4.1. The Results of Microencapsulation Containing Different Polymers
The hydrophilic substances encapsulated in polymeric microspheres are commonly released following a pattern of three main steps. First, the burst release phase, usually occurring during the first day and mainly determined by the drug in the surface, channels and pores of the microspheres, which were filled by the incubation media for a few hours at the beginning of the trail. Secondly, the slow release phase, releasing few or no drug at all. The third and the last phase comprises a faster release of drug due to the erosion of particles. Occasionally, the release can occur in two steps and the profile shows an asymptotic pattern.
Several processes contribute to the release of the encapsulated drugs, such as diffusion through pores and channels, and exposure of drug molecules to the incubation media, due to the superficial erosion polymeric matrix. The cannels and pores are formed during the assembly of the particles or result from polymeric degradation. Therefore, factors influencing the release profile include the properties of the polymeric matrix, and the drug used in the structure of the microparticle, the encapsulation technique and the experimental conditions, as well as the coencapsulation of additives for several purposes. Emulsification solvent extraction/evaporation involves two steps. The first step requires emulsification of the polymer solution into an organic phase (
Figure 1). During the second step polymer solvent is extracted/evaporated, including polymer precipitation of microparticles. A polymer organic solution containing the dissolved drug is dispersed into microparticles, using a dispersing agent; the solvent is subsequently extracted/evaporated by increasing the temperature under pressure or by continuous stirring ( 13). The size can be controlled by adjusting the stir rate, type and amount of dispersing agent viscosity of organic and aqueous, and temperature ( 14). The preparatory parameters are summarized in Table 1. Higher actual drug loading were obtained by increasing the theoretical drug loading. In cases, the encapsulation efficiencies acrylate methacrylate copolymer as Eudragit RL and RS were 73.9-87.21% and the encapsulation efficiencies derivatives cellulose as EC and CAB were similar and greater than 90.64-95.9%. Depending on therapeutic requirements, microspheres with varying drug contents could therefore be prepared through variation of the theoretical drug loading.
Table 1 Effect of drug: polymer ratio, stirring rate, dispersing medium and non-solvent on the content, production yield and particle size with different type of polymers in theophylline microparticles
Formulations Emulsion method Polymer type Drug/polymer ratio Production yield, % ± SD Theoretical drug content, % ± SD Mean amount of drug entrapped, % ± SD Drug loading efficiency, % ± SD Mean particle size, μm ± SD F RS O/O E u d r a g i t 5 :1 81.7 ± 3.79 14.29 12.21 ± 0.04 87.21 ± 0.28 260.37 ± 1.69 RS F EC W/O/O EC 0.5 : 1 55.24 ± 1.19 33.33 29.53 ± 4.92 90.64 ± 1.32 757.01 ± 2.72 F CAB O/O CAB 0.75 : 1 45.4 ± 0.45 43 41.10 ± 0.40 95.9 ± 0.95 273.6 ± 1.73 F RL O/O E u d r a g i t 4 : 1 59.1 ± 0.65 80 59.1 ± 0.25 73.9 ± 0.16 372.4 ± 1.70 RL 4.2. TH Loading Efficiency Obtained From Single/Double Emulsion
4.2.1. Extraction/Evaporation Technique
The TH entrapment efficiency was calculated as a percentage percentage of drug entrapped ratio in the microspheres to the initial amount of drug added to the system. Results are indicated in
Table 1. It was found that the TH entrapment efficiency was rather low (< 73.9 % for Eudragit RL than Eudragit RS with 87.21%). The entrapment efficiency of Eudragit RS100 microspheres was higher than that of the Eudragit RL100 microspheres. This behavior can be explained on the basis of differences of the chemical structures and the % content of quaternary ammonium groups. Eudragit RL100 contains higher amount of quaternary ammonium groups (10%), which facilitates the diffusion of a part of entrapped drug to the surrounding medium during preparation of microspheres. Eudragit RS100 has a thick polymeric surface due to the lower amount of quaternary ammonium groups (5%), which restricts the migration of drug particles to the surrounding medium and also helps to sustain the drug. TH is slightly soluble in water and insoluble in the organic phase in which the EC, Eudragit RS and RL, CAB was dissolved. As a result, TH dispersed in the polymer solution will be extracted by the external phase. However, taken together, the entrapment efficiency of TH using the single emulsion preparation (O/O) was suitable (73.9-95.9%) for practical applications and that TH was found back almost quantitatively (90.64%) in double emulsion preparation with EC polymer. The results suggest that the single emulsion technique (O/O) is suitable for preparation of TH-loaded Eudragit RS, RL and CAB microspheres. Therefore, an alternative method, namely the double emulsion solvent extraction/evaporation method, was investigated to prepare TH-EC microspheres with a high loading efficiency. 4.3. Particle Size
The average particle size was determined by laser light scattering particle size analyzer (SALD-2101, Shimadzu, Japan). The microspheres were observed to be 260.37, 757. 273.6 and 372.47µ for Eudragit RS, EC, CAB and Eudragit RL respectively. The particle size of the microspheres obtained by acetonitrile and dichloromethane was much larger than those obtained by methanol and acetone/only acetone. It had been previously reported that using acetone as a co-solvent decreased the particle size (
15). In the current study, addition of acetone to methanol also decreased the size of TH-loaded Eudragit RS microspheres (260.37 µ). Acetone is water-miscible while dichloromethane is water-immiscible. Acetone is miscible with methanol as well as dichloromethane. Consequently, the addition of acetone to methanol increases water solubility of the halogenated solvents resulting in an extraction of the solvent by the external phase. Due to the solvent extraction, an interfacial turbulence occurs between the organic polymer phase and the external phase leading to the formation of small particles. 4.4. In vitro Release Studies
Dissolution rate of polymer coat determines the release rate of drug from the microcapsule when the coat is soluble in the dissolution fluid. Thickness of coat and its solubility in the dissolution fluid influence the release rate. The polymer coat of microcapsule acts as semi-permeable membrane and allows the creation of an osmotic pressure difference between the inside and the outside of the microcapsule and drives drug solution out of the microcapsule through small pores in the coat. The drug release behavior of microsphere formulations and tablet SR (200 mg) are shown in
Figure 3, respectively. TH in vitro release from microspheres containing EC, Eudragit RS, CAB and Eudragit RL exhibited initial burst effect which may be due to the presence of some drug particles on the surface of the microspheres. Table 2 shows the dissolution efficiency and difference factor values for microsphere formulations dissolution profiles and tablet SR. Dissolution efficiency and difference factor were used to compare the potential parameters and evaluate the dissolution profiles of different products. Comparison of various dissolution profiles is analyzed by several special measures including the dissolution Rel 2 (amount of drug release after 2h), Rel 8 (amount of drug release after 8h), efficiency (DE %) and the difference factor (F 1) ( 8). The difference factor is used to determine whether the test product is different to the reference products. An F1 value higher than 0% means that the average difference between both dissolution profiles is less than 15% at all sampling points indicating difference of the two products ( 16). The DE value for the total time profile of 1440 minute indicated higher dissolution efficiency for the F EC compared to commercial tablet SR and other microspheres. Further, F 1 (%32.59, %67.91, %19.91 and %30.44) for F EC, F RS, F CAB and F RL, respectively showed difference in the dissolution profiles between their microspheres and tablet SR. The difference between DE values at 1440 minutes was statistically significant (P < 0.05).
Microspheres with high loading efficiency (F CAB and F RL formulations) showed lower dissolution rate for Q2h (6.45% and 7.41%, respectively). Figure 3 and Table 2 indicated that the initial drugs release for some of microsphere formulations were slightly high (F RS and F EC). F CAB and F RL formulations showed the lowest burst release in comparison with theophylline SR. The burst release could be attributed to the presence of some TH particles on the surface of microspheres. When particles are prepared by O1/O2 or W/O1/O2 method, Water-soluble drugs do not have the tendency to migrate to the non-polar medium, thereby concentrating on the surface of the microspheres lead to burst effect. Moreover, the burst release could also be explained by the imperfect encapsulation of the drug inside microparticles, resulting from the unstable nature of the emulsion droplets during the solvent removal step. This potential instability may cause a part of the loaded drug to relocate at the microparticle surface, thereby would be rapidly released. Figure 3 also shows that in most cases a biphasic dissolution pattern existed, where pH of the dissolution medium was altered from 1.2 to 7.4. Comparing the drug release from microspheres containing 4 polymers ( Figure 3) showed that the release of drug from these microspheres (FCAB and FRL)was slower than that of microspheres containing F RS and F EC (25% and 22.24%, respectively). However, no significant difference was observed between the percentages of drug released at 8h (Q8) microspheres containing F RL and commercial tablet SR (P > 0.05). The first portion of the biphasic dissolution curves is due to TH dissolution which starts immediately after the beginning of the dissolution process. To release the drug in the second phase combination of the diffusion of the remaining dispersed drug into the bulk medium, formation of pores within the matrix due to the initial drug dissolution and swelling which enhances the permeability of the polymer to the drug might be involved ( 8). Figure 3 illustrates that different TH microspheres exhibited different dissolution profiles. In order to find out which release profiles was more suitable for oral administration, the release data were compared with those of commercial TH extended release formulations. The TH microspheres prepared in this study could be embedded into soft gelatin capsules for peroral administration. According to the US pharmacopoeia not less than 70-80% of the TH should be released within 8 h. The difference factor showed that microsphere formulations containing EC, CAB, Eudragit RL and RS and did not match the release profile of commercial formulations ( Table 2) and there was no significant similarity among these dissolution profiles (f1 = 19.91-67.91%). CAB has a low permeability to drug which results from its high intermolecular attraction. Hydrogen bonding between the hydroxyl groups of the carboxylic moiety and the carbonyl oxygen of ester group increases the degree of solidity of the polymer and decreases its porosity and permeability. However, Eudragit RL and RS are a copolymer of acrylic and methacrylic acid esters with a low and high content of quaternary ammonium groups. The ammonium groups present as salts promote permeability and act as a channeling agent for the entrance of the liquid medium through the floating microsphere wall, causing it to swell. Eudragit RL100 microspheres was a little higher than that of Eudragit RS100 microspheres because Eudragit RL100 contained higher amount of quaternary ammonium groups, which rendered it more permeable and accelrated the drug release as reported. These observations could be attributed to the fact that RS100 microspheres have thicker polymeric surface as compared to Eudragit RL100 microspheres. The thick polymeric barrier slows the entry of surrounding dissolution medium in to the microspheres and hence less quantity of drug leaches out from the polymer matrices of the microspheres exhibiting slow release with a lag time of 2 h. However, Eudragit RS 100 microspheres showed a three phase composition. First, an initial release due to the drug desorption from the particle surface; secondly, a lag time for a certain period, resulting from the diffusion of the drug into microsphere surface; and thirdly, a constant sustained release of the drug resulting from the diffusion through the polymer wall as well as its erosion. This facilitates the diffusion of the dissolved drug out of the microsphere into the dissolution medium. Thus, by varying the ratio of CAB, Eudragit RL, and RS in the TH microspheres, TH release rate can be controlled. CAB polymer exhibit slower rate of in vitro drug release initiated by lag time, which reduces the plasma drug fluctuations, as seen in conventional tablet dosage forms ( 8). Acrylic derivatives include insoluble polymers (EC, CAB) with varying degrees of permeability.
Figure 3 Cumulative percent release of theophylline from microspheres prepared with different type of polymers, and theophylline SR® tablet.
Table 2 Comparison of various release characteristics of theophylline from different type of polymers in microparticles formulations, and theophylline SR® Tablet
Formulation codeQ 2 a, % Q 8 b, % DE c T d 50%, h f1 e F EC 25 ± 2.10 91.87 ± 3.40 80.48 ± 4.21 4 32.59 ± 2.23 F RS 22.24 ± 1.16 68.27 ± 1.22 67.8 ± 3.55 3.5 67.91 ± 4.42 F CAB 6.45 ± 0.16 71.39 ± 2.06 69.39 ± 4.01 5 19.91 ± 1.23 F RL 7.41 ± 0.03 77.97 ± 1.17 72.36 ± 5.52 > 3 30.44 ± 3.67 Theophylline SR ® 12.89 ± 1.55 80.86 ± 5.73 73.72 ± 3.98 4
aAmount of drug release after 2h
bAmount of drug release after 8h
dDissolution time for 50% fractions