Development of Polymeric Blend Microspheres from Chitosan-Hydroxypropylmethyl Cellulose for Controlled Release of an Anti-Cancer Drug
Development of Polymeric Blend Microspheres from Chitosan-Hydroxypropylmethyl Cellulose for Controlled Release of an Anti-Cancer Drug
Journal of the Korean Chemical Society. 2013. Aug, 57(4): 439-446
Copyright © 2013, Korea Chemical Society
  • Received : January 14, 2013
  • Accepted : July 10, 2013
  • Published : August 20, 2013
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About the Authors
Lakshmi C. Narayana, Reddy
Department of Chemistry, Sri Krishnadevaraya University, Anantapur, India.
Rama Subba P., Reddy
Department of Chemistry, Yogi Vemana University, Kadapa, India.
Krishna K.S.V., Rao
Department of Chemistry, Yogi Vemana University, Kadapa, India.
M.C.S., Subha
Department of Chemistry, Sri Krishnadevaraya University, Anantapur, India.
Chowdoji K., Rao
Department of Polymer Science& Tech, Sri Krishnadevaraya University, Anantapur, India.

Chitosan (CS) and hydroxypropylmethyl cellulose (HPMC) blend microspheres were prepared by water-in-oil emulsion technique and were loaded with an anti-cancer drug 5-fluorouracil (5-FU). CS-HPMC microspheres were characterized by Fourier transform infrared spectroscopy to confirm the cross-linking reaction. Scanning electron microscopy (SEM) was also used to assess the surface morphology of particles prepared. The quantity of release of 5-FU from the microspheres have been studied in terms of blend composition and amount of cross-linking agent. Differential scanning calorimetry and X-ray diffraction techniques indicated a uniform distribution of 5-FU particles in microspheres, whereas SEM suggested the spherical structure of the microspheres with slight rough surface. The in vitro drug release indicated that the particle size and release kinetics depend upon blend composition, amount of cross-linking agent used and amount of 5-FU present in the microspheres.
Polymeric drug-loaded matrices have several inherent advantages over the conventional dosage forms in optimizing patient treatment regimes. Controlled release (CR) systems are capable of delivering drugs at constant rates over an extended period of time. 1 In these systems, the rate of drug release is controlled by the balance between the drug diffusion across the concentration gradient and the polymer relaxation rate as a result of the diffusion-controlled process due to swelling. 2, 3 Swelling CR systems can achieve zero-order release 25 by modifying the structure-property relationship of the device. For the effective control of drug release over an extended length of time, understanding different types of transport processes is important. 25
Chitosan (CS), a (1-4)2-amino-2-deoxy-b-D glucan, has structural characteristics similar to glycosaminoglycans. This polycationic biopolymer is generally obtained by alkaline deacetylation of chitin, which is the main component of the exoskeleton of crustaceans, such as shrimps. 6 Recently, CS has been used in the biomedical field because of its favorable characteristics such as good biocompatibility and has been reported to be useful for pharmaceutical preparations. 7, 8 Among some interesting applications, use as a drug carrier in drug delivery systems (DDSs) is especially noteworthy. 9, 10 But CS’s main disadvantages for using in DDSs are that it is insoluble in common organic solvents except for dilute acetic acid and has low mechanical properties and also a high dependency of its physical properties on pH. Therefore, in the case of using CS as a drug carrier, especially for oral administration, it is difficult to control the drug-release behavior under various identified pH values of the internal organs of the human body. So, there is quite a possibility that over releasing of the drug may bring about ill effects in the human body. Hydroxypropyl methyl cellulose (HPMC) is modified cellulose soluble in water. It is a popular carrier material for swellable matrix tablets. 11 The drug release from HPMC-tablets can be modified by changing variables such as the viscosity grade or the particle size of HPMC, 12, 13 or the HPMC: filler ratio or HPMC: drug ratio, 1416 or the solubility of the filler 17 or by the addition of other hydrophilic polymers. 18 Depending on the solubility of the drug, the drug is primarily released either by diffusion through a gelled layer or erosion of this layer. 19, 20
Encapsulation for drug in to the micro structures is the standard process in the drug delivery devises. There are many types of mucoadhesive drug delivery systems such as gels, patches, films, and tablets for oral, buccal, nasal, ocular, and topical routes. Compare to conventional dosage forms, mucoadhesive micro structured devices are advantageous due to their controlled drug release more patient compliance and minimization of dosage. 2025 Mucoadhesive polymers are helpful to develop the micro particles with both properties such as mucoadhesive nature and microencapsulation for enhancement of drug adsorption in the gastrointestinal. Hence, in this report we prepared the CS and HPMC microspheres by simple water-in-oil emulation technique with high water swellability and high mechanical properties. CS-HPMC blend matrix microspheres are used for controlled release of 5-fluorouracil at 7.4 pH.
Chitosan (CS) (Degree of deacylation was 86.8%) was purchased from Aldrich chemicals, USA. Hydroxypropylmethyl cellulose (HPMC) sample used in this study was purchased from Himedia chemicals, Mumbai, India. The AnalaR grade samples of light paraffin oil, glutaraldehyde (25% aqueous solution) (GA), acetic acid, tween-80, hydrochloric acid were purchased from s.d. fine chemicals, Mumbai, India. 5-fluorouracil (5-FU) was received from Himedia Laboratores, Pvt., Ltd., Mumbai, India.
- Preparation of Blend Microspheres
CS-HPMC blend microspheres were prepared by W/O emulsion water-in-oil emulsion technique. Briefly, CS was dissolved 2% acetic acid solution under constant stirring over night. To this solution, required amounts of HPMC solution were added and stirred well. A required amount of 5-FU was added and stirred to obtain a homogeneous solution. The drug-loaded polymer blend mixture was emulsified by liquid paraffin oil (100 mL) with 1% (w/v) tween-80 taken in a 500 mL beaker and stirred at 400 rpm (REMI Motors, Vasai, India). To this mixture, different amounts of GA and 1 mL of 0.1 M HCl were added. Microspheres formed were collected, washed with hexane and water alternately, dried at room emperature for 24 h and stored in a desiccator until for further experimentation. Totally, eight formulations were prepared and the assigned formulation codes are given in 1 .
Results % of encapsulation efficiency different formulations and the release kinetics parameters of k, n and r for different Formulation codes
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S.D: standard deviation calculated 95% accurately. E.E: Encapsulation Efficiency
- Estimation of Drug Loading and Encapsulation Efficiency
The 5-FU loaded microspheres (10 mg) were pulverized and incubated in 10 ml 0.02M phosphate buffer (pH = 7.4) at room temperature for 24 h. The suspension was then centrifuged at 6000 rpm for 30 min. The supernatant was assayed spectrophotometrically for 5-FU content at the wavelength of 270 nm. The results of % drug loading and encapsulation efficiency were calculated as following equations
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- In Vitro Release Studies
In vitro release studies have been carried out by performing the dissolution experiments using a tablet dissolution tester (Model DS-8000, LabIndia, Mumbai, India) equipped with eight baskets. Dissolution rates were measured at 37 ℃ under 100 rpm speed. Drug release from the microspheres was studied in an intestinal (7.4 pH phosphate buffer) fluid like atmosphere. At regular intervals of time, sample aliquots were withdrawn and analyzed by UV spectrophotometer (Model LabIndia-3000 + , Mumbai, India) at the fixed λ max value of 270 nm.
- Characterization
FTIR spectral measurements performed (Nicolet, Model Impact 410, USA) to confirm the cross-linking reaction between CS and HPMC microspheres. Polymeric microspheres finely ground with KBr to prepare pellets under a hydraulic pressure of 600 kg/cm 2 and spectra scanned between 4,000 and 450 cm −1 . SEM images of the microspheres were recorded using a Hitachi S520 scanning electron microscope (Japan) at the required magnification. Working distance range of 8.5−9.5 mm was maintained and the acceleration voltage used was 20 kV with the secondary electron image (SEI) as a detector. Differential scanning calorimetric (DSC) curves were recorded (TA instruments Model: STA, Q600 USA) to confirm the uniform distribution of the drug. The samples were weighed between 10 and 12 mg and were heated from 0 to 400 ℃ at a heating rate of 10 ℃/min in nitrogen atmosphere (flow rate of 100 mL/min). X-RD study helps to find the crystallinity of drug in the microspheres. X-RD patterns of the pure 5-FU (a), plain CS-HPMC microspheres (b) and drugloaded microspheres (c) were recorded using a Rigaku Geiger flex diffractometer equipped with Ni-filtered CuK radiation (λ=1.5418 Å) and shown in the 4 . Dried microspheres of uniform size were mounted on a sample holder and X-RD patterns were recorded in the range 2−80° at the speed of 5°/min.
In the ever growing category of biodegradable polymers developed for biomedical applications. Several reports on chitosan drug delivery formulations with cellulose derivatives are reported in the literature. Sudha et al. developed an interpenetrating polymer network blend microspheres composed with hydroxyethyl cellulose for controlled release of isoniazid. 26 Ahmed et al. also designed semi IPN based on hydroxyethyl cellulose grafted with poly acrylamide with chitosan microspheres for drug delivery application. 27 Chitosan and methyl cellulose based microsphere formulations also developed by Ajit et al. 28 However, the blend of chitosan and hydroxypropyl cellulose are advantageous because HPMC is the most important hydrophilic carrier material used for the preparation of oral controlled drug delivery system compared to other celluloses like hydroxyethyl cellulose and methyl cellulose. The most important characteristics of HPMC are high swellability, which has a significant effect on the release kinetics of an incorporated drug. Upon contact with water or biological fluid the latter diffuses into the device, resulting in polymer chain relaxation with volume expansion. 29, 30 Then, the incorporated drug diffuses out of the system. The objective of our study was to prepare 5-FU loaded microspheres for enhancement of bioavailability and to deliver effective concentration of drug at the site of action. Most of the oral drugs presently available in use fail to achieve required effective concentration in the interstainal tract. In this study chitosan, hydroxypropyl methylcelluloses are used to prepare microspheres. Microspheres using combination of these polymers were also tried and evaluated. These polymers used are regarded as safe by FDA and posses good mechanical properties which might help the oral delivery of microspheres to get for longer period and release the drug in sustained manner.
- Characterization of Microspheres
The FTIR Spectrum of pure CS (A) plain CS/HPMC microspheres (B) and drug loaded CS/HPMC Polymer (C) are indicated in 1 . In case of CS, a broad band at 3435 cm −1 is attributed to −OH stretching vibrations. Band at 2982 cm −1 N−H stretching vibrations and the peak at 2960 cm −1 represent aliphatic C−H stretching vibration. Three bands observed at 1649, 1594 and 1379 cm −1 indicate amide-I, amide-II and amide-III, respectively. In case of placebo microspheres, all the bands of both CS and HPMC were observed in addition to a new band observed at 1031 cm −1 , which confirmed the C−N stretching vibration of the acetyl group, which is formed due to the reaction of GA with hydroxyl groups of HPMC. Thus, FTIR confirms the crosslinking reaction of GA with CS and HPMC. The crosslinking reaction between CS and HPMC showed in 1 . In case of drug loaded microspheres all the bands that were observed in plain microspheres indicating the chemical stability of microspheres after encapsulation of drug into the polymer matrices.
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FTIR curves of pure chitosan (A), plain CS-HPMC blend microspheres (B), drug loaded CS-HPMC microspheres.
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Schematic crosslinking chemistry of GA crosslinked CS-HPMC blend.
2 shows SEM micrographs of 5-FU loaded HPMC/Chitosan microspheres. In all cases, the particles were almost spherical in nature and aggregated with rough surfaces, as is evidenced by SEM photographs shown in 2 . Visibly porous structures with the presence of no drug crystals were present on the surfaces of the microspheres.
DSC scans of pure drug (curve-a), plain microspheres (curve-b) and drug loaded microspheres (curve-c) in 3 showed a systemic trend in their glass transition temperatures. 5-FU shows a sharp endothermic peak was observed at 285.16 ℃ due to polymorphism and melting, but in case of 5-FU loaded microspheres, no characteristic peak was observed at 285.16 ℃, suggesting that 5-FU is molecularly dispersed in the blend microspheres. The most intensive peak of 5-FU is observed at 2θ of 38° suggesting its crystalline nature. But, this peak does not exist in drug loaded semi-IPNs plain CS-HPMC micrograms and in the, indicating that the drug is masked at uniform molecular level distribution in the polymer matrix. These X-RD curves are displayed in 4 .
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Scanning electron micrographs (A) and (B) both are CS-HPMC blended microspheres.
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DSC thermograms of pure drug (a), placebo microspheres (b), and 5-FU loaded microspheres.
- Encapsulation Efficiency
Results of % encapsulation efficiency (%EE) presented at 1 . Three different concentrations of 5-FU i.e., 5, 10 and 15 wt% were loaded in the polymeric microspheres during the crosslinking of microspheres. It shows increasing trends with increasing drug loading in the microspheres. For all formulations %EE noted from 42.2 to 59.0%. Such smaller values obtained are due to a hydrophilicity of drug, thus incorporating a lesser amount of 5-FU into microspheres. %EE increased with increasing amount of HPMC in the polymeric microspheres shown in 1 . For microspheres containing CS/HPMC ratios are 3:1, 1:1 and 1:3 and 10 wt% 5-FU with 5 mL of GA, encapsulation efficiencies were 46.8, 47.4 and 58.3%, respectively. For the ratios of 1:1 of chitosan and HPMC in the matrix, the results on extent of crosslinking on the size have increased, and then the %EE decreased. For microspheres crosslinked with 2.5, 5 and 7.5 mL of GA, encapsulation efficiencies is, respectively 59.0, 47.4 and 42.2%. Such a decreasing trend is due to an increase in crosslink density, because the microspheres will become rigid, thereby reducing the free volume spaces within the polymer matrix and hence, a reduction in encapsulation efficiency is observed.
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X-RD spectra of (A) pure drug, (B) pristine CS-HPMC microspheres and (C) drug loaded CS-HPMC microspheres.
- Release Kinetics Parameters of Different Formulations
Drug release kinetics was analyzed by plotting cumulative release data versus time and by fitting these data to the exponential equation of the type. 31
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Here, Mt/Mα represents the fractional drug release at time t , k is a constant characteristic of the drug-polymer system, and n is an empirical parameter characterizing the release mechanism. Using the least squares procedure, we have estimated the values of n and k for all the seven formulations, and these values are given in 1 . If n = 0.5, the drug diffuses and releases from the polymer matrix following a Fickian diffusion. If n > 0.5, an anomalous or non-Fickian type drug diffusion occurs. If n = 1, a completely non-Fickian or case II release kinetics operative. The intermediary values ranging between 0.1 and 0.5 are attributed to the Fickian diffusion type transport.
The values of k and n have shown a dependence on the extent of cross-linking, % drug loading, and HPMC content of the matrix. Values of n for microspheres prepared by varying the amount of HPMC in the microspheres of ratios of 3, 1 and 1/3 by keeping 5-FU (10%) and GA (5 ml) constant, ranged from 46.4 to 58.3%, leading to the drug diffuse and release from the polymer matrix following almost non-Fickian (or) case-II type diffusion.
- Effect of Cross-Linking Agent
The % cumulative release data vs time plots for varying amounts of GA i.e., 2.5, 5.0 and 7.5 mL at the fixed amount of the drug (5%) are displayed in 5 . The % cumulative release is quite fast and large at the lower amount of GA (i.e., 2.5 mL), whereas the release is quite slower at higher amount of GA (i.e., 7.5 mL). The cumulative release is somewhat smaller when lower amount of GA was used. At higher concentration of GA, polymeric chains become rigid due to the contraction of micro voids, thus decreasing % cumulative release of 5-FU through the polymeric matrices. As expected, the release becomes slower at higher amount of GA, but becomes faster at lower amount of GA.
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% of cumulative release of 5-FU at different amounts of drug content: (▲) 5% 5-FU; (■) 10% 5-FU; (●) 15% 5-FU.
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% of cumulative release of 5-FU at different amounts of cross linking agents: (▲) 2.5 mL GA; (●) 5 mL GA; (■) 7.5 mL GA.
- Effect of Drug Loading Content
6 . shows the release profiles of 5-FU-loaded chitosan-HPMC microspheres at different amount of drug loadings. Release data showed that formulations containing the highest amount of drug (15%) displayed fast and higher release rates than those formulations containing a small amount of 5-FU. A prolonged release was observed for the formulation containing lower amount of 5-FU. In other words, with a decreasing amount of drug in the matrix, it is noticed that the release rate becomes quite slower at the lower amount of drug in the matrix, and this is due to the availability of more free void spaces through which lesser number of drug molecules will transport. For all formulations the 5-FU release was observed upto 600 min in a controlled manner.
- Effect of HPMC Content
Effect of HPMC content on encapsulation efficiency and in vitro release of 5-FU was investigated. In vitro release profiles of 5-FU from formulations prepared by taking different amounts of HPMC and 20% of 5-FU are shown in 7 . Faster release rates were observed from formulations prepared with higher amount of HPMC (i.e., 80%) observed than those formulations prepared using a lower amount of HPMC i.e., 40%. About 98% of the drug was released within the first 6 h from formulations prepared with higher amount of HPMC; where as only 77% of 5-FU was released within 5 h from formulations prepared with a lower amount of HPMC. Faster drug release observed from formulations prepared with higher particles.
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% of cumulative release of 5-FU at different ratios of CS-HPMC content: (▲) 3:1; (■) 1:1; (●) 1:3.
Crosslinked chitosan (CS)-hydroxy propylmethylcellulose (HPMC) blend microspheres were prepared by waterin-oil emulsion technique. 5-Fluorouracil was successfully loaded into these microspheres and encapsulation efficiency was found to be 42.2 to 59.0%, depending upon the blend composition, crosslinking agent used and the amount of drug loading. The FTIR analyses were carried out to confirm the crosslinking of CS-HPMC blend microspheres. Scanning electron micrographs of the microspheres showed the formation of non-uniform spherical microspheres. X-RD and DSC studies on the microspheres indicated a molecular level dispersion of the drug in the semi-IPN matrix. The in vitro dissolution studies performed in pH 7.4 buffer medium have shown that release of 5-fluorouracil is dependent upon the amount of drug loaded, polymer composition and crosslinking. 5-fluorouracil is released in a sustained and controlled release manner from the blend microspheres up to 10 h.
Authors Dr. K.S.V. Krishna Rao and Mr. P. Rama Subba Reddy highly thankful to the Department of Science and technology (DST), New Delhi, India, (DST No.SR/FT/CS-047/2009) for a financial support. And the publication cost of this paper was supported by the Korean Chemical Society.
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