle heating, but the production of plain PHB microspheres using the solvent evaporation technique is a straightforward method giving the highest yield.31 To induce pH sensitivity in the PHB microspheres, we blended PHB with another pH-sensitive polymer, namely, CAP. However, CAP is not soluble in DCM, but it is completely soluble in DCM/IPA mixtures, and the reason to blend PHB with CAP is to prevent the release of the drug at the acidic pH of the stomach to facilitate a maximum drug release in the colon. Results in terms of percentage yields of the microspheres for PHB and pH-sensitive microspheres of PHB are shown in Tables 1 and 2, respectively. Using the solvent evaporation method, PHB-based microspheres were obtained in yields up to 90%, but for 5-FU-loaded plain PHB microspheres, the yield was 83% (i.e., probably lower because of the heterogeneous 10416


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Table 1. Effect of Initial Percentage Drug Loading on Mean Particle Size, Percentage Encapsulation Efficiency (%EE), and Percentage Yield of Plain PHB Microspheres at PHB Concentration of 20 mg/mL in 1:80 Volume Ratio of Dispersed to Continuous Phases formulation

5-FU loading

mean particle

code

(mg)

size (μm) ( SD

F1 F2

18 24

75 ( 25 74 ( 31

50.08 ( 4.01 83 ( 1.7 43.60 ( 1.04 83.3 ( 3.0

F3

30

71 ( 18

39.14 ( 3.14 83 ( 2.3

EE (%)

yield (%)

Table 2. Effect of Polymer Blend (PHB/CAP) Composition on Mean Particle Size, Percentage Encapsulation Efficiency (%EE), and Percentage Yield of PHB/CAP Blend Microspheres formulation PHB/CAP blend codes ratio (w/w)

EE (%)

mean particle size (μm) ( SD yield (%)

B1

2:1

45.69 ( 3.46

48 ( 19

98 ( 1.06

B2

1:1

47.70 ( 3.22

47 ( 16

98 ( 0.88

B3

1:3

53.10 ( 4.01

44 ( 11

98 ( 1.01

dispersion formed while incorporating 5-FU). To obtain compatible blends of PHB and CAP, we maintained an appropriate weight percentage of blend polymer and composition of the DCM/IPA mixture. For instance, if the amount of CAP is increased or if the volume of IPA is decreased, then the blend components are incompatible (i.e., phase separation occurs). To avoid this problem, we dissolved 50 mg of PHB in 3 mL of DCM and mixed it with a solution containing 50 mg of CAP dissolved in 3 mL of a mixture of DCM and IPA (2:1 composition). If the volume of IPA is increased, as a result of a high solvent diffusion rate, smaller particles can be obtained with a reduction in percentage yield. To minimize the escape of CAP from the solvent mixture during the emulsification stage, a few drops of 0.1 N HCl were added to the PVA solution to make the dispersion medium slightly acidic to achieve >90% yield of the blend microspheres. 3.1. Morphological Characteristics of the Microspheres. SEM images of microspheres of both PHB and polymeric blends of PHB with CAP (1:1) are displayed in Figure 2. Figure 2A shows the image of placebo PHB microspheres taken at 300 magnification, whereas the SEM image of 5-FU-loaded PHB microspheres taken at 160 magnification is shown in Figure 2B. Placebo microspheres of the PHB/CAP blend and 5-FU-loaded blend microspheres are shown in parts C and D, respectively, of Figure 2. At lower magnification, SEM images were taken in such a way that the maximum number of particles is visible such that both shapes and sizes can be observed. Some images were recorded at higher magnifications to observe surface properties. The microspheres are spherical and free-flowing, with no particle aggregation. Morphological differences between the microspheres prepared from plain PHB and polymer blend are quite evident from the SEM images taken at higher magnification. Ridges can be seen on the surface of the microspheres prepared from both PHB (see Figure 2B,E) and the blend (Figure 2D,F). It is obvious that the ridges are more prominent in the blend microspheres than the plain PHB microspheres, because of the intermingling of the CAP chains with the PHB chains.

Figure 2. SEM images of (A) placebo PHB microspheres at 300 magnification, (B) 30% (w/w) 5-FU-loaded PHB microspheres at 160 magnification, (C) placebo PHB/CAP (1:1) blend microspheres at 800 magnification, (D) 30% (w/w) 5-FU-loaded PHB/CAP (1:1) blend microspheres at 400 magnification, (E) 30% (w/w) 5-FU-loaded PHB microspheres at 800 magnification and (F) 30% (w/w) 5- FU-loaded PHB/CAP (1:1) blend microspheres at 2000 magnification.

SEM images were also recorded after the completion of dissolution experiments. Figure 3 shows the effect of 5-FU release from the 5-FU-loaded PHB and blend microspheres. Specifically, panels AC of Figure 3 are images of 5-FU-loaded PHB microspheres after dissolution at 250, 600, and 5000 magnifications, respectively, and panels DF of Figure 3 are images of 5-FU-loaded blend microspheres after dissolution at 140, 800, and 5000 magnifications, respectively. The PHB microspheres remained intact even after 24 h of drug release, whereas the microspheres prepared from the blend of PHB and CAP developed some fine pores, as seen in Figure 3E,F. This could be due to the dissolution of CAP present in the blend in alkaline medium (phosphate buffer of pH 7.4), leading to a higher release of 5-FU from the blend microspheres in alkaline medium compared to acidic medium. The mean particle sizes of the 5-FU-loaded plain PHB and blend microspheres are summarized in Tables 1 and 2, respectively, which shows that the blend microspheres are much smaller than the plain PHB microspheres. The mean particle size of placebo PHB microspheres ranges from 97 ( 13 to 101 ( 09 μm, whereas that of 5-FU-loaded PHB microspheres ranges from 10417



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Figure 4. FTIR spectra of (a) PHB, (b) CAP, and (c) placebo PHB/ CAP (1:1) blend microspheres.

Figure 3. SEM images of 30% (w/w) 5-FU-loaded PHB microspheres after 24 h of release at (A) 250, (B) 600, and (C) 5000 magnifications and of 30% (w/w) 5-FU-loaded PHB/CAP (1:1) blend microspheres after 24 h of release at (D) 140, (E) 800, and (F) 5000 magnifications.

43 to 105 μm (i.e., lower values). The mean diameter of placebo microspheres of the blend is ∼45 ( 14 μm, whereas the mean diameter of 5-FU-loaded microspheres of the blend is around 2967 μm. The reduction in mean particle size of the blend microspheres compared to the PHB microspheres might be due to the presence of IPA in the polymer solution. IPA, being watermiscible, leads to the increased escape of solvent due to diffusion into the dispersion medium, causing a reduction in particle size. 3.2. Fourier Transform Infrared Spectroscopy. FTIR spectra of PHB, CAP, and placebo microspheres of PHB/CAP blend are shown in Figure 4. The peak for PHB at 1725 cm1 is assigned to CdO stretching, whereas those at 2854, 2927, and 2966 cm1 are assigned to C—H stretching, and the peak at 3435 cm1 is assigned to the end OH groups of PHB. Similar observations were reported before by Barud et al.32 for PHB. In the case of CAP, a broad peak at 1745 cm1 refers to CdO stretching, the peaks at 2925 and 2855 cm1 refer to C—H stretching, and the peak at 3431 cm1 refers to O—H stretching. The peak at 1072 cm1 refers to C—O—C stretching, whereas that at 743 cm1 refers to ortho-substituted aromatic compound. Placebo microspheres of PHB/CAP blend show peaks related to both PHB and CAP, and no blend incompatibility was observed.

FTIR spectra of 5-FU, 5-FU-loaded PHB microspheres, and 5-FU-loaded blend microspheres are shown in Figure 5. In the case of 5-FU-loaded PHB microspheres, characteristic peaks of 5-FU at 3134 cm1 due to NH stretching, at 1731 and 1666 cm1 due to CdO stretching, and at 1248 and 814 cm1 due to CH in-plane and CH out-of-plane deformation are observed together with the characteristic peaks of PHB. The 5-FU-loaded microspheres of PHB/CAP blend show additional peaks related to 5-FU, indicating the absence of drugpolymer interactions in both 5-FU-loaded PHB microspheres and 5-FUloaded microspheres of the blend systems. 3.3. Differential Scanning Calorimetry. DSC curves of PHB, CAP, and PHB/CAP (1:1) blend placebo microspheres are shown in Figure 6. Sharp peaks at 176 °C (Tm1) and 299 °C (Tm2) are related to PHB melting and decomposition, respectively.32 A broad endothermic peak at 75.3 °C in the DSC curve of CAP is due to the presence of moisture. Comparatively, a sharp peak observed at 179 °C corresponding to its glass transition temperature,33 followed by the broad peaks at 231 and 275 °C are due to decomposition of CAP. Placebo microspheres of PHB/CAP blend show a peak at 174 °C, which is lower than both Tm1 of PHB and Tm of CAP, indicating a shift in the Tm1 of PHB, due to blending of the polymers. Another peak observed at 266 °C is lower than Tm2 of PHB, but both these peaks indicate the influence of the blend composition on the crystallization of PHB. Figure 7 shows the DSC traces of pure 5-FU, 5-FU-loaded PHB microspheres, and 5-FU-loaded PHB/CAP (1:1) blend microspheres. A sharp endothermic peak observed at 285 °C for pure 5-FU corresponds to its melting point. The DSC curve of 5-FU-loaded PHB microspheres shows melting endotherms at 10418



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Figure 7. DSC profiles of (a) pure 5-FU, (b) 30% (w/w) 5-FU-loaded PHB microspheres, and (c) 5-FU-loaded PHB/CAP (1:1) blend microspheres.

Figure 5. FTIR spectra of (a) 5-FU, (b) 30% (w/w) 5-FU-loaded PHB microspheres, and (c) 30% (w/w) 5-FU-loaded PHB/CAP (1:1) blend microspheres.

Figure 6. DSC profiles of (a) PHB, (b) CAP, and (c) PHB/CAP (1:1) blend placebo microspheres.

176 and 288 °C, indicating a slight lowering of the Tm2 value of PHB, suggesting the probable presence of drug in amorphous form within the microspheres. The DSC curve of 5-FU-loaded PHB/CAP blend microspheres shows endothermic peaks at 174 and 266 °C. However, no detectable endotherm corresponding to 5-FU is observed, suggesting that the drug is mixed at a molecular level in the microspheres. 3.4. Thermogravimetric Analysis. Results of the thermal behavior (TG curves) of plain PHB, plain CAP, and PHB/CAP (1:1) blend placebo microspheres are shown in Figure 8. The

Figure 8. TGA curves for the thermal decomposition of (a) CAP, (b) PHB, and (c) PHB/CAP (1:1) blend microspheres.

curve obtained for CAP (Figure 8a) shows three significant events of weight loss with increasing temperature. The first event occurred at 43 °C, which rose to 92 °C, due to a gradual weight loss of about 7% as a result of loss of moisture. The second event of weight loss occurred at 173 °C and continued to 228 °C with 34.5% weight loss, followed by the third event, which started at 331 °C and continued to 367 °C. During this phase, a weight loss of 49% was observed due to CAP pyrolysis, and around 10% residue was observed as carbonaceous matter. The TG curve for PHB (Figure 8b) shows only one event of weight loss starting at 272 °C that continues to 292 °C with a significant weight loss of 10419



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Figure 10. In vitro release profiles of 5-FU from PHB microspheres loaded with (Δ) 50%, (0) 40%, and (O) 30% (w/w) 5-FU at 37 °C.

Figure 9. XRD curves of (a) placebo PHB/CAP (1:1) blend microspheres and (b) 30% (w/w) 5-FU-loaded PHB/CAP (1:1) blend microspheres.

94% due to the thermal decomposition of PHB. The TG curve of the PHB/CAP blend microspheres (Figure 8c) containing a 1:1 ratio of PHB/CAP showed a composite of the events observed for CAP and PHB. A slight weight loss was observed starting around the ambient temperature due to residual solvent, followed by a significant weight loss of 90% starting at 249 °C that lasted up to 264 °C, which, in turn, was followed by another event of weight loss starting at 322 °C that lasted up to 353 °C, but with no residue after the analysis. 3.5. X-ray Diffraction. XRD was carried out to determine the state of 5-FU after formulation. The XRD of placebo and 5-FUloaded microspheres of the blend of PHB/CAP (1:1) are shown in panels a and b, respectively, of Figure 9. In the case of placebo PHB/CAP blend microspheres, two sharp peaks are observed at 2θ = 13.5° and 16.9°, but broader peaks are observed at 2θ = 19.9°, 21.5°, 22.5°, 25.5°, and 27.15°. These diffraction lines can be attributed to orthorhombic PHB lattice.34 The CAP is noncrystalline in nature,35 and even in PHB/CAP blend microspheres, no peaks related to CAP are observed. The 5-FU-loaded microspheres of PHB/CAP blend showed all of the peaks at the same 2θ values as observed in the case of placebo blend microspheres, but with much lower intensity. This fact, together with the absence of peak at 2θ = 28°, which is the characteristic peak of 5-FU, indicates the molecular-level dispersion of 5-FU within the polymeric matrix.35 3.6. Drug Loading and Encapsulation Efficiency. Results of percentage encapsulation efficiencies (EEs) for 5-FU-loaded PHB microspheres are presented in Table 1, wherein the theoretical drug loading and percentage EE values show a greater influence on the initial loading of 5-FU. As the initial amount of 5-FU in the microspheres increased from 18 to 30 mg per unit total dry weight of the polymer, the actual drug loading also increased from (9.01 ( 1.01)% to (11.74 ( 2.06)%, but the EE values decrease from (50.1 ( 4.0)% to (39.1 ( 3.1)%. The high DL values with a corresponding reduction in percentage EE might be due to the increased drug concentration in the

Figure 11. In vitro release profiles at 37 °C of 5-FU (30% theoretical loading) from PHB/CAP blend microspheres with compositions of (Δ) 1:3, (0) 1:1, and (O) 2:1.

microspheres, which might create a concentration gradient of the drug between polymer walls and the outer aqueous phase, leading to a greater loss of drug during solvent evaporation. The water-soluble 5-FU is somewhat difficult to encapsulate by the traditional oil/water (o/w) solvent evaporation method, but the speed of stirring influences the percentage EE values. The higher the speed of stirring during particle production, the smaller the particles with a reduced percentage DL. Hence, a moderate speed was maintained during preparation of the formulations. The 5-FU-loaded blend microspheres were prepared using three different ratios of PHB/CAP blends, namely, 2:1, 1:1, and 1:3 at 30% (w/w) of initial drug loading prepared by w/o/w solvent evaporation technique. The effect of polymer blend ratio on the percentage EE of 5-FU is presented in Table 2, wherein a higher percentage EE of 5-FU is observed at the PHB/CAP blend ratio of 1:3. With increasing concentration of CAP in the blend, a rapid formation of microspheres (hardening process) occurs, thereby minimizing the escape of 5-FU from the microspheres to increase the percentage EE. 10420



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Figure 12. Release mechanism of 5-FU from the PHB/CAP blend microspheres.

Table 3. Correlation Coefficients, r2, from Different Equations Used for 5-FU-Loaded Formulations KorsemeyerPeppas formulation codea

zeroth-order

first-order

Higuchi

HixsonCrowell

n

r2

F1 F2

0.997 0.994

0.998 0.999

0.999 0.999

0.997 0.994

1.195 0.747

0.994 0.997

F3

0.730

0.764

0.772

0.730

0.766

0.772

B1

0.996

0.999

0.999

0.996

0.942

0.996

B2

0.948

0.925

0.928

0.948

0.464

0.995

B3

0.992

0.960

0.981

0.992

2.522

0.995

a

F1, F2, and F3 are 5-FU-loaded PHB microspheres with 30%, 40%, and 50% theoretical drug loading, respectively, and B1, B2, and B3 are 5-FU-loaded PHB/CAP blend microspheres with compositions of 2:1, 1:1, and 1:3, respectively, with 30% theoretical drug loading.

3.7. In Vitro Drug Release. Results of in vitro release along with the error bars from the plain PHB microspheres are shown in Figure 10 for 2 h in simulated gastric medium, followed by the simulated intestinal medium for up to 24 h. Around 20% of 5-FU was released at the end of 2 h in acidic medium, followed by 70% release at the end of 24 h in alkaline medium. The release rate of 5-FU increases with increase in drug loading to the extent of 70% in 24 h. This could be due to the increase in concentration of the drug in the microspheres, thus developing a high concentration gradient, resulting in a fast release. However, nearly 30% of the 5-FU was not released within 24 h, because PHB forms a dense matrix. Similarly, in a previous study31 on PHB loaded with piroxicam prepared by the o/w emulsion solvent evaporation technique, ∼70% of the drug was released in 24 h. In our study, PHB was blended with a pH-sensitive CAP polymer to restrict the release of 5-FU at acidic pH to enhance its release at alkaline pH. Release data for PHB/CAP blend microspheres loaded with 5-FU with various blend ratios of PHB/CAP are presented in Figure 11. Around 12% of 5-FU was released in acidic medium at the end of 2 h, followed by around 72% release in alkaline medium in 24 h. As the CAP concentration increases [0.5%, 1%,

and 3% (w/w) CAP], the release rate also increases until leveling of at 71% and 72%. The blend microspheres, namely, formulations B2 and B3, showed a delayed release response in alkaline medium, facilitating the CR of 5-FU. Here, the addition of CAP to PHB makes the microspheres more porous upon exposure to alkaline pH as the CAP dissolves. This is schematically illustrated in Figure 12, which shows the possible mechanism of 5-FU release from the PHB/CAP blend microspheres. A dense polymer network is formed as a result of blending of PHB with CAP to form microspheres that remained intact in acidic medium, but allowed CAP to start leaching out of the matrix in alkaline medium, giving a porous structure. This mechanism is also supported by SEM images of the microspheres after dissolution, indicating a porous structure (Figure 3E,F). Porous microspheres are thus produced after the complete dissolution, suggesting that the incorporation of CAP into PHB produces microspheres that restrict the release of 5-FU at acidic pH, but with a maximum release at alkaline pH, due to the formation of a loose porous network. 3.8. Model Fitting. All of the release data were fitted to various empirical equations using PCP Disso v 3 software to evaluate the regression coefficients, r2. The values of r2 for 30%, 40%, and 50% 10421


Industrial & Engineering Chemistry Research (w/w) 5-FU-loaded PHB formulations, as well as all of the PHB/ CAP blend formulations, as analyzed by empirical equations are summarized in Table 3. A regression coefficient of 0.999 was observed for the Higuchi equation for formulation F1, indicating that drug release follows a square root of time relationship. However, an r2 value of 0.999 was observed for the Higuchi and first-order equations in the case of formulation F2, whereas for formulation F3, r2 = 0.772 for both the Higuchi and Korsemeyer Peppas equations, indicating a burst release. An r2 value of 0.999 approaching unity was observed for the first-order and Higuchi equations in the case of the B1 formulation, indicating that drug release follows a square root of time relationship for this matrix. An r2 value of 0.995 was observed for the KorsemeyerPeppas equation for formulation B2. For the latter, an n value of 0.46 was observed, suggesting a Fickian or quasi-Fickian mode of transport. The r2 = 0.995 was observed for the Korsemeyer Peppas equation with n > 1 for formulation B3, indicating that the drug release follows super case II transport.

4. CONCLUSIONS Poly(3-hydroxybutyrate) blend microspheres with a pH-sensitive property, induced by the presence of CAP, were prepared to investigate the effect of the concentration of CAP on the 5-FU release profile. Microspheres of PHB/CAP blends were successfully loaded with 5-FU, giving encapsulation efficiencies of 4257%. FTIR data for the individual polymers, drug, placebo matrix, and 5-FU-loaded blend microspheres suggest the absence of drugpolymer interactions. DSC data indicated the dispersion of 5-FU particles at a molecular level in the polymer matrix. TGA confirmed the thermal behavior and compatibility of the polymers in the blend. A molecular-level dispersion of 5-FU particles in the blend matrix was confirmed by XRD. The in vitro release profiles of 5-FU from the blend microspheres were compared with that of PHB microspheres to observe a higher release of 5-FU at alkaline pH than at acidic pH, suggesting the potentiality of PHB/CAP blend with a pH-sensitive character for the CR of 5-FU. The matrix released 5-FU mostly in the intestine, suggesting its potential for colon delivery. When analyzed with empirical equations, the in vitro release profiles suggested widely varying release patterns, depending on the type of equation employed. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (A.R.K.), aminabhavi@yahoo. com (T.M.A.). Tel.: +91-836-2448540. Fax: +91-836-2467190.

’ ACKNOWLEDGMENT K.C. acknowledges financial support from the Council of Scientific and Industrial Research [CSIR, Grant 21/(0760)/ 09/EMR-II], New Delhi, India, for a research fellowship. T.M.A. thanks CSIR for the award of Emeritus Scientist. ’ REFERENCES (1) Mundargi, R. C.; Babu, V. R.; Rangaswamy, V.; Patel, P.; Aminabhavi, T. M. Nano/micro technologies for delivering macromolecular therapeutics using poly(D,L-lactide-co-glycolide) and its derivatives. J. Controlled Release 2008, 125, 193. (2) Liang, H. F.; Chen, S. C.; Chen, M. C.; Lee, P. W.; Chen, C. T.; Sung, H. W. Paclitaxel-loaded poly(γ-glutamic acid)-poly(lactide)

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and Cellulose Acetate Phthalate for Colon Delivery of 5-Fluorouracil

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