Encapsulation of an antifungal agent within biodegradable polymers: composition effect

4274 | P a g e J a n u a r y 2 9 , 2 0 1 6 Encapsulation of an antifungal agent within biodegradable polymers: composition effect Rima Kassab, Dima Moussa, Paolo Yammine* 1 Department of Chemistry, Faculty of Sciences, University of Balamand, Tripoli, Lebanon rima.kassab@balamand.edu.lb dima.moussa@balamand.edu.lb paolo.yammine@balamand.edu.lb Corresponding author: paolo.yammine@balamand.edu.lb ABSTRACT


INTRODUCTION
Much research has been focused recently on using biodegradable polymeric microspheres as novel drug delivery systems. Thermoplastic aliphatic polyesters such as Polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) are most commonly used in these applications owing to their biodegradability, biocompatibility, mechanical strength, and nontoxicity [1][2][3][4] (Figure 1). Medical uses of these polymers in drug delivery involve the microencapsulation of small dug molecules as well as large biomolecules as proteins, peptides, and DNA/RNA [5][6][7][8][9]. With new diseases and infections emerging worldwide, the work in the medical field is concentrating on finding suitable drug treatment methods that are highly effective and less costing. Among these, the hydrophobic small drug molecules constitute a significant class which could be extremely helpful in polymeric delivery systems. However, their use poses a great challenge in order to reach the best encapsulation and release rates. These characteristics are affected by several factors such as drug properties, its solubility in aqueous and organic media, its stability in vivo, and drug-polymer interactions [10].
Routes of drug administration mainly depend on drug properties in addition to the type of delivery system. Yet, the oral route of administration remains the most commonly used, since it is simple, painless, and dosing of the medication can be easily adjusted or terminated [10,11]. Drug delivery systems prepared to be used in oral administration must enable the protection of the drug from enzymatic degradation and remaining body organs from undesirable side effects.
Many studies have been done to encapsulate hydrophobic drugs in polymeric microspheres of PLA and PLGA. These could be prepared by different methods, among which, the emulsion solvent evaporation technique is the most widespread. However, there are several preparative variables that could affect drug encapsulation and release rates obtained; most importantly the drug solubility within the polymeric mixture [12].
Amphotericin B (AmB), an antifungal hydrophobic drug, is chosen as a model drug to be encapsulated in polymeric microspheres of PLA and PLGA. This is done by changing the ratio of lactide and glycolide content in the polymeric mixture to evaluate the resulting effects on prepared microspheres.f drug content, particle size, morphology, interaction between drug and polymer, stability, drug release and cytotoxicity.

Methods
AmB-loaded PLA and PLGA microspheres were synthesized using o/w emulsion solvent evaporation method. It consisted on preparing different microspheres formulations by varying the ratios of PLA and PLGA each time in the polymeric mixture and fixing the quantity of drug used, as shows Table 1. This was done to check the effect of lactide and gycolide content, in addition to the effect of drug solubility in this polymer solution on drug encapsulation and release.
Typically, 500 mg of polymer mixture containing PLA and PLGA at different ratios and 70 mg drug were dissolved in an organic solution of 14:6 ml DCM:MeOH. This organic phase was added to an aqueous phase containing the surfactant Tween 80. The whole mixture was stirred for 6 hours at 1400 rpm using a mechanical stirrer to allow solvent evaporation and microspheres formations. These were collected by filtration after washing with water and methanol, finally oven dried. J a n u a r y 29, 2 0 1 6 -Fourier Transform Infrared (FT-IR) study: analysis was carried out on a FT-IR spectrometer (Frontier NIR, Perkin Elmer, USA) in order to investigate the possible chemical interactions between the drug and the polymeric matrix. Samples were mixed with IR grade KBr salt and pressed into pellets using a manual hydraulic press for scanning.
-In vitro drug release study: The In vitro release study is carried out in PBS solution (0.2 M, pH 7.4). 25 mg microspheres are introduced in small vial containing 25 ml PBS, used as release medium, and maintained at 37°C. At different time intervals, 5 ml of the release medium is withdrawn and replaced with fresh solution. This is evaluated for its drug content at 409 nm. Table 2 shows that higher values of DE and DL were observed for formulations with higher PLA ratios, and thus higher lactide content in the polymeric mixture. This could be explained by the fact that PLA is more hydrophobic than PLGA due to the presence of the side methyl groups, as illustrated in Figure 1 [6,13,14]. As a result, the increased size of hydrophobic region facilitates the incorporation of AmB which is hydrophobic in character [15]. In addition, the increase in hydrophobicity with increase in lactide content results in better solid-state solubility of AmB in the hydrophobic polymeric matrix 12 . These reasons explain the higher DE and DL values for F1 to F3 compared to the other formulations. 10.2 0.45 125 J a n u a r y 29, 2 0 1 6 Table 1 also gives that the average particle size for the prepared microspheres. It ranges between 125 and 300 µm. This indicates that the variation in PLA and PLGA ratios in the polymeric mixture, and thus increasing in lactide content, doesn't have any influence on particle size. It is rather the procedure used and changing the speed of mixing that leaves a direct impact on the particle size [16,17]. Moreover, SEM analysis presented in Figure 2 showed mcirospheres that are spherical in shape and porous in structure.

Fig 2: SEM photos of microspheres at (a) 100x and (b) 500x magnifications
Concerning in vitro drug release, Table 3 shows that the release rate is slower for formulations having the highest %DL values. These are characterized by a higher burst release after 1 hr 30 min in comparison to the other formulations that have a more prolonged or extended release, as shows Figure 3. In addition, the release rate increases as %DL decreases. At higher %DL, higher drug quantity requires more time to be released [18,19].

Fig 3: Drug release profile of AmB microspheres formulations
In additoon, the release rate is directly affected by the ratio of PLA and PLGA in the polymeric mixture. For formulations with higher lactide content, they have a slower release rate. This could be explained by the affinity of hydrophobic AmB to the more hydrophobic lactide-rich polymer [13,14].
Besides, polymer characteristics as the solid-state polymer-drug solubility have an influence on drug release and drug distribution between the polymeric phase and the aqueous release medium [12]. Higher polymer-drug solubility results in 500PLGA J a n u a r y 29, 2 0 1 6 lower release rates. This is due to the higher hydrophobicity of the polymer containing more lactide, and to lower diffusion of the hydrophobic drug to the external release medium [20].
Lastly, figure 4 reveals the results of polymer-drug physico-chemical compatibility studies. FT-IR spectra were recorded for AmB, PLA/PLGA blank microspheres, and PLA/PLGA AmB-loaded microspheres. For PLA/PLGA blank microspheres, characteristic peaks are noted at 2900-3000 cm -1 for aliphatic C-H stretching, at 1750 cm -1 , for C=C alkene stretching, at 1450 cm -1 for aliphatic C-H bending, and at 1080-1170 cm -1 for C-O ester stretching [21,22]. These peaks are also present in the spectrum of loaded microspheres. For AmB, several characteristic peaks appear at 1690 cm -1 for C=C alkene, at 2940 cm -1 for C-H methyl stretching, and the broad bank at 3300-3400 cm -1 for -OH alcohol and -NH2 amine groups [23,24]. This broad band which is absent in the spectrum of blank polymeric microspheres is present in the case of drugloaded microspheres. This indicates the presence of both drug and polymer intact in drug-loaded microspheres and the absence of any chemical interaction between the two during microencapsulation procedure.

CONCLUSION
The emuslion solvent evaporation technique is one of the most widespeard and successful methods for preparing drug delivery systems based on microspheres ready for oral administration. Polymeric Amphotericin B-loaded microspheres of Polylactic acid and Poly(lactid-co-glycolic acid) were formulated by varying the ratio of the two polymers in the organic mixture. This factor had a great influence on drug encapsulton and release. As the lactide or polylactic acid content increased, Amphotericin B loading and encapsulation also increased. In addition, the drug release was found to be slower with more lactide. Particle size obtained was not influenced and was more homogenous for all formulations which is expected in the case of solvent evaporation which also yields spherical and porous microspheres. Finally, FT-IR analysis revealed that both the drug and the polymeric mixture remained intact following microencapsulation.