of Triamcinolone
Acetonide-loaded
Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
(PHBHx) Microspheres
CEMBAYRAM ANDEMIRBAKIDENKBAS*
Hacettepe University, Chemistry Department, Biochemistry Division Beytepe, Ankara, Turkey
EBRUKILIC¸AY AND BAKIHAZER
Karaelmas University, Chemistry Department, Physical Chemistry Division, Zonguldak, Turkey
HASANBASRIC¸AKMAK
Ministry of Health, Atatu¨rk Training Hospital, Ophthalmology Clinics, Ankara, Turkey
ISAONODA
The Procter and Gamble Company,West Chester, Ohio, USA
ABSTRACT: Triamcinolone acetonide loaded in poly(3-hydroxybutyrate-co-3 hydroxyhexanoate) (PHBHx) microspheres were prepared to treat cystoid macular oedema (CMO) and acute posterior segment inflammation associated with uveitis. The PHBHx microspheres were prepared by solvent evaporation technique using methylene chloride as the solvent and aqueous poly(vinyl alcohol) emulsifier as the dispersion medium. The PHBHx micro-spheres obtained were well formed with narrow size distribution; the average size prepared ranged from 40–200 mm depending on the formulation used.
*Author to whom correspondence should be addressed. E-mail: [email protected]
Figures 1 and 3–6 appear in color online: http://jbc.sagepub.com
The stirring rate of the dispersion medium, emulsifier concentration, and polymer/solvent ratio parameters were varied to determine their effect on the size and size distribution of the PHBHx microspheres. Increasing the stirring rate and emulsifier concentration decreased the size and the size distribution of the microspheres, while increasing the polymer/solvent ratio caused the opposite effect. The polymer/drug ratio was the most effective parameter for controlling drug loading and release properties. More than 90% of the loaded drug was released within the first 24 h; after that, the release rate was slower for all formulations.
KEY WORDS: triamcinolone acetonide, PHBHx, cystoid macular oedema, uveitis, microspheres, controlled drug delivery, controlled drug release.
INTRODUCTION
P
eriocular injections of long-acting corticosteroids have been used to deliver high drug concentrations to the posterior eye for cystoid macular oedema (CMO) and other posterior segment inflammatory changes associated with uveitis. Several techniques have been used, including, subconjunctival, sub-Tenon’s capsule, trans-septal, orbital floor, and retrobulbar injections [1–6]. However, the success of each technique has been limited and each is associated with complications, including, the injection itself, blepharoptosis, orbital fat herniation, globe perforation, and even retinal and choroidal vascular occlusion [1–9]. More recently, intravitreal corticosteroid injections [10–12] and intravi-treal corticosteroid implants [13,14] were investigated. Unfortunately, these methods risk associated complications, such as, vitreous hemor-rhaging, retinal detachment, suprachoroidal placement of implant, and endophthalmitis [13,15,16].In this research, we planned to utilize a trans-Tenon’s retrobulbar infusion of controlled release form of triamcinolone to treat CMO and acute posterior segment inflammation associated with uveitis. In this case, the repetition amount and the other risks will be reduced due to the reservoir nature of the proposed system.
Periocular injections of triamcinolone acetonide has been traditionally used for the treatment of macular edema secondary to inflammation or following intraocular surgery (typically cataract surgery) [17,18]. To achieve a higher intraocular concentration of corticosteroid to treat retinal disease, the intravitreal injection of triamcinolone acetonide was first studied in the 1970s, in an animal model, as a pharmacological adjunct to prevent the formation of proliferative scar tissue (prolif-erative vitreoretinopathy) and improve the out come following retinal detachment surgery [19]. Since then, pure triamcinolone acetonide and
in Kenalog has been shown to have an acceptable safety profile in animal [20,21]. Subsequent to the safety profile of intravitreal Kenalog in animal models, intravitreal triamcinolone in the form of Kenalog has been clinically used for a variety of retinal diseases, such as age-related macular degeneration [22–24].
Polyhydroxyalkanoates (PHA) is a family of biopolyesters that are produced by specific bacteria [25,26]. PHA have good biodegradable and biocompatible properties; thus, are suitable materials for biomedical applications. Most of the biomedical studies on PHA were focused on poly(3-hydroxybutyrate) (PHB), the first member of the PHA family [27]. PHB was found to have potential applications as an implant material owing to its biocompatibility, but experienced difficulties due to its crystallinity and brittleness [28]. A newer member of PHA family, poly(3-hydroxybutyrate-co-3 hydroxyhexanoate) (PHBHHx), is cur-rently being produced in large scale [29]. PHBHx is more ductile and processable than PHB because it has a propyl side chain, which reduces the crystallinity and melting temperature [30]. Studies have demonstrated that PHBHHx, a microbially synthesized polyester with good biodegradability and biocompatibility, as well as strong mechanical properties, consequently, is very promising for biomedical applications [31]. In this study, PHBHx was selected as a carrier in microsphere form for the sustained release of triamcinolone acetonide.
MATERIAL AND METHODS
Materials
PHBHx copolymer was obtained from Procter and Gamble Company, USA. The triamcinolone acetonide active agent (containing Kenacort and BSS Balanced Salt Solution) was supplied by Atatu¨rk Training Hospital Ophthalmology Clinics. PVA (Mw 72,000, Fluka, USA) was used as an emulsifier and methylene chloride (Merck, Germany) as the solvent. All other chemicals were of highest purity available and were used without further purification.
Preparation of Microspheres
PHBHx particles in the size range of 40–200 mm were produced by solvent evaporation [32]. In a typical procedure, 0.25 g of the PHBHx polymer were dissolved in 5 mL of methylene chloride and added dropwise using a dossage pump (Sage, UK) at a 1 mL/min flow rate to
50 mL of distilled water (the dispersion phase) containing 0.250 g of the emulsifier (PVA, Fluka, USA), while the medium was stirred with a mechanical stirrer at 2000 rpm at room temperature. The organic solvent was evaporated under vacuum conditions for 2 h. The spherical particles were separated from the aqueous medium by centrifugation at 4000 rpm for 10 min and washed twice with distilled water. The microspheres obtained were dried and kept in a dessicator for further experimentation.
Morphological Evaluations
Morphological evaluations of PHBHx polymeric particles were made on a scanning electron microscope (SEM, JEOL, Japan). A 100 mL aqueous suspension of particles was dried on a metal support under vacuum at room temperature for 4 h and then coated with gold to obtain the SEM micrographs.
Size and Size Distributions
The size and size distribution of the PHBHx polymeric particles were determined from the micrographs taken with an optical microscope (Olympus, Japan). The particle diameters on the micrographs (each containing about at least 40 particles) were measured and average sizes with standard deviations were evaluated by using an image analysis software (Image-Pro, USA). The effects of the organic solvent/polymer ratio, emulsifier concentration and stirring rate on the average particle size, and size distribution were investigated.
Effective Parameters on Size and Size Distributions Stirring Rate
One of the most effective parameters in the preparation of polymeric microspheres was the stirring rate of the dispersion medium. The stirring rates of 500, 1000, and 2000 rpm were used in the formation of the PHBHx microspheres. The other parameters (5 mL of solvent, 250 mg of PVA as an emulsifier and 50 mL of distilled water as the dispersion medium) were kept constant.
Emulsifier Concentration
The emulsifier causes a reduction in the surface tension differences between the polymer and aqueous media. Therefore, to determine the
effect on the microsphere sizes and size distributions, the concentration used in this study was from 1 to 10 mg PVA/mL in distilled water as the dispersion medium.
Polymer/solvent Ratio
Polymer concentration is another effective parameter to change the size and size distribution, due to the required energy change for dif-ferent viscosities or concentration of the polymer in dispersion medium. To probe the polymer concentration effect, dilute (25 mg PHBHx/mL solvent) and concentrated (250 mg PHBHx/mL solvent) polymer solutions were added dropwise into the dispersion medium during the preparation of polymeric microspheres. In most cases, the medium concentration used was 50 mg PHBHx/mL solvent to obtain micro-sphere samples.
Drug Loading Studies
Triamcinolone acetonide corticostereoid was dispersed in the PHBHx polymer solution. The drug/polymer ratios by mass were prepared as 1/100, 2/100, and 4/100 (w/w), respectively. The mixture was sonicated in a water bath for 30 min and then added dropwise into the dispersion medium consisting of distilled water and poly(vinyl alcohol) as the emulsifier. The drug-loaded microspheres were isolated from the dispersion medium in a silk filter and washed with distilled water twice and then dried at room temperature for further in vitro release analysis. Initial drug mass/initial PHBHx mass ratio was used to characterize thein vitro sustained release studies.
Drug Entrapment Efficiency
Drug loading ratio was calculated by using the following equation:
Drug loading ratio ¼ ðMass of drug in microspheres=
Mass of drug used in the formulationÞ 100
The mass of drug present in microspheres was calculated by sub-stracting the mass of free drug in the dispersion medium and washing water from the total mass of drug used in the formulation. The free triamcinolone acetonide was determined by UV spectro-photometry (UVmini 1240, Schimadzu, Japan), at the wavelength of 240.5 nm, where the maximum absorbance was observed and did not
interfered with other absorbant peaks. The amount of entrapped drug was correlated with the amount of total drug released in the in vitro release studies.
In vitro Release Studies
The release profiles of triamcinolone acetonide from PHBHx micro-spheres were determined in the 25 mL of BSS(Balanced Salt Solution) medium filled covered glass bottles in a shaking (50 rpm) water bath at 378C temperature. At scheduled time intervals, samples (2 mL) were taken for UV spectrophotometric measurement and replenished with fresh medium. The absorbance measurements were carried out at 240.5 nm taken in duplicate.
RESULTS AND DISCUSSION
Morphological Evaluations
The spherical PHBHx polymeric microspheres in the size range of 40–200 mm were produced by a solvent evaporation technique. The morphology of PHBHx microspheres were evaluated with SEM and optical micrographs. A SEM and optical micrograph of the PHBHx microspheres are given in Figures 1 and 2, respectively. The microspheres were well shaped spheres and the surface morphology,
as seen in Figure 2, appears to be textured with some pores turned towards to the inner part of the microspheres. Microsphere size and the size distributions depended on the preparation conditions and formulations.
Parameters Effecting Particle Size and Size Distribution We investigated the effects of the stirring rate, emulsifier concen-tration polymer/solvent ratio, and drug concenconcen-tration on the average particle size and size distribution (Table 1).
Table 1. Effective parameters on microsphere size and size distribution.
Sample no PHBHHx concentration (mg/mL) PVA concentration (mg/mL) Stirring rate (rpm) Microsphere diameter (mm) Effect of polymer concentration
1 250 5 2000 101 31
2 50 5 2000 41 11
3 25 5 2000 39 12
Effect of emulsifier concentration
2 50 5 2000 41 11
4 50 1 2000 67 23
5 50 10 2000 36 9
Effect of stirring rate
2 50 5 2000 41 11
6 50 5 1000 136 22
7 50 5 500 211 44
20kv ×1,100 10 µm Kirikkale
Figure 2. SEM micrograph of PHBHx microspheres.
Stirring Rate
The effect of stirring rates of the dispersion medium evaluated were at 500, 1000, and 2000 rpm, respectively. Microsphere size versus stirring rate effects are shown in Figure 3. The size values increased by decreasing the stirring rate as expected, as the higher stirring rate dispersed the polymeric solution into the smaller droplets [33–35]. Emulsifier Concentration
The emulsifier regulated the surface effects between the polymer and the aqueous media in the preparation of polymeric microspheres by solvent evaporation. Increasing the emulsifier concentration caused a reduction in the surface tension differences between the polymer and aqueous media. In this study, the emulsifier concentration was changed in the range of 1–10 mg PVA/mL distilled water as the dispersion medium. The size and size distribution values of the PHBHx micro-spheres prepared are given in Figure 4. The microsphere size was decreased significantly by increasing the emulsifier concentration as found our earlier studies [36].
Polymer/Solvent Ratio
Polymer concentration is another effective parameter that is used to change particle size and size distribution, based on the energy change for different viscosities or concentrations of the polymer in the
Microsphere size ( µ m) 0 50 100 150 200 250 2000 rpm 1000 rpm 500 rpm 211 ± 44 136 ± 22 41 ± 11 Stirring rate (rpm)
dispersion medium. To probe the polymer concentration effect, dilute (25 mg PHBHx/mL solvent) and concentrated (250 mg PHBHx/mL solvent) polymer solutions were added dropwise to the dispersion medium during the preparation of polymeric microspheres. Shown in Figure 5 are the size and size distribution values obtained by using different polymer/solvent ratios. As seen in this figure, the size of microspheres was increased by increasing the polymer/solvent ratio; the higher polymer/solvent ratio produced more viscous solutions, hence it was more difficult to disperse the polymer solution which is similar to the results obtained in related studies [32, 35–38].
0 10 20 30 40 50 60 70 1 mg/mL 5 mg/mL 10 mg/mL Microsphere size ( µ m) Emulsifier concentration (mg/mL) 67 ± 23 41 ± 11 36 ± 9
Figure 4. Effect of emulsifier concentration on PHBHHx microsphere size.
0 20 40 60 80 100 120 250 mg/mL 50 mg/mL 25 mg/mL Microsphere size ( µ m) Polymer/solvent ratio (mg/mL) 101 ± 31 41 ± 11 39 ± 12
Drug Loading and In vitro Release Experiments
In the drug encapsulation and in vitro release experiments, initial drug/polymer ratio by mass was evaluated as a parameter that affected the drug release rate. The drug / polymer ratios by mass were prepared as 1/100, 2/100, and 4/100 (w/w) and the encapsulation efficiency determined; the release data are given in Table 2 and Figure 6, respectively, for all ratios. The higher the initial drug/polymer ratio, the greater amount and rate of released drug (Table 2).
In addition, the drug release rate, based on the percentage of the released drug in a period of time, is faster in the higher drug load/mass ratio as seen in the Figure 6. On the other hand, for most of the formulations, more than 90% of the total amount of drug was released within 24 h. After that period of time, a very slow release rate was observed.
CONCLUSIONS
In this study, triamcinolone acetonide-loaded PHBHx microspheres were prepared by solvent evaporation with sizes ranging from 40 to 200 mm by changing the preparation conditions (stirring rate of the
0 20 40 60 80 100 0 5 10 15 20 25 30 Time (h) Cumulative release (%) 1/100 2/100 4/100 Initial drug/polymer ratio by mass
1/100 2/100 4/100
Figure 6. In vitro release profile of triamcinolone acetonide-loaded PHBHx microspheres. Table 2. Drug encapsulation yield.
Initial drug by mass/polymer
by mass (w/w) 1/100 2/100 4/100
dispersion medium, emulsifier concentration and polymer/solvent ratio). Stirring rate affects the supply of the required energy for the formation of polymeric droplets during the procedure, and the size of the microspheres decreased with increasing the stirring rates. With more emulsifier, the size and the size distribution of the microspheres were decreased due to the reduced surface tension differences in the medium. The polymer/solvent ratio increased the size and the size distribution which was related to viscosity increases in the polymer solution. The optimization of these parameters has enabled us to prepare PHBHx microspheres with specific sizes and size distribution. Triamcinolone acetonide was dispersed into the polymer solution prior to the pre-paration of microspheres. The drug was 90% released within the first 24 h, then at a slower rate. The release profile can be adjusted by changing the preparation of conditions, such as, the initial drug/polymer ratio and the particle size distribution can be controlled by emulsifier and polymer concentrations and stirring rate. PHBHx microspheres are promising vehicles for the controlled release of drugs for a number of treatments.
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