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Preparation and optimization of voriconazole microemulsion for ocular delivery
Colloids and Surfaces B: Biointerfaces 117 (2014) 82–88
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Preparation and optimization of voriconazole microemulsion for ocular delivery Rakesh Kumar, V.R. Sinha ∗ UIPS, Panjab University, Chandigarh 160014, India
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 1 February 2014 Accepted 6 February 2014 Available online 15 February 2014 Keywords: Voriconazole Microemulsion Phase diagram Optimization Ocular
a b s t r a c t Optimized microemulsions (o/w type) of voriconazole were formulated for efficient ocular delivery. Optimized batches were selected through construction of phase diagrams following stability studies. No significant physiochemical interactions were found between the drug and excipient (oil and surfactant/co-surfactant) as confirmed by H NMR and FTIR studies. Drug content was found between 53 and 72% depending on size and composition. Selected microemulsion batches exhibited shear thinning properties with acceptable viscosities. Globule size analyzed by zetasizer as well as TEM images of selected batches were found within the desired range (<200 nm). In vitro release studies of microemulsions exhibited sustained release property (>70% in 12 h). Ex vivo permeation study also supported the enhanced drug flux through cornea from microemulsions. Based on size, surfactant/co-surfactant concentration, viscosity, drug content and release studies, the microemulsion batch ME-10 was selected for future in vivo studies. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Topical application is one of the most widely preferred routes of administration for treating ocular infections/diseases as about 90% of the marketed ophthalmic formulations are in the form of eye drops. Features like patient compatibility, tolerability, the easier production method and economical cost make eye drops acceptability wider [1,2]. However, the meager bioavailability (<5%) of conventional eye drops (suspensions, solutions, etc.) is one of the major concern that could be ameliorated through the application of novel pharmaceutical approaches [3]. Application of nanotechnology-based formulations (like nanosuspension, nanoemulsion/microemulsion, solid lipid nanoparticle etc.) in the field of ophthalmology offers several beneficial features like improved solubility (for lipophilic drugs), targeted delivery and controlled release of therapeutic agents [4–7]. Microemulsions are one of the interesting and promising sub-micron carriers for ocular drug delivery. These are transparent, single optically isotropic and thermodynamically stable dispersions of water, oil and amphiphilic compounds (surfactant and co-surfactant) [8]. Microemulsions offer several advantages
∗ Corresponding author. Tel.: +911722534101. E-mail address: Sinha [email protected] (V.R. Sinha). http://dx.doi.org/10.1016/j.colsurfb.2014.02.007 0927-7765/© 2014 Elsevier B.V. All rights reserved.
like improved drug-loading and bioavailability (facilitating transcorneal penetration) with acceptable biocompatibility (due to presence of physiological lipids/oils) over polymeric nanoformulations. Microemulsion of many ocular drugs like ofloxacin, timolol, tacrolimus, everolimus and prednisolone were successfully prepared with sustained effect and better penetrability [9–13]. Voriconazole (VCZ), C16 H14 F3 N5 O, a second generation antifungal agent possesses phenomenal characteristics like broadspectrum activity, activity against resistant fungal species, and acceptable tolerability [14–16]. Almost 100% in vitro susceptibility was observed against various fungal isolates associated with keratitis and endophthalmitis. Moreover, studies suggested an excellent efficacy of VCZ against several ocular mycoses following topical administration [17–19]. Peng et al. fabricated PLGA [Poly (lactide-co-glycolide)] nanoparticles loaded with VCZ to improve agglomeration and antifungal efficacy in mice (renal tissue) [20]. Similarly, Sinha et al. exhibited excellent pulmonary deposition of VCZ following inhalational delivery of PLGA nanoparticles [21]. El-Hadidy et al. demonstrated significant enhancement in antifungal activity of VCZ microemulsion in comparison to drug supersaturated solution for topical effect (dermal and transdermal) [22]. So far, no topical (ocular) formulation of VCZ has been available in the market, though several researchers supported the need of effective topical delivery. This article proposes the formulation and optimization of o/w microemulsions for ocular delivery of VCZ.
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2. Experimental 2.1. Materials VCZ was a gift sample from Lifecare Innovations Pvt. Ltd. (Gurgaon, India). Oleic acid (OA), isopropyl myristate (IPM), tween 80, propylene glycol, cotton seed oil, castor oil liquid paraffin, span 80 were purchased from S.D. Fine Chemicals (Mumbai, India). Plurol oleique was a gift sample from Gattefosse India (Mumbai, India). Dialysis membrane (MW cut-off 12–14 kDa) was purchased from HiMedia Laboratories (Mumbai, India). All other chemicals used in the study were of analytical reagent grade. 2.2. Methods 2.2.1. HPLC analysis of VCZ A reversed phase HPLC method was developed and validated for analysis of VCZ. The HPLC (Shimadzu, Kyoto, Japan) instrument was equipped with a model series LC-10 ADVP pump, SCL-10 AVP system controller, DGU-12 A Degasser, Rheodyne 7725i injector with a 20-L loop and a SPD-10 AVP ultraviolet-visible detector. Separation and quantitation were made on a C18 reverse phase (250 mm × 4.6 mm (internal diameter), 5-m Inertsil ODS-3) column that was operated at 40 ◦ C. The mobile phase comprises a mixture of methanol and 0.1% acetic acid (pH 4) in the ratio of 70:30 (v/v), which was run at a flow rate of 1 mL/min. The eluents were analyzed spectrophotometrically at 256 nm. The retention time of VCZ was obtained at 5.45 min. The method developed was validated for linearity, precision and accuracy. A linear standard plot was obtained with R2 = 0.9985. For precision (inter- and intra-day), the relative standard deviation was found below 2% while the %recovery (accuracy) was found between 98.84 and 99.65%. Limit of detection (LOD) and limit of quantitation was found as 0.1 and 0.3 g/mL, respectively. 2.2.2. Equilibrium solubility studies for VCZ Drug solubility in non-aqueous components is an important parameter for development of effective ophthalmic preparations. Solubility studies of VCZ were performed by mixing an excess amount of drug with various non-aqueous solvents (oils and surfactants) using a water bath shaker at room temperature (37◦ ± 2 ◦ C) for 72 h to reach equilibrium. The equilibrated samples were centrifuged at 3000 rpm for 15 min. The supernatant was filtered through a 0.45 membrane filter and the amount of VCZ solubilized was analyzed using UV–visible spectrophotometer at 256 nm. 2.2.3. Construction of pseudo-ternary diagrams For construction of each phase diagram, oil and specific Smix ratio (surfactant:co-surfactant) were mixed carefully in diverse weight ratios from 9:1 to 1:9 (% w/w). Each weight ratio mixture was gradually titrated with distilled water and visual appearance was noted down for clear and easily flowable o/w microemulsions. 2.2.4. Stability evaluation of microemulsion and dispersibility test 2.2.4.1. Thermodynamic stability. Various thermodynamic stability tests like heating–cooling cycle, centrifugation and freeze–thaw cycle were performed on selected regions of microemulsions. Formulations that passed these stress tests were further subjected to dispersibility test. 2.2.4.2. Dispersibility test. This test assesses the efficiency of selfemulsification for VCZ microemulsions. 1 mL of each formulation was added to 500 mL of water at 37 ± 0.5 ◦ C and the in vitro emulsification rates as well as appearance of microemulsions was graded visually.
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2.2.5. Physiochemical interactions To determine the presence and extent of interactions of VCZ with different components of microemulsions (oil and surfactant/co-surfactant) NMR and FTIR spectroscopy was used. H NMR measurements were performed on NMR spectrometer (Bruker Avance III 400 MHz, Wageningen, Netherlands) and the chemical shifts were determined using D2 O as internal locking agent. FTIR spectra were obtained using FTIR spectrophotometer (Spectrum Two, PerkinElmer, USA). Transmittance (%T) was recorded in the spectral region of 500–4500 cm−1 using a resolution of 4 cm−1 and 40 scans. 2.2.6. Globule size, polydispersity index (PDI) and drug content The average globule size and PDI of microemulsions were determined by photon correlation spectroscopy. Measurements were made using Zetasizer 1000 HS (Malvern Instruments, Worcestershire, UK), wherein light scattering was monitored at 25 ◦ C at a 90o angle. Drug content of microemulsions was analyzed by UV–visible spectrophotometer (Shimadzu 1700, Japan) at 256 nm. 2.2.7. Viscosity, pH and conductivity The viscosity of undiluted microemulsions was determined using Brookfield DV-II+ viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA 02346, USA) using spindle no. 27 at 25 ± 0.5 ◦ C. The pH and conductivity of nanoformulations were determined using a pH meter (CyberScan pH 510, Eutech Instruments) conductivity meter (ELICO, CM 180) at 25 ± 0.5 ◦ C. 2.2.8. Surface morphology and structure Transmission electron microscopy (Hitachi, Tokyo, Japan) was used to carry out morphological and structural examination of drug-loaded microemulsions on H7500 machine operating at 100 kV capable of point-to-point resolution. 0.5 mL droplet of the formulation stained with 0.5% aqueous solution of phosphotungstic acid was directly positioned on the copper electron microscopy grids. Combinations of different bright-field imaging at increasing magnification were used to reveal the form and size of the microemulsions. 2.2.9. In vitro drug release A volume of 2.0 mL of the microemulsion was enclosed in a dialysis bag (cellulose membrane, mw cut-off 12400) and incubated in 40 mL release medium at 37◦ ± 0.5 ◦ C under mild agitation in a water bath. Simulated phosphate buffer saline (PBS, pH 7.4) was used as the release medium. At predetermined time intervals, 500 L of the samples were withdrawn from the incubation medium and analyzed spectrophotometrically at 256 nm. After sampling, 500 L of fresh medium was added in the incubation medium. 2.2.10. Ex vivo permeation study All animal experiments were carried out after approval of the protocol by the Institutional Animal Ethical Care committee (IAEC), Panjab University, Chandigarh, India, and conducted according to the Indian National Science Academy (INSA) guidelines for the use and care of experimental animals. The ex vivo permeation study was performed on the excised goat eye collected within 30 min after sacrifice from the slaughter house. Fresh excised cornea was tied to one side of the open tube (donor compartment) in such a way that its epithelial surface faced the donor compartment. The tube was submerged carefully in a beaker containing 40 mL of PBS, pH 7.4 (receptor compartment). Phosphate buffer was stirred at 50 rpm and maintained at 37◦ ± 0.5 ◦ C. About 2.01 cm2 corneal surface area was exposed to the donor cell and made available for drug permeation. Samples (0.5 mL) were withdrawn from the receptor
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cell at regular intervals for 12 h and analyzed by HPLC. Permeation flux was calculated as the ratio of drug permeation rate from the corneal tissue and the cross-sectional area of the tissue. For hydration study, each cornea (after 12 h) was weighed, and soaked in methanol, dried overnight at 90 ◦ C and reweighed. The corneal hydration was calculated from the difference in weight [23]. The amount of drug retained within the corneal tissue was determined using HPLC after rinsing (with PBS) and homogenizing the tissue. 2.2.11. Statistical analysis Simple analysis of variance (one-way ANOVA, GraphPad Prism 5) was used to determine statistically significant differences between the results and values with p < 0.05 were considered statistically significant as analyzed by the Dunnett multiple comparison test. 3. Results and discussion 3.1. Solubility studies and component selection Based on solubility studies (Table 1), oleic acid and isopropyl myristate was selected as the oil carrier while tween 80 (HLB = 15) and propylene glycol (HLB = 11.6) was selected as surfactant and cosurfactant, respectively, for formulation of o/w microemulsion. The microemulsion was prepared by drop-wise addition of the required
Table 1 VCZ solubility in various non-aqueous (oily) components. Solubility in mg/mL (mean ±SD, n = 3) Oil Oleic acid Isopropyl myristate Cotton seed oil Castor oil Liquid paraffin Surfactant/co-surfactant Tween 80 Propylene glycol Plurol Oleique Span 80
36.95 ± 1.34 14.04 ± 2.12 12.53 ± 1.13 4.54 ± 0.98 3.24 ± 2.11 26.86 ± 2.11 36.55 ± 1.24 4.82 ± 1.73 1.23 ± 1.17
amount of water into the pre-mixed solution of oil, surfactant and co-surfactant under magnetic stirring. For each oil phase, two separate phase diagrams were constructed comprising different Smix ratio (tween 80: propylene glycol) i.e., 1:1 and 2:1 (Fig. 1). These pseudo-ternary phase diagrams (consisting oil, Smix and water) demonstrated an extensive region of microemulsion formation. In addition, phase diagrams also help in determination of concentration range of components used for formulation of microemulsion.
Fig. 1. Optimization of microemulsion region using phase diagram.
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Table 2 Composition of optimized microemulsion batches. Phase diagram
Microemulsion batches
VCZ (%)
Oil (%) OA
Smix (%):Propylene glycol IPM
1:1
2:1
Water (%)
I
ME-1 ME-2 ME-3
0.1 0.1 0.1
5 5 10
– – –
50 55 55
– – –
45 40 35
II
ME-4 ME-5 ME-6
0.1 0.1 0.1
5 5 10
– – –
– – –
45 50 50
50 45 40
III
ME-7 ME-8 ME-9
0.1 0.1 0.1
– – –
5 5 10
35 40 50
– – –
60 55 40
IV
ME-10 ME-11 ME-12
0.1 0.1 0.1
– – –
5 5 10
– – –
35 40 50
60 55 40
3.2. Formulation selection and stability studies 3.2.1. Thermodynamic stability From each phase diagram, points having different concentration of oil with minimal Smix concentration were selected. The selected formulations were subjected to different thermodynamic stress tests as mentioned earlier. 3.2.2. Dispersibility test Those formulations, which remained stable after exposure to thermodynamic variations, were taken for dispersibility tests (based on emulsification rate and appearance). Formulations from different phase diagrams (I–IV) that passed dispersibility test in Grade A (rapid and clear) and B (rapid and bluish white) were selected for further study, as these formulations will remain stable when dispersed at targeted sites. For grades A and B there was no drug precipitation upon dilution, demonstrating that the microemulsions formed were capable of keeping VCZ solubilized. While formulations graded as C (moderate and milky white), D (gradual and grayish-white) and E (gradual with the presence of large oil globules) were discarded out due to the possibility of phase separation on dilution, which could lead to precipitation of poorly soluble drug (data given in supplementary files). Finally, the optimized formulations (from different phase diagrams) were selected to perform further studies (Table 2). 3.3. Physiochemical interaction No significant interactions were found between VCZ and the excipient (oil and surfactant/co-surfactant) used in microemulsions as confirmed by H NMR and FTIR spectra. H NMR spectra showed almost negligible interaction except a slight chemical shift in proton of the OH group of VCZ. On the other hand, IR spectra
of microemulsions retain all important peaks of drug like OH (3200–3600 cm−1 ), C H alkane (2800–3000 cm−1 ), C C aromatic (1400–1600 cm−1 ), C N aryl (1250–1360). 3.4. Globule size, polydispersity index (PDI) and drug content Globule size of microemulsions was found in the range of 160–280 nm (Table 3). It was observed that globule size enhances on increasing oil concentration (keeping surfactant concentration constant) while size reduces on enhancing surfactant concentration (keeping oil concentration constant). The polydispersity index (PDI) of the microemulsions was found below 0.4 which confirmed narrow size distribution of oil droplets. Generally, for narrow distribution PDI ranges from 0.01 to 0.5 while samples with very broad size distribution have PDI > 0.7 [24]. The drug content of all batches was found >50% which varied in the range of 53–72% (Table 3). To ensure minimal size variations and narrow distribution, the nanoformulations were filtered through a 0.45 membrane filter. This procedure separates out the larger globules/precipitates that may be the reason for lower/varying drug content. 3.5. pH, viscosity and conductivity The pH values of microemulsions were varied from the range 6.11 to 6.95 (Table 3). The eye has limited buffering capacity as the ocular comfort ranges from pH 6.6 to 7.8 for topical formulations [25]. Since the pH of therapeutic substances applied as eye drops vary from 3.5 to 8.5, the pH values of the prepared microemulsions could be counted in the tolerable/acceptable range [26]. Apart from pH, viscosity is another important parameter to assess the efficiency of formulation (low viscosity causes immediate drainage, hence reduces bioavailability) and its compatibility
Table 3 Physiochemical properties of VCZ microemulsions (Mean ± SD, n = 3). Microemulsion batches
Size (nm)
PDI
Drug content (%)
Viscosity (cP)
pH
Conductivity (S)
ME-1 ME-2 ME-3 ME-4 ME-5 ME-6 ME-7 ME-8 ME-9 ME-10 ME-11 ME-12
210.8 ± 1.23 165.2 ± 1.67 280.2 ± 2.45 213.4 ± 1.89 190.1 ± 3.44 268.1 ± 2.56 226.8 ± 3.15 180.8 ± 2.66 243.1 ± 2.98 168.3 ± 3.17 160.6 ± 2.11 213.7 ± 1.93
0.293 ± 0.01 0.28 ± 0.01 0.34 ± 0.03 0.404 ± 0.02 0.411 ± 0.02 0.208 ± 0.06 0.303 ± 0.04 0.409 ± 0.02 0.184 ± 0.03 0.323 ± 0.06 0.346 ± 0.03 0.055 ± 0.02
64.52 ± 0.26 66.31 ± 0.31 53.16 ± 2.21 66.32 ± 1.11 72.14 ± 0.64 54.23 ± 0.71 64.21 ± 0.29 65.23 ± 0.62 57.31 ± 0.44 67.31 ± 2.13 68.24 ± 0.74 61.43 ± 2.21
8.32 ± 0.21 8.99 ± 0.33 13.55 ± 0.17 11.67 ± 0.24 14.02 ± 0.11 16.08 ± 0.34 7.16 ± 0.26 9.89 ± 0.33 13.83 ± 0.17 6.38 ± 0.25 11.31 ± 0.29 16.03 ± 0.23
6.94 ± 0.32 6.82 ± 0.36 6.11 ± 0.21 5.78 ± 0.32 6.95 ± 0.16 6.51 ± 0.33 6.62 ± 0.23 6.03 ± 0.21 6.89 ± 0.14 6.35 ± 0.13 6.15 ± 0.21 6.89 ± 0.11
115.4 ± 2.23 114.5 ± 1.54 112.7 ± 2.78 108.2 ± 3.22 119.5 ± 2.06 117.4 ± 3.11 118.6 ± 2.14 111.3 ± 3.02 113.4 ± 1.78 102.1 ± 2.34 105.6 ± 3.05 104.7 ± 2.79
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Fig. 2. (a) Viscosity graph of selected microemulsion batches; (b) Effect of dilution on conductivity of microemulsion.
to eye (high viscosity results in blurred vision and blinking pain). The viscosity value of microemulsions (at 100 rpm) varied from 5.16 to 16.03 cP which fulfills the criteria for effective eye drops (<20 cP). Fig. 2(a) exhibited non-Newtonian flow (shear thinning) of microemulsions i.e. viscosity reduces at higher shear rate and vice versa. Such type of flow is usually preferred for ophthalmic formulations as during blinking the shear rates are very high and hence these shear-thinning fluids exhibit low viscosity. While during the period of inter blink during which tear drainage occurs, the high viscosity of these fluids leads to reduced drainage and a concurrent increase in precorneal residence time [27]. Conductivity different formulations was shown in Table 3 that ranged from 100.4 to 119.5 S. Such higher conductivity values confirm the existence of o/w microemulsions (w/o formulations generally exhibit lower conductivity). Fig. 2(b) exhibited the effect of dilution (% water) on the conductivity plot of microemulsions. The conductivity chart follows a bell-shaped curve with percolation transition, i.e. changes in conductivity with composition. Conductivity plot exhibited an increase in conductance with dilution i.e. system changes from insulator to a conductor. This phenomenon could be explained by the inter droplet interaction, which results in transfer of charge from one drop to another (increased concentration of discrete droplets). At a certain point (of dilution), conductivity of the formulations get declined that might be due to enhanced viscosity because of clustering of nanodroplets (decreased concentration of discrete droplets) [28]. 3.6. Surface morphology and structure TEM images showed spherical oil globules of microemulsions with diameters below 200 nm which appear darker with bright surroundings (Fig. 3). The results were in agreement with the globule size analysis performed by zetasizer. Nanodroplets were randomly dispersed without any agglomeration thorough out the field as observed under TEM.
Fig. 4. In vitro release profile of selected microemulsion batches (mean ±SD, n = 3).
3.7. In vitro drug release Optimized microemulsion batches (globule size < 200 nm with lower Smix concentration) each from different phase diagrams were selected for in vitro release study in phosphate buffered saline (pH 7.4) for 12 h (Fig. 4). The release profiles of microemulsions were compared with the drug suspension (VCZ + Smix ). All the microemulsions batches showed burst release (>25%) in the first 0.5 h followed by controlled release (>70%) for the next 12 h. In contrast, the drug suspension exhibited about 95% release within 6 h. To examine the mechanism of release from microemulsions system, data were fitted to various mathematical models like zero order, first order, Higuchi, Korsemeyer–Peppas and Hixson–Crowell model. Among the kinetic models employed, Higuchi model showed a better fit as R2 value was comparatively
Fig. 3. TEM images of microemulsions (ME-2 and ME-10).
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Fig. 5. (a) Flux of VCZ microemulsions through excised cornea; (b) Amount of VCZ retained within cornea (mean ±SD, n = 3). (** P ≤ 0.01; statistically significant as analyzed by Dunnett multiple comparison test)
higher than other release models (data not shown). The values of n (release exponent) in the Peppas model were found below 0.5 that confirmed Fickian release of VCZ form the formulations. The value of n in the Peppas model is used to characterize different release mechanisms. For nanospheres, values of 0.43, 0.43 < n < 0.85 and 0.85 are related to Fickian diffusion (case I transport), anomalous, and case II transport (zero order release), respectively [29].
3.8. Ex vivo permeation study The nanoformulations exhibited about 3-fold higher permeation of VCZ through the excised cornea in comparison to the drug suspension after 12 h (Table 3). VCZ flux was found significantly higher for microemulsions in contrast with the drug suspension (Fig. 5 (a)). A statistically significant drug accumulation in corneal tissue was found for microemulsions when compared with VCZ suspension (Fig. 5(b)). These results support the enhanced permeation properties of VCZ in the form of microemulsions. Moreover, the increased drug permeation and/or accumulation could be further justified by the permeation enhancing properties of oleic acid and isopropyl myristate used in the preparation of microemulsions [30]. The excised cornea exposed to VCZ formulations had shown quite acceptable values for hydration levels, confirming the good corneal integrity (Table 3). Generally, the ocular damage is assessed by determination of hydration level of the cornea. For a healthy cornea, the hydration level should be in the range between 76 and 80% [31].
4. Conclusion VCZ microemulsions were successfully developed and characterized using different parameters. An improved property of VCZ microemulsions was observed in comparison to its suspension form. With respect to various parameters studied (like smaller size, minimum surfactant concentration, desired viscosity with excellent release and permeation), the batch ME-10 was selected as the most appropriate and optimized batch that could be studied for in vivo experiments in future to establish its potential as an effective and efficient ophthalmic formulation.
Acknowledgment The authors are thankful to University Grant Commission (UGC), New Delhi, India for providing financial support to this project.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Preparation and optimization of voriconazole microemulsion for ocular delivery Rakesh Kumar, V.R. Sinha ∗ UIPS, Panjab University, Chandigarh 160014, India
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 1 February 2014 Accepted 6 February 2014 Available online 15 February 2014 Keywords: Voriconazole Microemulsion Phase diagram Optimization Ocular
a b s t r a c t Optimized microemulsions (o/w type) of voriconazole were formulated for efficient ocular delivery. Optimized batches were selected through construction of phase diagrams following stability studies. No significant physiochemical interactions were found between the drug and excipient (oil and surfactant/co-surfactant) as confirmed by H NMR and FTIR studies. Drug content was found between 53 and 72% depending on size and composition. Selected microemulsion batches exhibited shear thinning properties with acceptable viscosities. Globule size analyzed by zetasizer as well as TEM images of selected batches were found within the desired range (<200 nm). In vitro release studies of microemulsions exhibited sustained release property (>70% in 12 h). Ex vivo permeation study also supported the enhanced drug flux through cornea from microemulsions. Based on size, surfactant/co-surfactant concentration, viscosity, drug content and release studies, the microemulsion batch ME-10 was selected for future in vivo studies. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Topical application is one of the most widely preferred routes of administration for treating ocular infections/diseases as about 90% of the marketed ophthalmic formulations are in the form of eye drops. Features like patient compatibility, tolerability, the easier production method and economical cost make eye drops acceptability wider [1,2]. However, the meager bioavailability (<5%) of conventional eye drops (suspensions, solutions, etc.) is one of the major concern that could be ameliorated through the application of novel pharmaceutical approaches [3]. Application of nanotechnology-based formulations (like nanosuspension, nanoemulsion/microemulsion, solid lipid nanoparticle etc.) in the field of ophthalmology offers several beneficial features like improved solubility (for lipophilic drugs), targeted delivery and controlled release of therapeutic agents [4–7]. Microemulsions are one of the interesting and promising sub-micron carriers for ocular drug delivery. These are transparent, single optically isotropic and thermodynamically stable dispersions of water, oil and amphiphilic compounds (surfactant and co-surfactant) [8]. Microemulsions offer several advantages
∗ Corresponding author. Tel.: +911722534101. E-mail address: Sinha [email protected] (V.R. Sinha). http://dx.doi.org/10.1016/j.colsurfb.2014.02.007 0927-7765/© 2014 Elsevier B.V. All rights reserved.
like improved drug-loading and bioavailability (facilitating transcorneal penetration) with acceptable biocompatibility (due to presence of physiological lipids/oils) over polymeric nanoformulations. Microemulsion of many ocular drugs like ofloxacin, timolol, tacrolimus, everolimus and prednisolone were successfully prepared with sustained effect and better penetrability [9–13]. Voriconazole (VCZ), C16 H14 F3 N5 O, a second generation antifungal agent possesses phenomenal characteristics like broadspectrum activity, activity against resistant fungal species, and acceptable tolerability [14–16]. Almost 100% in vitro susceptibility was observed against various fungal isolates associated with keratitis and endophthalmitis. Moreover, studies suggested an excellent efficacy of VCZ against several ocular mycoses following topical administration [17–19]. Peng et al. fabricated PLGA [Poly (lactide-co-glycolide)] nanoparticles loaded with VCZ to improve agglomeration and antifungal efficacy in mice (renal tissue) [20]. Similarly, Sinha et al. exhibited excellent pulmonary deposition of VCZ following inhalational delivery of PLGA nanoparticles [21]. El-Hadidy et al. demonstrated significant enhancement in antifungal activity of VCZ microemulsion in comparison to drug supersaturated solution for topical effect (dermal and transdermal) [22]. So far, no topical (ocular) formulation of VCZ has been available in the market, though several researchers supported the need of effective topical delivery. This article proposes the formulation and optimization of o/w microemulsions for ocular delivery of VCZ.
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2. Experimental 2.1. Materials VCZ was a gift sample from Lifecare Innovations Pvt. Ltd. (Gurgaon, India). Oleic acid (OA), isopropyl myristate (IPM), tween 80, propylene glycol, cotton seed oil, castor oil liquid paraffin, span 80 were purchased from S.D. Fine Chemicals (Mumbai, India). Plurol oleique was a gift sample from Gattefosse India (Mumbai, India). Dialysis membrane (MW cut-off 12–14 kDa) was purchased from HiMedia Laboratories (Mumbai, India). All other chemicals used in the study were of analytical reagent grade. 2.2. Methods 2.2.1. HPLC analysis of VCZ A reversed phase HPLC method was developed and validated for analysis of VCZ. The HPLC (Shimadzu, Kyoto, Japan) instrument was equipped with a model series LC-10 ADVP pump, SCL-10 AVP system controller, DGU-12 A Degasser, Rheodyne 7725i injector with a 20-L loop and a SPD-10 AVP ultraviolet-visible detector. Separation and quantitation were made on a C18 reverse phase (250 mm × 4.6 mm (internal diameter), 5-m Inertsil ODS-3) column that was operated at 40 ◦ C. The mobile phase comprises a mixture of methanol and 0.1% acetic acid (pH 4) in the ratio of 70:30 (v/v), which was run at a flow rate of 1 mL/min. The eluents were analyzed spectrophotometrically at 256 nm. The retention time of VCZ was obtained at 5.45 min. The method developed was validated for linearity, precision and accuracy. A linear standard plot was obtained with R2 = 0.9985. For precision (inter- and intra-day), the relative standard deviation was found below 2% while the %recovery (accuracy) was found between 98.84 and 99.65%. Limit of detection (LOD) and limit of quantitation was found as 0.1 and 0.3 g/mL, respectively. 2.2.2. Equilibrium solubility studies for VCZ Drug solubility in non-aqueous components is an important parameter for development of effective ophthalmic preparations. Solubility studies of VCZ were performed by mixing an excess amount of drug with various non-aqueous solvents (oils and surfactants) using a water bath shaker at room temperature (37◦ ± 2 ◦ C) for 72 h to reach equilibrium. The equilibrated samples were centrifuged at 3000 rpm for 15 min. The supernatant was filtered through a 0.45 membrane filter and the amount of VCZ solubilized was analyzed using UV–visible spectrophotometer at 256 nm. 2.2.3. Construction of pseudo-ternary diagrams For construction of each phase diagram, oil and specific Smix ratio (surfactant:co-surfactant) were mixed carefully in diverse weight ratios from 9:1 to 1:9 (% w/w). Each weight ratio mixture was gradually titrated with distilled water and visual appearance was noted down for clear and easily flowable o/w microemulsions. 2.2.4. Stability evaluation of microemulsion and dispersibility test 2.2.4.1. Thermodynamic stability. Various thermodynamic stability tests like heating–cooling cycle, centrifugation and freeze–thaw cycle were performed on selected regions of microemulsions. Formulations that passed these stress tests were further subjected to dispersibility test. 2.2.4.2. Dispersibility test. This test assesses the efficiency of selfemulsification for VCZ microemulsions. 1 mL of each formulation was added to 500 mL of water at 37 ± 0.5 ◦ C and the in vitro emulsification rates as well as appearance of microemulsions was graded visually.
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2.2.5. Physiochemical interactions To determine the presence and extent of interactions of VCZ with different components of microemulsions (oil and surfactant/co-surfactant) NMR and FTIR spectroscopy was used. H NMR measurements were performed on NMR spectrometer (Bruker Avance III 400 MHz, Wageningen, Netherlands) and the chemical shifts were determined using D2 O as internal locking agent. FTIR spectra were obtained using FTIR spectrophotometer (Spectrum Two, PerkinElmer, USA). Transmittance (%T) was recorded in the spectral region of 500–4500 cm−1 using a resolution of 4 cm−1 and 40 scans. 2.2.6. Globule size, polydispersity index (PDI) and drug content The average globule size and PDI of microemulsions were determined by photon correlation spectroscopy. Measurements were made using Zetasizer 1000 HS (Malvern Instruments, Worcestershire, UK), wherein light scattering was monitored at 25 ◦ C at a 90o angle. Drug content of microemulsions was analyzed by UV–visible spectrophotometer (Shimadzu 1700, Japan) at 256 nm. 2.2.7. Viscosity, pH and conductivity The viscosity of undiluted microemulsions was determined using Brookfield DV-II+ viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA 02346, USA) using spindle no. 27 at 25 ± 0.5 ◦ C. The pH and conductivity of nanoformulations were determined using a pH meter (CyberScan pH 510, Eutech Instruments) conductivity meter (ELICO, CM 180) at 25 ± 0.5 ◦ C. 2.2.8. Surface morphology and structure Transmission electron microscopy (Hitachi, Tokyo, Japan) was used to carry out morphological and structural examination of drug-loaded microemulsions on H7500 machine operating at 100 kV capable of point-to-point resolution. 0.5 mL droplet of the formulation stained with 0.5% aqueous solution of phosphotungstic acid was directly positioned on the copper electron microscopy grids. Combinations of different bright-field imaging at increasing magnification were used to reveal the form and size of the microemulsions. 2.2.9. In vitro drug release A volume of 2.0 mL of the microemulsion was enclosed in a dialysis bag (cellulose membrane, mw cut-off 12400) and incubated in 40 mL release medium at 37◦ ± 0.5 ◦ C under mild agitation in a water bath. Simulated phosphate buffer saline (PBS, pH 7.4) was used as the release medium. At predetermined time intervals, 500 L of the samples were withdrawn from the incubation medium and analyzed spectrophotometrically at 256 nm. After sampling, 500 L of fresh medium was added in the incubation medium. 2.2.10. Ex vivo permeation study All animal experiments were carried out after approval of the protocol by the Institutional Animal Ethical Care committee (IAEC), Panjab University, Chandigarh, India, and conducted according to the Indian National Science Academy (INSA) guidelines for the use and care of experimental animals. The ex vivo permeation study was performed on the excised goat eye collected within 30 min after sacrifice from the slaughter house. Fresh excised cornea was tied to one side of the open tube (donor compartment) in such a way that its epithelial surface faced the donor compartment. The tube was submerged carefully in a beaker containing 40 mL of PBS, pH 7.4 (receptor compartment). Phosphate buffer was stirred at 50 rpm and maintained at 37◦ ± 0.5 ◦ C. About 2.01 cm2 corneal surface area was exposed to the donor cell and made available for drug permeation. Samples (0.5 mL) were withdrawn from the receptor
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cell at regular intervals for 12 h and analyzed by HPLC. Permeation flux was calculated as the ratio of drug permeation rate from the corneal tissue and the cross-sectional area of the tissue. For hydration study, each cornea (after 12 h) was weighed, and soaked in methanol, dried overnight at 90 ◦ C and reweighed. The corneal hydration was calculated from the difference in weight [23]. The amount of drug retained within the corneal tissue was determined using HPLC after rinsing (with PBS) and homogenizing the tissue. 2.2.11. Statistical analysis Simple analysis of variance (one-way ANOVA, GraphPad Prism 5) was used to determine statistically significant differences between the results and values with p < 0.05 were considered statistically significant as analyzed by the Dunnett multiple comparison test. 3. Results and discussion 3.1. Solubility studies and component selection Based on solubility studies (Table 1), oleic acid and isopropyl myristate was selected as the oil carrier while tween 80 (HLB = 15) and propylene glycol (HLB = 11.6) was selected as surfactant and cosurfactant, respectively, for formulation of o/w microemulsion. The microemulsion was prepared by drop-wise addition of the required
Table 1 VCZ solubility in various non-aqueous (oily) components. Solubility in mg/mL (mean ±SD, n = 3) Oil Oleic acid Isopropyl myristate Cotton seed oil Castor oil Liquid paraffin Surfactant/co-surfactant Tween 80 Propylene glycol Plurol Oleique Span 80
36.95 ± 1.34 14.04 ± 2.12 12.53 ± 1.13 4.54 ± 0.98 3.24 ± 2.11 26.86 ± 2.11 36.55 ± 1.24 4.82 ± 1.73 1.23 ± 1.17
amount of water into the pre-mixed solution of oil, surfactant and co-surfactant under magnetic stirring. For each oil phase, two separate phase diagrams were constructed comprising different Smix ratio (tween 80: propylene glycol) i.e., 1:1 and 2:1 (Fig. 1). These pseudo-ternary phase diagrams (consisting oil, Smix and water) demonstrated an extensive region of microemulsion formation. In addition, phase diagrams also help in determination of concentration range of components used for formulation of microemulsion.
Fig. 1. Optimization of microemulsion region using phase diagram.
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Table 2 Composition of optimized microemulsion batches. Phase diagram
Microemulsion batches
VCZ (%)
Oil (%) OA
Smix (%):Propylene glycol IPM
1:1
2:1
Water (%)
I
ME-1 ME-2 ME-3
0.1 0.1 0.1
5 5 10
– – –
50 55 55
– – –
45 40 35
II
ME-4 ME-5 ME-6
0.1 0.1 0.1
5 5 10
– – –
– – –
45 50 50
50 45 40
III
ME-7 ME-8 ME-9
0.1 0.1 0.1
– – –
5 5 10
35 40 50
– – –
60 55 40
IV
ME-10 ME-11 ME-12
0.1 0.1 0.1
– – –
5 5 10
– – –
35 40 50
60 55 40
3.2. Formulation selection and stability studies 3.2.1. Thermodynamic stability From each phase diagram, points having different concentration of oil with minimal Smix concentration were selected. The selected formulations were subjected to different thermodynamic stress tests as mentioned earlier. 3.2.2. Dispersibility test Those formulations, which remained stable after exposure to thermodynamic variations, were taken for dispersibility tests (based on emulsification rate and appearance). Formulations from different phase diagrams (I–IV) that passed dispersibility test in Grade A (rapid and clear) and B (rapid and bluish white) were selected for further study, as these formulations will remain stable when dispersed at targeted sites. For grades A and B there was no drug precipitation upon dilution, demonstrating that the microemulsions formed were capable of keeping VCZ solubilized. While formulations graded as C (moderate and milky white), D (gradual and grayish-white) and E (gradual with the presence of large oil globules) were discarded out due to the possibility of phase separation on dilution, which could lead to precipitation of poorly soluble drug (data given in supplementary files). Finally, the optimized formulations (from different phase diagrams) were selected to perform further studies (Table 2). 3.3. Physiochemical interaction No significant interactions were found between VCZ and the excipient (oil and surfactant/co-surfactant) used in microemulsions as confirmed by H NMR and FTIR spectra. H NMR spectra showed almost negligible interaction except a slight chemical shift in proton of the OH group of VCZ. On the other hand, IR spectra
of microemulsions retain all important peaks of drug like OH (3200–3600 cm−1 ), C H alkane (2800–3000 cm−1 ), C C aromatic (1400–1600 cm−1 ), C N aryl (1250–1360). 3.4. Globule size, polydispersity index (PDI) and drug content Globule size of microemulsions was found in the range of 160–280 nm (Table 3). It was observed that globule size enhances on increasing oil concentration (keeping surfactant concentration constant) while size reduces on enhancing surfactant concentration (keeping oil concentration constant). The polydispersity index (PDI) of the microemulsions was found below 0.4 which confirmed narrow size distribution of oil droplets. Generally, for narrow distribution PDI ranges from 0.01 to 0.5 while samples with very broad size distribution have PDI > 0.7 [24]. The drug content of all batches was found >50% which varied in the range of 53–72% (Table 3). To ensure minimal size variations and narrow distribution, the nanoformulations were filtered through a 0.45 membrane filter. This procedure separates out the larger globules/precipitates that may be the reason for lower/varying drug content. 3.5. pH, viscosity and conductivity The pH values of microemulsions were varied from the range 6.11 to 6.95 (Table 3). The eye has limited buffering capacity as the ocular comfort ranges from pH 6.6 to 7.8 for topical formulations [25]. Since the pH of therapeutic substances applied as eye drops vary from 3.5 to 8.5, the pH values of the prepared microemulsions could be counted in the tolerable/acceptable range [26]. Apart from pH, viscosity is another important parameter to assess the efficiency of formulation (low viscosity causes immediate drainage, hence reduces bioavailability) and its compatibility
Table 3 Physiochemical properties of VCZ microemulsions (Mean ± SD, n = 3). Microemulsion batches
Size (nm)
PDI
Drug content (%)
Viscosity (cP)
pH
Conductivity (S)
ME-1 ME-2 ME-3 ME-4 ME-5 ME-6 ME-7 ME-8 ME-9 ME-10 ME-11 ME-12
210.8 ± 1.23 165.2 ± 1.67 280.2 ± 2.45 213.4 ± 1.89 190.1 ± 3.44 268.1 ± 2.56 226.8 ± 3.15 180.8 ± 2.66 243.1 ± 2.98 168.3 ± 3.17 160.6 ± 2.11 213.7 ± 1.93
0.293 ± 0.01 0.28 ± 0.01 0.34 ± 0.03 0.404 ± 0.02 0.411 ± 0.02 0.208 ± 0.06 0.303 ± 0.04 0.409 ± 0.02 0.184 ± 0.03 0.323 ± 0.06 0.346 ± 0.03 0.055 ± 0.02
64.52 ± 0.26 66.31 ± 0.31 53.16 ± 2.21 66.32 ± 1.11 72.14 ± 0.64 54.23 ± 0.71 64.21 ± 0.29 65.23 ± 0.62 57.31 ± 0.44 67.31 ± 2.13 68.24 ± 0.74 61.43 ± 2.21
8.32 ± 0.21 8.99 ± 0.33 13.55 ± 0.17 11.67 ± 0.24 14.02 ± 0.11 16.08 ± 0.34 7.16 ± 0.26 9.89 ± 0.33 13.83 ± 0.17 6.38 ± 0.25 11.31 ± 0.29 16.03 ± 0.23
6.94 ± 0.32 6.82 ± 0.36 6.11 ± 0.21 5.78 ± 0.32 6.95 ± 0.16 6.51 ± 0.33 6.62 ± 0.23 6.03 ± 0.21 6.89 ± 0.14 6.35 ± 0.13 6.15 ± 0.21 6.89 ± 0.11
115.4 ± 2.23 114.5 ± 1.54 112.7 ± 2.78 108.2 ± 3.22 119.5 ± 2.06 117.4 ± 3.11 118.6 ± 2.14 111.3 ± 3.02 113.4 ± 1.78 102.1 ± 2.34 105.6 ± 3.05 104.7 ± 2.79
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Fig. 2. (a) Viscosity graph of selected microemulsion batches; (b) Effect of dilution on conductivity of microemulsion.
to eye (high viscosity results in blurred vision and blinking pain). The viscosity value of microemulsions (at 100 rpm) varied from 5.16 to 16.03 cP which fulfills the criteria for effective eye drops (<20 cP). Fig. 2(a) exhibited non-Newtonian flow (shear thinning) of microemulsions i.e. viscosity reduces at higher shear rate and vice versa. Such type of flow is usually preferred for ophthalmic formulations as during blinking the shear rates are very high and hence these shear-thinning fluids exhibit low viscosity. While during the period of inter blink during which tear drainage occurs, the high viscosity of these fluids leads to reduced drainage and a concurrent increase in precorneal residence time [27]. Conductivity different formulations was shown in Table 3 that ranged from 100.4 to 119.5 S. Such higher conductivity values confirm the existence of o/w microemulsions (w/o formulations generally exhibit lower conductivity). Fig. 2(b) exhibited the effect of dilution (% water) on the conductivity plot of microemulsions. The conductivity chart follows a bell-shaped curve with percolation transition, i.e. changes in conductivity with composition. Conductivity plot exhibited an increase in conductance with dilution i.e. system changes from insulator to a conductor. This phenomenon could be explained by the inter droplet interaction, which results in transfer of charge from one drop to another (increased concentration of discrete droplets). At a certain point (of dilution), conductivity of the formulations get declined that might be due to enhanced viscosity because of clustering of nanodroplets (decreased concentration of discrete droplets) [28]. 3.6. Surface morphology and structure TEM images showed spherical oil globules of microemulsions with diameters below 200 nm which appear darker with bright surroundings (Fig. 3). The results were in agreement with the globule size analysis performed by zetasizer. Nanodroplets were randomly dispersed without any agglomeration thorough out the field as observed under TEM.
Fig. 4. In vitro release profile of selected microemulsion batches (mean ±SD, n = 3).
3.7. In vitro drug release Optimized microemulsion batches (globule size < 200 nm with lower Smix concentration) each from different phase diagrams were selected for in vitro release study in phosphate buffered saline (pH 7.4) for 12 h (Fig. 4). The release profiles of microemulsions were compared with the drug suspension (VCZ + Smix ). All the microemulsions batches showed burst release (>25%) in the first 0.5 h followed by controlled release (>70%) for the next 12 h. In contrast, the drug suspension exhibited about 95% release within 6 h. To examine the mechanism of release from microemulsions system, data were fitted to various mathematical models like zero order, first order, Higuchi, Korsemeyer–Peppas and Hixson–Crowell model. Among the kinetic models employed, Higuchi model showed a better fit as R2 value was comparatively
Fig. 3. TEM images of microemulsions (ME-2 and ME-10).
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Fig. 5. (a) Flux of VCZ microemulsions through excised cornea; (b) Amount of VCZ retained within cornea (mean ±SD, n = 3). (** P ≤ 0.01; statistically significant as analyzed by Dunnett multiple comparison test)
higher than other release models (data not shown). The values of n (release exponent) in the Peppas model were found below 0.5 that confirmed Fickian release of VCZ form the formulations. The value of n in the Peppas model is used to characterize different release mechanisms. For nanospheres, values of 0.43, 0.43 < n < 0.85 and 0.85 are related to Fickian diffusion (case I transport), anomalous, and case II transport (zero order release), respectively [29].
3.8. Ex vivo permeation study The nanoformulations exhibited about 3-fold higher permeation of VCZ through the excised cornea in comparison to the drug suspension after 12 h (Table 3). VCZ flux was found significantly higher for microemulsions in contrast with the drug suspension (Fig. 5 (a)). A statistically significant drug accumulation in corneal tissue was found for microemulsions when compared with VCZ suspension (Fig. 5(b)). These results support the enhanced permeation properties of VCZ in the form of microemulsions. Moreover, the increased drug permeation and/or accumulation could be further justified by the permeation enhancing properties of oleic acid and isopropyl myristate used in the preparation of microemulsions [30]. The excised cornea exposed to VCZ formulations had shown quite acceptable values for hydration levels, confirming the good corneal integrity (Table 3). Generally, the ocular damage is assessed by determination of hydration level of the cornea. For a healthy cornea, the hydration level should be in the range between 76 and 80% [31].
4. Conclusion VCZ microemulsions were successfully developed and characterized using different parameters. An improved property of VCZ microemulsions was observed in comparison to its suspension form. With respect to various parameters studied (like smaller size, minimum surfactant concentration, desired viscosity with excellent release and permeation), the batch ME-10 was selected as the most appropriate and optimized batch that could be studied for in vivo experiments in future to establish its potential as an effective and efficient ophthalmic formulation.
Acknowledgment The authors are thankful to University Grant Commission (UGC), New Delhi, India for providing financial support to this project.
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