Triamcinolone acetonide–Eudragit^® RS100 nanofibers and nanobeads: Morphological and physicochemical characterization

Abstract

Context and objective: The aim of the present research was to fabricate triamcinolone acetonide (TA)-Eudragit^® RS100 nanostructures using the electrospraying method. Materials and methods: The physicochemical properties of the electrosprayed formulations as well as drug release patterns were assessed. The particle size and morphology were evaluated using scanning electron microscopy. X-ray crystallography and differential scanning calorimetry were also conducted to investigate the crystallinity and polymorphic alterations of the drug in the formulations. Probable chemical interactions between the drug and the carrier during the preparation process were analyzed using FT-IR spectroscopy. The drug release kinetic was also considered to predict the release mechanism. Results and discussion: Increasing the concentration of injected polymer solution resulted in the formation of more fibers and fewer beads, with the particle diameter ranging from 60 nm to a few micrometers based on the drug: polymer ratio. The drug crystallinity was notably decreased during the electrospraying process; however, no interaction between drug and polymer was detected. The electrosprayed formulations with 1:10 drug: polymer ratio showed an almost similar drug release rate compared to the pure drug, while those with 1:5 ratio revealed slower release profiles. The release data were best fitted to the Weibull model, so that the corresponding shape factor values of the Weibull model were less than 0.75, indicating the diffusion controlled release mechanism. Conclusion: Our findings revealed that TA loaded Eudragit^® RS100 nanofibers and nanobeads were properly prepared by the electrospraying method, which is a simple, surfactant-free and cost effective technique for producing drug: polymer nanostructures.

Keywords::

• electrospraying
• Eudragit^® RS100
• nanobeads
• nanofibers
• triamcinolone acetonide

Introduction

The concept of novel drug delivery has rapidly grown and attracted the interest of scientists from different fields of science. Targeted and controlled delivery of the drugs, decreased toxicity and side effects as well as patient compliance are a few among many of the advantages of novel drug delivery systems (Prisant et al. 1992, Reddy 2000, Midhun et al. 2011). Nanocarriers, as one of the major novel drug delivery systems, have been investigated and benefited from for more than 20 years, thanks to their outstanding features such as high surface area and increased solubility (Kaparissides et al. 2006, Kocbek et al. 2006, Snopczynski et al. 2009, Akhgari et al. 2013). Different techniques have been developed to prepare nanoparticles including emulsification solvent evaporation (Javadzadeh et al. 2010), template synthesis (Krishna Sailaja et al. 2011), aerosol flow reactor (Eerikäinen et al. 2003), supercritical fluid, nanoprecipitation and electrospraying (Rao and Geckeler 2011, Sailaja et al. 2011, Sode et al. 2013). Electrospraying is rapidly growing as a popular technology for the fabrication of submicron polymeric structures containing different molecules. In comparison with traditional production techniques, electrospraying has the potential to fabricate reproducible particles with regulated particle size distribution and morphology (Bock et al. 2012). The device used for the electrospraying of polymers is simply composed of three main parts: a high voltage DC power supplier, syringe and collector. In brief, this method takes advantage of a high voltage electrical field (approximately 10–30 KV) to induce electrical charge on the surface of a polymer solution. The dropped solution would then form a liquid jet due to electrostatic repulsion forces and fly towards the grounded collector screen. The solvent evaporates by its movement to the collector, and depending on the solution's viscosity, nanofibers or nanobeads take shape on the collector (Doshi and Reneker 1995, Cui et al. 2006, Adibkia et al. 2007b, Yu et al. 2009). It was reported that the characteristics of the electrosprayed particles were also related to other parameters like drug: polymer ratio, flow rate, voltage and the distance between needle and collector (Pham et al. 2006, Martins et al. 2008, Bhardwaj and Kundu 2010). Therefore, aside from the simplicity and cost effectiveness of this technique, electrospraying also has the advantage of managing the morphology and physicochemical properties of the product by optimizing the operating conditions, thus making it applicable on an industrial scale. Electrospraying and electrospinning have already been used for biomedicine, optical electronics (Wang et al. 2002), tissue engineering scaffolds (Luu et al. 2003), protective clothing (Subbiah et al. 2005) and filtration (Rajesh and Natarajan 2009). In contribution to the pharmaceutical industry, this method was applied to prepare nanoformulations of diclofenac sodium(Shen et al. 2011), ketoprofen (Yu et al. 2013), salicylic acid (Nguyen et al. 2012), lidocaine hydrochloride (Ignatova et al. 2013) ibuprofen (Pires et al. 2013, Shi et al. 2013) naproxen (Canbolat et al. 2014) and doxycycline (Chaturvedi et al. 2013). Eudragit^® RS100 is a cationic copolymer of poly (ethylacrylate, methyl–methacrylate and chlorotrimethyl– ammonioethyl methacrylate). This polymer is almost insoluble at physiologic pH; however, its hydrophilic characteristics make the polymer become a little swollen. The presence of quaternary ammonium groups accounts for the polycationic nature of Eudragit^® RS100, making it a suitable carrier to encapsulate the anionic drugs and also attach to the negatively charged cells (Adibkia et al. 2007a, Loveymi et al. 2012). Thus, Eudragit^®RS100 seems to be an appropriate polymer for prolonged and localized delivery of a desired medicine to some physiologic fluids. Eudragit^® RS100 has been previously applied in the delivery of anti-inflammatory drugs to the eye (Pignatello et al. 2002b, Adibkia et al. 2007b, 2011). Triamcinolone acetonide (TA) is a synthetic corticosteroid with a potent anti-inflammatory effect. It is administered as an injectable suspension for the treatment of different inflammatory conditions including arthritic disorders, macular edema and diabetic retinopathy (Beer et al. 2003, Moshfeghi et al. 2003). TA nanoformulations have been prepared using emulsification/solvent diffusion (Sabzevari et al. 2013), high pressure homogenization (Araujo et al. 2010, Araújo et al. 2011), interfacial polymerization (Krause et al. 1986) and o/w emulsion- solvent evaporation (Kadam et al. 2012); however, to the best of our knowledge TA-Eudragit^® RS100 nanoparticles have not been prepared by the electrospraying method. In the present work, TA-Eudragit^® RS100 nanofibers and nanobeads were formulated and the physicochemical characteristics of the nanoformulations were assessed.

Materials and methods

Materials

TA was purchased from Crystal Pharma (Spain). Eudragit^® RS100 from Degussa (Darmstadt, Germany) was received as a gift from Akbarieh Co. (Tehran, Iran). All other chemicals were of analytical grade or the best grade available.

Preparation of the nanofibers and nanobeads

A custom-designed electrospraying system was used for preparation of the TA–Eudragit^® RS100 electrosprayed samples. The electrospraying apparatus was equipped with a high-voltage DC power supply (Fanavaran Nano-Meghyas, Iran). The polymer and drug were co-dissolved in ethanol at room temperature with drug: polymer ratios of 1:5 and 1:10, so that the polymer solution concentrations were 10, 15 and 20% (w/v). The solutions were dropped through a 5 mL syringe attached to a circular-shaped polyethylene capillary tube with an inner diameter of 0.1 mm and an injection rate of 5 ml/h. The applied voltage was controlled in 25 kV between the spraying needle and grounded Teflon screen. The distance between the nozzle and collector was adjusted to10 cm.

Scanning electron microscopy

A scanning electron microscope (SEM; Tescan, Brno Czech) operating at 20 KV was used to characterize the shape and surface morphologies of the electrosprayed samples. Prior to examination, the samples were mounted on aluminum specimen stubs and coated with gold under argon atmosphere by means of a sputter coater.

Differential scanning calorimeter

DSC60 (Shimadzu, Japan) was used to evaluate the thermal behavior of the drug and polymer in the formulations. The accurately weighed samples (5 mg) were placed in aluminum pans and sealed. Thermograms were achieved at a scanning rate of 20°C/min, covering a temperature range from 25 up to 250°C. Aluminum oxide and indium powders were employed as reference and standard, respectively. The thermograms were recorded and analyzed via TA60 software.

X-ray powder diffraction

X-ray diffractograms of the pure drug, polymer, physical mixture and the electrosprayed formulations were obtained using an automated diffraction system (Siemens, Model D5000, Germany). Measurements were carried out under exposure of Cu Kα radiation of wavelength 1.5405A^°, at 40 kV, 30 mA. The scanning rate was 0.06° min − 1, over the range of 2θ = 5–35°.

Fourier transformed infrared spectroscopy

The fourier transformed infrared spectroscopy (FT-IR) analysis was performed to verify the possibility of a chemical interaction between drug and polymer. The analysis was conducted with the FT-IR spectrophotometer (Shimadzu 43000, Shimadzu) by the KBr disk method (2 mg sample in 200 mg KBr). The scanning range was 400–4000 cm^{− 1} and the resolution was 2 cm^{− 1}. The spectra were obtained by averaging 32 scans.

Dissolution study and drug release kinetic

The release rates of TA from the physical mixtures, pure drug and prepared nanoformulations with the drug: polymer ratios of 1:5 and 1:10, were determined using the USP apparatus II, paddle stirrer. The dissolution medium was 300 ml phosphate buffer (pH = 6.8) and the temperature was controlled at 37°C with continuous orbital mixing (50 rpm). All the examined formulations contained an equivalent of 20 mg of TA. At specified time intervals, 3 ml of media was taken by syringe and filtered through a cellulose acetate membrane (Whatman, UK) with the pore diameter of 20 nm. The volume removed was replaced with fresh buffer and the concentration of TA was assayed at 254 nm using a UV spectrophotometer (Shimadzu, Kyoto, Japan). The amount of TA in the samples was calculated using a calibration curve. The cumulative amount of the TA released was calculated for 8 h and plotted against time. Data obtained from in vitro release studies were fitted into nine common kinetic models including: zero order, first order, Higuchi, power law, Hixson-Crowell, Square root of mass, three seconds root of mass, the Weibull and Linear probability (Wagner 1969, Costa and Sousa Lobo 2001, Barzegar-Jalali 2008), to predict the mechanism of drug release from the prepared formulations . The squared correlation coefficient (RSQ) and percent error (PE) were presented for each model as well.

Results and discussions

Particle size and morphology

In the current study, the electrospraying process was employed to encapsulate TA, a water-insoluble drug, into Eudragit^® RS100. SEM images of the prepared nanoformulations are shown in Figure 1. The results of this study as well as previous works indicated that polymer solution concentration was the key factor in the morphology of the fabricated nanostructures (Huang et al. 2003, Pham et al. 2006). In lower concentrations, the formulations were mainly composed of discrete beads and a few fibers, with the bead size varying from about 60 nm in the drug: polymer ratio of 1:5 to a few micrometers in the ratio of 1:10. Nevertheless, in higher polymer solution concentrations (20%), more fibers and fewer beads were observed. The length of the fibers was several micrometers and the fiber diameter depended on the drug: polymer ratio. Fiber diameter was about 70 nm in a drug: polymer ratio of 1:5 and increased several fold in the drug: polymer ratio of 1:10. Formation of the beads at low concentrations (10%) of polymer solution is explained by the fact that the surface tension of the liquid is stronger than its viscoelastic forces, which initiates the instability of the liquid jet and breaks the jet into separate droplets. Conversely, when the polymer concentration is high enough, the surface tension drops below the critical instability amount and so the liquid jet is preserved, resulting in the formation of the fibers (Yu et al. 2006). Therefore, polymer viscosity becomes the most important process parameter which affects the fiber size. Surface morphology of the electrosprayed nanostructures pointed out that larger beads were obtained in the 1:10 ratio. The concavities present on the surface of the beads were possibly due to the solvent evaporation whereas smaller beads were nearly spherical with smooth surfaces. In the case of fibers, it was noted that the formulations with drug: polymer ratio of 1:10 had smoother surfaces in comparison with the 1:5 ratio. Similar results were reported for collagen (Huang et al. 2001), cellulose acetate (Liu and Hsieh 2002), PLGA (Kim et al. 2005), and polyethylene oxide (Frey and Li 2007) by other researchers.

Figure 1. SEM images for TA-Eudragit^® RS100 electrosprayed samples (a) and (b): samples with the drug: polymer ratio of 1:5 and solution concentrations of 10% (a) and 20% (b). (c) and (d): samples with the drug: polymer ratio of 1:10 and solution concentrations of 10% (c) and 20% (d).

Differential scanning calorimeter

Differential scanning calorimeter (DSC) thermograms of pure TA, Eudragit^® RS100, physical mixture as well as drug loaded nanofibers and nanobeads are presented in Figure 2. The large endothermic peak at about 290°C in the TA thermogram corresponded to its melting point, indicating that the intact TA was in a crystalline anhydrous state. The small endothermic peak at around 65°C represented the Eudragit^® RS100 glass transition temperature. The characteristic endothermic peak in the drug thermogram disappeared in the physical mixture due to dilution effect of the polymer and/or the interaction between drug and polymer, induced by heating. Thermograms of the electrosprayed samples exhibited no clear peaks which could be explained by the decrease in drug crystallinity and/or solvation of the drug in the melted carrier and/or the heat-induced interaction between drug and polymer (Adibkia et al. 2007b, Javadzadeh et al. 2010). Comparable results were reported for itraconazole electrospuns (Verreck et al. 2003), as well as other size reduction processes including cogrinding of gliclazide (Barzegar-Jalali et al. 2010b), quasi-emulsion solvent diffusion of azithromycin and clarithromycin (Mohammadi et al. 2010, 2011), and solvent evaporation/extraction of piroxicam and naproxen (Adibkia et al. 2007b, 2011).

Figure 2. DSC thermograms of TA; Eudragit^® RS100; PM 1:5 (physical mixture with the drug: polymer ratio of 1:5) and formulations with the drug: polymer ratio of 1:5 prepared using 10% (F1) and 20% (F2) solution concentrations.

X-ray powder diffraction

X-ray diffractograms of pure TA, polymer, physical mixture of drug and polymer and the prepared nanoformulations are demonstrated in Figure 3. The pure drug spectrum exhibited sharp peaks at about 2°θ: 9, 14, 17 and 24 degrees that were characteristic of the crystalline structure of TA whereas the Eudragit^® RS100 diffractogram contained no distinctive peaks due to its amorphous nature. The decreased intensity of the TA peaks in the physical mixture spectrum was explained by the dilution effect mentioned before. The X-ray powder diffraction (XRPD) analysis of prepared nanofibers and nanobeads revealed no clear peaks due to drug amorphization (Pignatello et al. 2002a, Adibkia et al. 2007c). Therefore, the XRPD findings confirmed the DSC results. Similar results have previously been reported for the AZI-PLGA nanoparticles prepared by the quasi-emulsion solvent diffusion technique (Jasanada et al. 2002, Barzegar-Jalali et al. 2010a, Mohammadi et al. 2010).

Figure 3. X-Ray diffractograms of TA; Eudragit^® RS100; PM 1:5 (physical mixture with the drug: polymer ratio of 1:5); and formulations with the drug: polymer ratio of 1:5 prepared using 10% (F1) and 20% (F2) solution concentrations.

Fourier-transform infrared spectroscopy

Possible interactions between the drug and polymer were evaluated by an fourier-transform infrared spectroscopy (FT-IR) analysis. The FT-IR spectra of the pure drug, physical mixture and the prepared formulations are displayed in Figure 4. The TA spectrum, with the empirical formula of C₂₄H₃₁FO₆, contained characteristic bands including the bands in the range of 3650–3200 cm^{− 1} for the OH stretch, below 3000 cm^{− 1} for the C–H stretch of sp2 and sp3 carbons, at about 1700 cm^{− 1} for the C = O stretch, in the range of 1600–1690 cm^{− 1} for the C = C stretch, in the range of 1000–1300 cm^{− 1} for the C–O stretch of ether and at about 1050 cm^{− 1} for the C–F stretch. Comparing the spectra for the prepared formulations with those for the physical mixture and pure drug clarified the maintenance of major bands as well as the absence of new ones. These findings revealed the lack of any chemical interaction between the drug molecules and polymer during the electrospraying process. Similar results were reported for TA-loaded nanolipid carriers (Araujo et al. 2010) and TA-PLGA microparticles (da Silva-Junior et al. 2009).

Figure 4. FT-IR spectra of TA; Eudragit^® RS100; PM 1:5 (physical mixture with the drug: polymer ratio of 1:5); and formulations with the drug: polymer ratio of 1:5 prepared using 10% (F1) and 20% (F2) solution concentrations.

Dissolution study and drug release kinetic

The drug release profiles of the pure TA, physical mixtures, as well as the prepared nanofibers and nanobeads are presented in Figure 5. The release pattern for pure drug and physical mixtures exhibited no noteworthy difference. The time at which 50% of drug was released (t_50%) together with Q_15min and Q_30min (percent drug dissolved within 15 and 30 min respectively) were also determined for all of the samples (Table I).

Figure 5. Drug release profiles for the pure drug (TA); physical mixtures (PM) and the prepared electrosprayed samples (ES). The values 1:5 and 1:10 indicate drug: polymer ratio. The values of 10%, 15% and 20% represent the total solution concentrations.

Table I. Model-independent parameters of the different samples.

Sample	t50% (min)	Q15min (%)	Q30min (%)
TA	57.3	19.10 ± 0.19	35.57 ± 2.20
PM 1:5	57.5	21.26 ± 0.73	37.00 ± 0.26
PM 1:10	45.2	27.24 ± 0.48	45.28 ± 2.32
ES 1:5 10%	360.0	26.07 ± 2.59	35.50 ± 3.82
ES 1:5 15%	360.0	18.76 ± 0.13	26.67 ± 0.38
ES 1:5 20%	227.0	22.86 ± 2.84	27.36 ± 1.40
ES 1:10 10%	45.0	31.95 ± 0.67	47.45 ± 2.69
ES 1:10 15%	31.0	34.46 ± 1.13	48.46 ± 1.66
ES 1:10 20%	30.0	34.58 ± 2.18	40.98 ± 2.54

PM and ES stand for physical mixture and electrosprayed samples, respectively. 1:5, 1:10 represent TA: Eudragit^® RS100 ratio. 10%, 15% and 20% signify the applied solution concentration. t_50% indicates the time at which 50% of drug was released. Q_15min and Q_30min show the percent of drug dissolved within 15 and 30 min, respectively.

The electrosprayed formulations with the drug: polymer ratio of 1:10 revealed a release rate comparable with pure TA; however, the corresponding dissolution curves were composed of a biphasic release pattern; namely, the initial burst release within the first hour and the subsequent plateau release. The biphasic release profile is a common phenomenon in many nano drug delivery systems (Leo et al. 2000). The burst release could be explained by the high surface area as well as the accumulation of the drug molecules on the superficial layers of the particles, whereas the plateau phase is elucidated by the dissolution and diffusion of the drug from the inner layers (Yu et al. 2009, Mohammadi et al. 2010, Midhun et al. 2011). The faster release rate in the case of 1:10 drug: polymer ratios may partially contribute to a more homogeneous and finer dispersion of drug molecules in the polymer matrix. This finding was similarly reported for diclofenac- Eudragit^® RS100 nanoparticles (Barzegar-Jalali et al. 2012). Contrarily, the drug: polymer ratio of 1:5 led to a slower release pattern in comparison with pure TA and physical mixtures, in which the drug entrapment in the polymer matrix was probably the dominant mechanism of drug release. Similar results were obtained for the Naproxen-Eudragit^® RS100 nanoparticles (Adibkia et al. 2011).

A kinetic study of drug release could be helpful in obtaining one or two physically meaningful parameters that are used for comparative purposes and in correlating release parameters with important factors such as bioavailability. In addition, a kinetic parameter can be used to assess the influence of formulation factors on the drug release (Barzegar-Jalali et al. 2008). According to the RSQ and PE values (Table II), it was concluded that the Weibull was the best fitting model. The value of the exponent β in this model is an important indicator to estimate the drug release mechanism through the polymer matrix. The values of β ≤ 0.75 specify Fickian diffusion, while β values in the range of 0.75–1 determine a combined mechanism of Fickian diffusion and swelling-controlled release (Adibkia et al. 2007b). As indicated in Table II, the resulting β values were less than 0.75, signifying that diffusion was the dominant mechanism of drug release from the electrosprayed samples.

Table II. Parameters of the different kinetic models; squared correlation coefficients (RSQ); and percent error (PE) for the formulations F1–F6.

Model name and equation		F1 ES 1:5 10%	F2 ES 1:5 15%	F3 ES 1:5 20%	F4 ES 1:10 10%	F5 ES 1:10 15%	F6 ES 1:10 20%
Zero order F = k0t	K0	0.03	0.05	0.05	0.05	0.04	0.04
	RSQ	0.695	0.780	0.840	0.636	0.485	0.776
	PE	17.32	28.81	14.45	19.84	22.02	12.32
First order 1 n (1 − F) = − k t	Kf	0.06	0.08	0.08	0.11	0.07	0.09
	RSQ	0.763	0.842	0.888	0.729	0.558	0.856
	E	16.34	27.29	12.85	18.19	21.20	10.98
Higuchi	kH	0.12	0.16	0.15	0.18	0.14	0.15
	RSQ	0.850	0.921	0.953	0.812	0.666	0.911
	PE	12.13	16.39	7.02	14.15	17.46	7.32
Power law ln F = ln kp + plnt	kp	0.36	0.28	0.35	0.49	0.47	0.47
	p	0.23	0.37	0.25	0.24	0.22	0.19
	RSQ	0.931	0.929	0.968	0.919	0.828	0.969
	E	7.39	11.91	5.15	11.03	15.98	4.51
Hixson-Crowell	k1/3	0.02	0.02	0.02	0.03	0.02	0.02
	RSQ	0.740	0.823	0.873	0.699	0.533	0.831
	PE	16.65	27.87	13.37	18.87	21.42	11.35
Square root of mass	k1/2	0.02	0.03	0.03	0.04	0.03	0.03
	RSQ	0.730	0.813	0.865	0.684	0.521	0.818
	PE	16.81	28.03	13.64	18.94	21.55	11.58
Three seconds root of Mass	k2/3	0.03	0.04	0.04	0.04	0.03	0.04
	RSQ	0.718	0.802	0.857	0.668	0.509	0.804
	PE	16.98	28.29	13.90	19.22	21.69	11.82
Weibull ln[-ln(1-F)] = βlntd+βlnt	β	0.28	0.44	0.32	0.35	0.30	0.28
	td	18.81	11.04	12.34	2.66	3.97	4.42
	RSQ	0.949	0.952	0.970	0.944	0.855	0.977
	PE	6.20	9.40	4.96	6.52	10.88	3.36
Linear probability Z = Z0 + qt	Z0	− 0.53	− 0.80	− 0.60	− 0.21	− 0.21	− 0.26
	q	0.09	0.14	0.12	0.13	0.10	0.11
	RSQ	0.653	0.690	0.814	0.641	0.478	0.777
	PE	17.67	28.84	14.75	19.30	21.78	12.01

ES stands for electrosprayed samples. 1:5 and 1:10 represent TA: Eudragit^® RS100 ratio. 10%, 15% and 20% signify the applied solution concentration. F denotes fraction of drug released up to time t. k₀, k_f, k_H, p, k_P, k_1/3, k_1/2, k_2/3, t_d, β, Z₀ and q are parameters of the models. Z is the probit of fraction of drug released at any time. Z₀ is the value of Z when t = 0 and t = 1.

Conclusion

The SEM results revealed that TA-Eudragit^® RS100 nanobeads and nanofibers could be fabricated using the electrospraying method. However, the size and morphology of the particles depended on some parameters, specifically the concentration of the polymer solution applied, and the drug: polymer ratio, which could be optimized to produce the ideal formulation for intended applications. DSC and XRPD studies pointed out that drug crystallinity was notably decreased during the preparation process. The FT-IR spectra revealed no chemical interaction between TA and Eudragit^® RS100 during the electrospraying procedure. Dissolution studies demonstrated a biphasic release profile for the formulations with drug: polymer ratio of 1:10. Slower release patterns were observed for the formulations with the drug: polymer ration of 1:5. The kinetic study suggested that the Weibull was the best fitting model and that diffusion was the dominant mechanism of drug release.

Acknowledgement

The authors would like to thank the Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. This article is based on a thesis submitted for a Pharm D degree in Faculty of Pharmacy, Tabriz University of Medical Sciences, and Tabriz, Iran.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.