Materials
Ketoconazole (KTZ) was procured from a local pharmaceutical industry (Velite Pharmaceuticals, Ludhiana, Punjab, India). Phospholipon 90G and compritol 888 ATO (CATO) were obtained gift samples from Lipoid (Germany) and Gattefosse (France), respectively. Polyethylene glycol 400 (as cosolvent) and tween 80 (as surfactant) were received from CDH, Mumbai (India). Millipore water was used as aqueous solvent wherever required in the study. Fluorescence dye (fluorescein sodium) was purchased from Sigma Aldrich (Mumbai, India). All chemicals were of analytical grade.
Methods
Preliminary screening studies
Several batches of blank formulations were prepared to screen excipients such as solid lipid (Compritol® 888 ATO), surfactant (tween 80), cosolvent (polyethylene glycol 600) (PEG 600), and stabilizer (phospholipon 90G (P90G). Taguchi design was applied for factors and levels. Several variables (run cycles, speed and stirring time) of high pressure homogenization (HPH) technique were optimized to get stable SLNs (overnight benchtop stability).
Formulation method to load ketoconazole in solid lipid nanoparticles (KTZ-SLNs)
Preliminary study was carried out to select excipients and their levels (low and high). Based on benchtop stable product, the higher and the lower levels of factors were decided to feed as input parameters in experimental design software (Design Expert). The lipid organic phase was composed of CATO, PEG-600, and KTZ (fixed amount) which was heated to melt at 75°C. Similarly, the aqueous phase contains tween-80 and P90G previously set at the same temperature. The aqueous phase was stirred at high speed (1000 rpm) using stirrer (WiseTis, HG-15D, Daihan, Korea). The hot organic phase was slowly added to the aqueous phase under constant stirring to result in a coarse emulsion. The prepared coarse emulsion was passed through a high pressure homogenizer (HPH) (EmulsiFlex-C3, Avestin, Canada), at 1000 bar pressure for 7 cycles. The formed o/w emulsion was cooled to room temperature to achieve KTZ loaded SLNs (2% w/v). Thus, several batches of formulations were formulated as per dictated in experimental design (central composite design). In case of fluorescein sodium dye probed KTZ-SLNs, the same procedure was adopted and except dye was dissolved in aqueous phase.
Optimization process
Experimental protocol was designed to evaluate the critical factors and their significant levels to get the most robust formulation with optimal content of lipid (CATO) and tween 80 (surfactant). A central composite design (CCD) with α=1.414 was run in the Design Expert (version 8.0.1 Stat-Ease Inc. USA) [7]. CATO (X1) and tween 80 (X2) were selected as independent variables (factors). Similarly, mean particle size (Y1), %EE (Y2) and total drug content (Y3) were responses (dependent variables). In optimization process, total 13 runs were experimented at 5 levels (-α, -1, 0, +1, +α). A general polynomial mathematical quadratic equation was generated to quantify and establish a correlation between the independent (X) and dependent variables (Y):
Where Y is dependent variable with two coefficients (β1 & β2) of factors (X1 & X2). β0 is an intercept. β3 is a coefficient of interaction between factors X1 and X2, whereas β4 and β5 are the coefficients of quadratic terms “X1” and “X2”, respectively. Positive and negative signs indicate synergistic and antagonistic effect of factors on the response, respectively. ANOVA (analysis of variance) provides parameters (F, p, and r2 values) to validate the model applied for optimization process using the experimental design.
Characterization of KTZ loaded suggested SLNs formulations
Measurement of particle size, polydispersity index, and zeta potential
Particle size, size distribution and surface charge are critical factors to control in-vitro and in-vivo performance of product. Particle size and PDI were measured using photon correlation spectroscopy (PCS) technique which is based on the principle of light diffraction phenomenon. The sample was previously diluted with water (50 fold) for analysis (Beckman Coulter, Delsa™ Nano C, USA). Zeta potential of KTZ- SLNs dispersion was measured without sample dilution (Beckman Coulter, Delsa™ Nano C zetasizer, USA) at 25ºC and the electric field strength of 23.2 V/cm. Experiments were replicated for mean and standard deviation (n = 3).
Percent total drug content (%TDC) and entrapment efficiency (%EE)
KTZ-SLNs formulations contain 2%w/v of KTZ. Formulation (1 mL) was dissolved in chloroform: methanol mixture (2:1). The mixture of organic solvents were able to dissolve and disrupt solid lipid of tailored KTZ-SLNs. The mixture was filtered and the content of KTX was quantified using validated HPLC method. % EE was determined by dialyzing KTZ-SLN dispersion (1 mL) in a dialysis membrane (14K Da MW cut-off) immersed in 50 mL ethanol and stirred magnetically. After 1h KTZ-SLNs were removed from the bag, disrupted with suitable quantity of chloroform: methanol mixture (2:1) and amount of drug was determined by HPLC. The dialysate was decided based on the assumption that the entire quantity of unentrapped can dissolve in a suitable quantity (50 mL) in an appropriate time (≥1h) to accurately determine amount of unentrapped drug.
Desirability function
The desirability parameter was used to identify and evaluate the optimized formulation by experimental design. Mathematically, this is a numerical function parameter to identify possible interaction between factors. Moreover, it depends upon the set conditions of optimization process such as goal and importance given to each dependent and independent variables. The value of desirability function varies from zero to unity. Zero indicates the model is not fit and out of optimization whereas the value approaching to unity indicate the best fit of the model applied for optimization. The significant terms (p < 0.05) were chosen for final equations. The model was considered to be the best fit when the actual correlation coefficient (r2) value was close to the adjusted correlation coefficient (adjusted r2). Selected formulations of KTZ-SLNs were prepared from the design space and used as checkpoints to assess the prognostic behaviour of the developed mathematical model.
Preparation of ketoconazole suspension (KTZ-SUS)
KTZ suspension (KTZ-SUS) was prepared by method described before with slight modification [8]. An accurately weighed amount of KTZ was dispersed in water containing 1% w/v of tween 80 as surfactant and sodium salt of carboxymethyl cellulose (0.1%w/v) as suspending agent. The drug was rigorously stirred for 60 min in the aqueous phase to obtain a stable suspension with optimal consistency. Final strength of the suspension was equivalent to commercial product (2% w/v). This product was used in the further studies as control.
Thermal behaviour of the formulations
The thermal behaviours (fusion temperature and fusion enthalpy) of pure and formulations were assessed using a differential scanning calorimeter (DSC). A weighed amount (2 mg) of the samples (Lipid, KTZ, KTZ-SLNs, Blank SLNs) was placed in an aluminium pan and heated at a fixed heating rate (10ºC/min) till 300°C using DSC (821e Mettler Toledo, Switzerland). The generated thermograms were analysed, and marked for the values of any significant shift or disappearance/appearance of new peaks. The calorimeter was calibrated by pure Indium (melting point) for nitrogen flow and heating rate. Nitrogen gas was used at a purging rate of 50 mL /min.
Compatibility study using Fourier Transform Infra-red (FT-IR)
To negate any chemical interaction of the drug with explored excipients, the sample alone (KTZ) and formulations (KTZ-SLNs, and placebo SLNs) were subjected for FT-IR analysis. The FT-IR spectrometer (Agilent Technologies 630 Cary) was run for the sample using pellet method. A small amount of the sample was physically mixed with KBr followed by pellet formation. The pellet was processed for characteristic peaks using Micro Lab software. The samples were scanned over the range of 4000- 400 cm-1.
Solid state behaviour using powder X-ray diffraction (XRD) method
The prepared SLNs formulations were solid in nature and considered for improved solubility of the drug in solid matrix. In general, crystalline materials exhibit characteristic peaks in XRD graph. Therefore, it was required to assess solid state behaviour of the developed formulation. This was confirmed by analysing the nature of formulated nanoparticles using XRD (XPERT-PRO, PANalytical, Netherlands). KTZ-SLNs and blank SLNs dispersions were lyophilized prior for the analysis. The test sample was exposed to CuKα radiation (45 kV, 40 mA) with scanning angle ranged between 5° and 50°. The values of 2θ and scanning step time were 0.017° and 25s, respectively. Pure drug, lyophilized KTS-SLNs, blank SLNs (without drug) were analysed.
Surface morphology analysis
Surface morphology of prepared solid lipid nanoparticles (KTZ-SLNs) was examined by high resolution transmission electron microscopy (HR-TEM) and field emission scanning electron microscopy (FE-SEM). Prior to observation under HR-TEM and FE-SEM, KTZ-SLNs were diluted (50 X) with distilled water. The procedure for FE-SEM observations including placing the KTZ-SLNs dispersion on Nucleopore Track-Etch membrane and drying at room temperature. Dried membrane was attached to the silicon wafer using double sided carbon tape followed by sputter coating with gold under FE-SEM (FE-SEM SU8000, Hitachi, Japan). For HR-TEM KTZ-SLNs was stained (0.2% w/v of phosphotungstic acid) during 5 min in phosphate buffer at pH 6.8. Then, the excess phosphotungstic acid was removed using a filter paper. The stained sample of KTZ-SLNs was spread over carbon coated copper grid and was observed under HR-TEM (H-7500, Hitachi, Japan) at a voltage of 200 kV, for morphology (shape and size).
Release behaviour and mechanism
In-vitro release pattern of optimized formulation was studied using a dialysis membrane as per reported method [9-10]. A fixed volume (1 mL) of KTZ-SLNs and KTZ-SUS containing 20 mg of KTZ was loaded in the dialysis membrane (molecular weight cut-off of 12KDa). The dialysis membrane was soaked in water for 12 h before experiment. The dialysis membrane containing sample was suspended in a release medium (phosphate buffer solution, pH 7.4). Sink condition was maintained using dimethyl sulfoxide (DMSO). Sampling (2 mL) was carried out at various time points (1, 2, 4, 8, 12, 16, 24, 48, and 72 h). The withdrawn volume was replaced with fresh release medium (equal volume) at each time points. The sample withdrawn was filtered and analysed using validated HPLC method at λmax of 210 nm. Analysis was replicated for mean and standard deviation (n=6). Finally, various mathematical models were applied to investigate release mechanism (zero order, first order release, Higuchi model and Korsmeyer- Peppas model).
Drug permeation and deposition studies: Ex vivo performance across rat skin
Permeation potential and drug deposition were carried out using Franz diffusion cells as per reported method [11]. The optimized SLNs formulation was compared against drug suspension and marketed product. For this, rat skin (abdominal) was made free of hairs using digit trimmer without making any surgical cuts or injury. The excise and trimmed skin was placed between two chambers of Franz diffusion cell such that the upper layer faces the formulation and inner layer towards the receptor medium (PBS, pH 7.4). The receptor chamber was filled of release medium (30 mL) and set at 32±1°C under constant stirring using teflon coated magnetic bead [12]. The test sample (0.5 mL containing 10 mg of KTZ) was placed over exposed skin (available surface area of 2.1 cm2). Three samples (KTZ-SLNs, KTZ-SUS and KTZ-MKT) were studied separately under similar experimental conditions. The donor chamber was properly covered with paraffin film to avoid loss of solvent or dryness of the sample. The sampling was performed at different time points (0.5, 1, 2, 4, 6, 8, 12, 24, 48 and 72 h) followed by replacing equivalent volume of withdrawn sample with fresh release medium. Notably, DMSO (5%) was added to the receptor medium to maintain sink condition. The withdrawn sample was filtered using membrane filter (0.2 µm). The permeated amount of the drug across the skin was estimated using HPLC. Several permeation parameters (cumulative amount of drug permeation, permeation flux, and enhancement ratio) were calculated [13]. After completion of permeation study, the skin samples were removed and washed with running water for drug deposition (retention) study.
Skin retention studies
This study is an extension of the skin permeation study. After completion of the permeation study, the skin was washed with running water to remove adhered treated sample. Then, the skin was sliced into small pieces using surgical scissor and placed in a solution containing methanol and chloroform (2:1 ratio) [14]. The drug deposited or retained in the skin was extracted over 12 h under constant stirring at room temperature. Then, they were filtered and the filtrate was centrifuged (9000 rpm) for 15 min to get the supernatant. The obtained supernatant was analysed for the permeated amount of the drug using HPLC. The study was repeated for mean and standard values.
Dermatokinetics: An in-vivo study
The animal study was carried out in Wistar albino rats weighing about 250-300 g of both sexes. They were get issued from Institutional animal house UIPS (University Institute of Pharmaceutical Sciences) (Approved as regd. No. 45/GO/ReBiBt/S/99/CPCSEA). All of the animals were housed in conditioned room with free access of food and water as per guideline. Animals were randomly selected and grouped (n=6 per group) as per treatment schedule. The body surface of rats was properly inspected for any possible injury and abnormality. The dorsal surface was used to locate a site of application by making three areas (3 cm2) free of hairs. Each group received all three formulations at labelled location on the dorsal site. After 24 h of shaving, formulations (KTZ-SLNs, KTZ-SUS and KTZ-MKT), were applied with equivalent concentration and dose strength. Rat was ethically sacrificed at varied time points (2, 6, 12, 24, 48 and 72 h) for dermatokinetic study. Three skin samples were excised from the applied site and washed water to remove adhered content. Then, the skin samples were sliced into small pieces to extract the drug content by dispersing in a mixture of chloroform and methanol (2:1). The mixture was homogenized after 8 h and filtered. The filtrate was centrifuged to get a supernatant. The supernatant was used to estimate the extracted amount of the drug by HPLC method. The data obtained was fitted into one compartment open model. For dermatokinetics profile, the drug concentration versus time profile was estimated presented as a graph using a PK solver (version 1.1). Several dermatokinetics parameters such as area under the curve (AUC0–72 and AUC0-∞), the maximum drug concentration reached in the skin layer (Cmax), the time required to attain Cmax as Tmax were assessed.
Fluorescence microscopy study on human dermatome skin (EpiDermTM)
This was conducted to visualize the permeated KTZ tailored in SLNs using EpiDermTM as a skin model (MatTek Corporation, Ashland, USA). Fluorescein probed SLNs (F-SLNs) was prepared as per method discussed before. Approximately 30 μL of 2 % aqueous solution of fluorescein (aqueous solution) was taken as control and topically applied to the surface of EpiDermTM. Fluorescein was excited at 470 nm and the fluorescent emission was detected at 515 nm. Several representative images of the treated skin were visualized for mechanistic evaluation using fluorescent microscopy (IX71 Olympus Inverted Microscope, Olympus, Tokyo, Japan) at 2 h and 24 h with a 10X magnification.
Vibrational spectroscopic imaging techniques in human skin
Skin treatment procedure
Flash-frozen human skin with thickness 4cm2 (T-SKN-FF2CM) purchased from licensed supplier (ZenBio Inc, USA) was used for this study. All of the skin samples used in this study were from the same donor. 2.5 cm x 2.5 cm piece of skin was cut and cleaned. Formulations (KTZ-SLNs and KTZ-SUS) were applied topically on the skin surface in excess. Product was massaged on the skin using a glass rod and allowed to sit for 5 min. Skin was placed on a Franz diffusion cell for 3 and 24 h at 32˚C. After 3 and 24 h, the excess product on the skin surface was gently blotted with a wet kimwipe. To evaluate product penetration inside the skin, sample preparations were used. Transverse skins sections (8 µm) were obtained using cryo-microtome and scanned by ATR-FTIR imaging to visualize product penetration inside the different skin layers. Skin cross-sections (8µm) were cut using a cryostat. These cross-sections were scanned by ATR-FTIR imaging to evaluate product penetration inside the epidermis. ATR-FTIR images of the cross sections were recorded with a Spotlight 400 System (Perkin Elmer Instruments, Shelton, Conn., USA), consisting of a FTIR spectrometer with a mercury-cadmium-telluride (MCT) focal plane array detector. Images were collected in reflective mode at a spectral resolution of 4 cm-1 and 4 scans accumulations in the mid-infrared (MIR) region between 4000 and 750 cm-1 with a spatial resolution of 6.25 x 6.25 µm at room temperature (24ºC). The ATR imaging accessory used a germanium crystal placed directly in contact with the skin samples. All the data were processed (baseline correction, generation of spectroscopic parameters) using GRAMS/AI (Thermo Fisher Scientific) or ISys software from Spectral Dimensions (Olney, MD).
Confocal Raman spectroscopy imaging
Skin was also scanned by confocal Raman spectroscopy to evaluate product penetration inside the stratum corneum and beyond in the epidermis. Human skin was treated for 24 h at 34◦C. After incubation, skin was placed in a home-built brass cell. Confocal Raman images were acquired with a WITec Alpha-3000R plus confocal Raman microscope (UIm, Germany) equipped with a 532nm laser. XZ images were taken for each sample. The XZ depth image was typically 50x30µm2 covering SC and upper viable epidermis (VE) region with 4 µm steps and a 20 second exposure time.
Stability studies
Photostability
It was conducted for KTZ-SLNs and KTZ-SUS as per ICH guidelines Q1B [15]. The freshly prepared samples were packed in amber coloured clear glass vial, labelled and recorded for further process. Each batch was exposed to illumination light of 1.2 million lux h and an integrated near UV energy (200 watt h/m2) for 10 days in a photostability chamber (Binder Gmbh, Germany).
Long term accelerated stability
A long term accelerated stability of the developed formulations (KTZ-SLNs) were conducted as per ICH Q1A guidelines [16] . A constant amount of each formulation was transferred to fresh and clean amber colour glass container. Three batches for each formulations were prepared, packed and labelled as per experimental schedule [(2-8 0C, 30 (±2°C)/65% (± 5%) RH and 40 (±2°C)/75% (±5%) RH]. The samples were withdrawn at 0, 30, 90, 180, and 360 days, and evaluated for particle size, % EE, and the drug content (%).