International Journal of PharmTech Research

CODEN (USA): IJPRIF ISSN : 0974-4304

Vol.2, No.2, pp 1112-1118, April-June 2010

Formulation and Evaluation Porous Microspheres of

5- Fluorouracil for Colon Targeting
S. C. Dhawale1, A. S. Bankar2, *M. N. Patro3
Govt. college of Pharmacy, Karad - 415 124 , Dist- Satara, Maharashtra, India

*Corres.author: nagarajupatro@gmail.com

ABSTRACT: The treatment of colon cancer has been aimed by approaches of oral drug administration. 5-Fluorouracil
is a candidate to be delivered orally to the colon; pH - sensitive polymers Eudragit S 100 and L 100 were used to prepare
microspheres by a simple oil /water emulsification process. Process parameters were analyzed in order to optimize the
drug loading and release profiles. In further attempts mixtures with Eudragit S100 and L100 were prepared to prolong
drug release. Scanning electron microscopy permitted a structural analysis. The solvent extraction was preferable over
solvent evaporation with a view to the encapsulation rate (extraction: 37%; evaporation: 19%) due to the hydrophilic
character of the drug while release pattern were nearly unchanged. Eudragit S100, pure or in mixture, was found to
retain drug release at pH 4.5 lower than 41% within 6 h. At pH 7.4, nearly immediate release (within 30 min) was
observed for pure S100, while mixtures enabled to prolong the release slightly. Analysis of the morphology led to an
inhomogeneous polymer distribution of S100 and L 100 throughout the particle core. However, the formulation proved
its applicability in-vitro as a promising device for pH-dependent colon delivery of 5-fluorouracil. The study was aimed
at develop the porous microspheres, which can control the drug release up to 6 h and hence it can prevent the acid
decomposition in stomach.
KEYWORDS: Colon delivery; colorectal cancer; Microspheres; 5-Fu.

INTRODUCTION Moreover, the so-called ‘streaming’ is in favor of the

Colon cancer is the second most cause of development of smaller drug carriers providing longer
death after lung cancer by cancer diseases. Many presence in the large bowel.
different drugs or drug combinations have been tested A colon specific drug delivery by microsphere
for a successful therapy. 5-Fluorouracil (5-FU) is a formulations has been aimed for several anti-
commonly applied anticancer drug in the treatment of inflammatory drugs [9–12]. For the preparation of
colon cancer [1]. At present, the standard regimen is an these microspheres the pH-sensitive polymers
intravenous bolus injection of 5-fluorouracil (5- FU) EudragitS and L were applied. Usually the preparation
modulated by folinic acid (leucovorin) [2, 3]. Only few methods consisted of use of O/O emulsification
approaches for an oral administration have been process where EudragitS or L are dissolved in acetone
described in literature. Recently, enzyme dependent / 2-propanol mixtures and emulsified in liquid paraffin
tablet-based systems have been proposed, which might [10–12].
allow an efficient treatment combined with a reduction These systems were developed for an oral drug
of adverse effects [4]. However, due to variations in delivery in the treatment of ulcerative colitis.
transit time throughout the colon, the drug release can However, the pH-dependent release properties of such
be incomplete when the colon specific tablet matrix is a system also seem to be applicable to a potential local
not readily disintegrated and the treatment will remain treatment in colon cancer. Therefore, this work aimed
insufficient. Especially, diarrhea has been observed as the development of an oral delivery system for 5-
of one of the major adverse effects and also a toxicity fluorouracil which allows the release of the anti cancer
limiting factor of the therapy [5–7]. This can turn oral drug locally in the colon in dependence of luminal pH.
5-FU treatment insufficient. A size reduction of the This may reduce the systemic toxicity which is mainly
carrier system might be an option in order to the limiting factor in the treatment of colon cancer and
circumvent those problems, since size dependent allows a local dose increase compared to existing
gastrointestinal retention has been observed earlier [8]. treatment strategies.
M.N.Patro et al /Int.J. PharmTech Res.2010,2(2) 1113

EXPERIMENTAL: electron microscope (JEOL JSM-T330A scanning

Materials and methods microscope, Tokyo, Japan) at 10 kV.
Materials: Differential scanning Calorimetry (DSC) analysis
5-FU was kind gift from Biochem [15]
Laboratories, Mumbai. EudragitS100 and EudragitL Differential scanning calorimetric (DSC)
100 gift sample from Lupin Research Park, Pune. analyses of the 5-Fu and polymer were carried out
Polyvinyl alcohol (M 20 000 Da, 80% hydrolyzed), by using differential scanning calorimeter equipped
methylene chloride and other chemicals were obtained with computer analyzer (Shimadzu TA–60
were of analytical grade. differential scanning calorimeter, Shimadzu
Corporation, Kyoto, Japan). Samples (3-7 mg) were
Methods: heated under nitrogen atmosphere on an aluminum
Formulation Design: pan at a heating rate of 10 °C/min over the
Formulations of 5-flurouracil microspheres temperature range of 30-300 oC.
containing different concentration of Drug: Eudragit Powder X- ray diffraction studies (PXRD) [16]
were prepared using Factorial design and grouped as Powder X-ray diffraction (PXRD) patterns
Batch A, B and C shown in Table 1. were traced employing X-ray diffractometer (Philips
Preparation of microspheres: [13] PW 1729, Analytical XRD, Holland) for the samples
The preparation of microspheres was based on using Ni filtered CuK(α) radiation (intensity ratio(α1/
an O/W emulsification solvent evaporation. It was α2): 0.500), a voltage of 40 KV, a current of 30 mA
optimized as follows: a total polymer was dissolved in and receiving slit of 0.2 inches. The samples were
8 ml methylene chloride and Ethanol (3:1). Then, 5- analyzed over 2q range of 5.010-39.990o with
FU crystals (diameter [D]: 32.763.9 mm) were scanning step size of 0.020 o (2q) and scan step time
suspended by ultrasonication in polymer solution. This of one second.
suspension was poured into 75 ml of 1% w/w PVA
and O/W type of emulsion was formed by extensive Production Yield and Percentage drug entrapment
stirring with a three blade propeller at 500 rpm. When [14]
solvent evaporation was applied, the system was kept Emulsion- Solvent evaporation method offers
under agitation until methylene chloride was a versatile, easy and practical method for
evaporated. After decantation, the microspheres were manufacturing of Microspheres. Production yield is
filtered (HVLP filter, Millipore, pore size 0.45 mm), expressed as percent yield with respect to initial
washed extensively with distilled water and amount of drug and polymer.
lyophilized overnight.
Scanning electron microscopy: [14]
The external and internal morphology of
microspheres was analyzed by scanning electron
microscopy (SEM). The microspheres were fixed on
supports with carbon-glue, and coated with gold using
a gold sputter module in a high-vacuum evaporator.
Samples were then observed with the scanning
Table 1: Formulation Design Using Factorial Design

Drug : Polymer Ratio Formulation Code Polymer

1:1 A1
1 : 1.5 A2 Eudragit S100
1:2 A3
1 : 2.5 A4
1:3 A5
1:1 B1
1 : 1.5 B2 Eudragit L100
1:2 B3
1 : 2.5 B4
1:3 B5
1:1 AB1
1 : 1.5 AB2 Eudragit S100 : L100
1:2 AB3 (50:50)
1 : 2.5 AB4
1:3 AB5
M.N.Patro et al /Int.J. PharmTech Res.2010,2(2) 1114

Solvent evaporation and extraction:

Entrapment was determined by taking a weighed
A smoother surface was obtained for microspheres
quantity of preformed microspheres (approximately 25
from the evaporation process, while the extraction
mg) with phosphate buffered of pH 7.4 (10 ml) was
procedure led to less regular shaped particles with a
added in a vial. The solution was stirred vigorously for
procedure led to less regular shaped particles with a
24 hrs with mechanical stirrer. Supernatant was
generally in line with results about other polymers
collected by centrifugation and drug content in
which was reported to be due to the rapid solvent
supernatant was determined by using UV
dislocation in case of using an extraction method
spectrophotometer at suitable wavelength (266nm).
[16,17]. The long term contact with the external
Efficiency of drug entrapment for each batch was
aqueous phase in the evaporation process might allow
calculated in term of percentage drug entrapment as
a prolonged diffusion of the hydrophilic 5-FU out of
per the following formula [14]
the organic phase during the preparation procedure and
subsequently cause an intense drug loss. The drug
% Drug entrapment= release showed generally similar behavior at the
different pH. 5-FU was retained efficiently inside the
Practical content C 100
microspheres when tested in in-vitro buffer systems at
Theoretical content
pH 1.2 and 4.5 maintaining at least about 70 and 65%
In vitro drug release [17, 18] of the initial drug load after 6 h of incubation. A
The drug release rate from microspheres was comparatively fast release was observed at pH 7.4,
determined using USP XXIII paddle type dissolution which delivered about 100% of the incorporated drug
apparatus (Lab India Disso 2000, Lab India Ltd., within 60 min.
Mumbai). A weighed amount of microspheres When comparing evaporation and extraction
equivalent to 50 mg drug was filled into a capsule (# 3) process, a slightly faster drug release occurred after
and placed in the basket. Hydrochloric acid buffer (pH solvent evaporation for pH below 6.8. Since usually
1.2), Acetate buffer (pH 4.5), Phosphate buffer pH 7.4 smoother particles have been reported to release
was used as the dissolution medium and maintained at slower [17], the closer location of the drug crystals to
37 ± 2°C at a rotation speed of 100 rpm. Perfect sink the particle surface might be a reason. The rather
conditions prevailed during the drug release studies. 5 hydrophilic 5-FU diffuses towards the external
ml sample was withdrawn at each interval, passed aqueous phase and due to the longer contact with the
through a 0.45 μm membrane filter (Millipore), and external aqueous phase during the evaporation process
analyzed by UV method to determine the such a location near the interface might be intensified.
concentration of drug present in the dissolution The drug release occurs in two step at this pH where
medium at 266 nm. The initial volume of the the matrix is persistent (dissolution of the drug crystals
dissolution fluid was maintained by adding 5 ml of in the particle matrix followed by the diffusion), the
fresh dissolution fluid after each withdrawal. higher drug load with the extraction method may,
therefore, also be responsible for a slower drug release.
RESULTS AND DISCUSSION Moreover, also the slight differences in the particle
Eudragit S100 and L 100 belong to the pH- diameter can have an influence on the drug release as
sensitive Eudragit group of polyacrylates, exhibiting a the microspheres after solvent evaporation are smaller,
dissolution threshold pH slightly above 7.2 [19]. These exhibiting a larger surface for an enlarged drug
properties are based on its structure, synthesized by diffusion.
copolymerization of methacrylic acid, methyl acrylate
and methyl methacrylate. One major advantage of the pH dependent drug release:
polymer is the fact that it can be dissolved in The pH-dependent approach appears to have the
methylene chloride. This makes the polymer lowest risk for treatment failure as compared to other
applicable to different microsphere preparation delivery strategies, especially for Crohn’s disease.
methods replacing the complicated oil / oil Thus, although there is a considerable benefit in the
emulsification process used in prior formulations [10– new drug delivery approaches utilizing
12]. It seems to be very advantageous to circumvent microparticulate approach. The colon based on pH-
the unpleasant handling of the external oil phase sensitive release is conventional and includes all
including the long-term solvent evaporation under possible drawbacks of this strategy.
vacuum and the complex washing steps producing a Phosphate buffer of pH 7.4, 0.1 N HCl and
distinct quantity of additional solvent waste. Phosphate buffer of pH 4.5 were used to evaluate the
decomposition of 5-FU. The results obtained from
M.N.Patro et al /Int.J. PharmTech Res.2010,2(2) 1115

dissolution studies of the optimised formulation are formulations showed a high drug content in final
graphically represented in Fig 1, Fig 2 and Fig 3. formulation as compared to limited drug loading for
any other sophisticated formulations like nano
Scanning electron microscopy: particles, emulsifications, liposomes, micelles etc.
The results obtained from SEM studies
confirmed the porous and spherical structure of Drug release studies
microspheres (fig 4). Moreover, morphology of In-vitro dissolution studies were performed
microspheres revealed that degree of porosity of in three different dissolution media. Hydrochloric
microspheres was dependant on the composition of acid (0.1 N), Acetate buffer (pH 4.5) Phosphate
Eudragit present in the microspheres. High content buffer (pH 7.4) were used for in-vitro drug
of Eudragit led to less porosity. There was no dissolution study. Phosphate buffer of pH 7.4 were
porosity appearing in microspheres with above 200 used as the colonic pH is around 6.8 to 7.4 and
mg of Eudragit in formulation, whereas less than 100 hydrochloric acid buffer of pH 1.2 was used to study
mg content of Eudragit resulted in loss of spherical whether the decomposition of 5-Fu takes place in
structure and mechanical strength. acidic pH or not. Microspheres containing 30 mg
equivalent of 5-Fu were subjected to this study to
Differential scanning Calorimetry (DSC) analysis maintain the sink condition throughout the study.
DSC was taken by scanning formulation from Dissolution studies were carried out over the period
30 to 300oC with heating rate of 2K/min. A sharp of 3h for hydrochloric acid buffer and for 6 hr for
curve at 283.20oC gives exact phase transition acetate buffer and phosphate buffer, which mimic
temperature and reveals stability of formulation at the gastric emptying time. All the prepared
37oC. microspheres showed better controlled release
profile. Drug to Eudragit ratio of 1:2 of Batch A
Powder X- ray diffraction studies (PXRD) shown 101% release within 6 h in phosphate buffer
X-ray diffraction pattern for pure drug (pH 7.4)
indicated crystalline form of 5-FU. Sharp peak The in-vitro drug release studies showed that
between 28.385o2θ to 28.525o2θ was characteristic of about 25 % to 30 % drug released in acidic pH (pH
5-FU. In drug loaded microsphere formulation, the 1.2), about 40 % to 45 % drug released at pH 4.5 and
sharp peaks of pure 5-FU was not observed. It 88 %to 101 % drug release at pH 7.4 (Colonic pH).
indicates that there may be partial or complete
transitions of crystalline state to amorphous state. CONCLUSION
A colon specific microparticulate drug
Production Yield and Percentage drug entrapment delivery system can be prepared by a simple and fast
Entrapment efficacy of drug loaded o/w emulsification method. A relatively high drug load
microspheres was found to be in the range of 24.67 - even for the hydrophilic drug 5-FU can be achieved. A
41.23 %. Entrapment efficacy was dependent on the pH-dependent release can be provided in a range of pH
composition of drug as well as Eudragit. Low 6.8, where the main drug load is retained, and pH 7.4,
content of Eudragit and high drug composition in where a fast dissolution of the carrier occurs. When
formulation led to reduce the entrapment of drug in polymer combinations were applied for the
microsphere formulations. Drug to Eudragit ratio of microsphere preparation (Eudragit S100 and Eudragit
1:2 yielded maximum entrapment efficacy of 41.23 L 100) at different mixing ratios, only minor
%. Whereas, minimum entrapment efficacy of 24.67 influences on particle size and drug load are observed.
% was observed with 1:3 ratio of drug to Eudragit. These simple polymer mixtures can prolong the release
However, 5-Fu being high solubility drug with low only for a relatively short period due to the exceptional
dose required high concentration of polymer and/or structural arrangements of the carrier system.
excipients in dosage form for better formulation However, this new formulation is a good candidate for
development. an application in oral delivery for colon cancer. It has
Drug content of all the batches were to be proven in-vivo whether these relatively small
found to be in the range of 24.67 % - 41.23 % so the property changes have an impact on the colon delivery.
M.N.Patro et al /Int.J. PharmTech Res.2010,2(2) 1116

Fig. 1: Comparative % drug release profile

of Batch A in Phosphate buffer, pH 7.4

Fig. 2: Comparative % drug release profile

of Batch A in Phosphate buffer, pH 7.4

Fig. 3: Comparative % drug release profile

of Batch A in Phosphate buffer, pH 7.4
M.N.Patro et al /Int.J. PharmTech Res.2010,2(2) 1117

Fig 4: SEM images of empty Microsphere Fig 5: SEM images of Microspheres of Batch A

Acknowledgements transit rate of different-sized model dosage

The authors would like to thank Biochem forms through the human colon and the effects
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GCOP, Karad for providing necessary facilities. core carriers of 5-aminosalicylic acid, Pharm.
Res., 1992, 9, 1128–1131.
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