3.1Preparation of Polymeric Nanoparticles
Zein-based NPs encapsulating MTX were formulated using an already reported evaporation method, after minor modifications [4, 32, 35]. Briefly, zein was dissolved in ethanol (90 % v/v), constituting the organic phase, while phosphate buffer saline (i.e., PBS, pH 7.4), containing the requisite amounts of MTX and Pluronic F-68 (PF-68) as the stabilizer, constituted as the aqueous phase. The aforesaid organic phase was added into the aqueous phase, drop-wise, with constant stirring, employing a magnetic stirrer (REMI, Mumbai, India) to allow the diffusion and evaporation of organic phase, and eventually, the formation of colloidal dispersion of zein NPs.
3.2 QTPP and CQAs
As per QbD approach, the quality target product profile (QTPP) for the proposed polymeric nanocarriers was firstly embarked upon with an aim to improve the quality attributes of zein NPs [36]. Different critical quality attributes (CQAs), like mean particle size (PS), zeta potential (ZP) and % entrapment efficiency (EE), were identified, and plausible cause-effect relationships among the chosen CQAs and probable method or material parameters were delineated using an Ishikawa fish-bone diagram. Consequently, an initial risk estimation matrix (REM) exercise involving all the potential factors influencing the QAs (PS, ZP and % EE) was conducted by assigning these to low, medium and high levels of risk [37-39].
3.3 Factor Screening Studies
Design Expert® software package (Version 11, Stat-Ease Inc., Minnesota,USA) was used to implement Taguchi design (TgD) with a seven-variable eight-run matrix in order to chalk out highly influential method and material parameters. Only the high- and medium-risk factors, previously identified using REM studies in Section 3.2, were considered for factor screening. Table 1 enlists the design matrix for various variables screened, along with their high and low levels, respectively [33, 40].
3.4 Optimization Studies on Zein NPs
The CMPs, i.e., amount of polymer (X1) and percentage of surfactant (X2), were systematically optimized using a nonlinear second-order central composite design (CCD). Table 2 summarises the design matrix, consisting of a total of thirteen experimental trials, along with pertinent factors and their corresponding levels.
3.4.1 Modelling data analysis, validation and optimum search
Subsequently, in order to fit the experimental data, a quadratic polynomial model was employed to optimize data analysis and model validation. Model evaluation was carried out using Pearson’s correlation coefficient (R) and lack-of-fit analysis. The 2D-contour plots and 3D-response surfaces were drawn and deciphered for ostensible factor-response relationship(s). Optimal solution was selected using mathematical desirability function by “trading-off” different CQAs, and subsequentlyby graphical optimization, delineating the “design space”region by following the criteria of maximal values of % EE and ZP, and minimal values of PS and % cumulative drug release. Six confirmatory formulations were prepared as the validation runs (ZNP 1-6) to evaluate the predictive ability of the evolved polynomial models and DoE methodology, using percentage of prediction error (%PPE) and linear correlation plots.
3.5 Characterization of Zein NPs
3.5.1 Mean Particle sizeand Zeta Potential
The magnitudes of meanparticle size as well as of zeta potential of the prepared NPs were measured at 25°C using a particle size analyser, (Zetasizer, ZS90; Malvern Instruments, UK). Dispersion of zein NPs was placed into a micro-electrophoreticcell, fixed at an angle of 90º.
3.5.2 Field Emission Scanning Electron Microscopy (FESEM)
Surface morphology of optimized NPs was examined using an FESEM system (SU-8010, Hitachi,Tokyo, Japan). A droplet of sample was coated with gold, and the microphotographs were captured at suitable operating conditions.
3.5.3 Transmission electron microscopy (TEM)
The prepared optimized formulation was further characterized employing TEM (Tecnai, Holland, Netherland), operating at suitable accelerating voltage. A droplet of sample was placed on a grid surface, followed by (negative) staining with 1% phosphotungustic acid. Following air-drying, the grid was exposed for imaging and the microphotographs were taken at appropriate magnification(s).
3.5.4 Encapsulation efficiency
The values of % EE of the prepared zein NPs were obtained by adopting a reported method [41, 42]. Briefly, 2 mL aliquots of the optimized prepared formulation were centrifuged (SorvallTM legendTMXTR, Thermo Fisher Scientific, Massachusetts, USA) at 10,000 rpm (11,200g) for10 minutes, and free drug in the supernatant was estimated at a λmax of 257 nm using a UV-Vis spectrophotometer (UV 3000+,Labindia, Mumbai, India).
3.5.5 In vitrodrug release studies
Drug release kinetics from the optimized NPswas investigated employing the dialysis bag (MWCO 10-12, KD, Himedia) method [43, 44]. Two millilitres of suspensions, each of the drug and its optimized NPs, were placed into the dialysis bag(s) and suspended in the receptor compartment constituting phosphate buffer (pH 7.4: 50 mL) over a water bath (Rivotek, Mumbai, India), maintained at 37°C. At regular intervals, 2 mL aliquots each of medium were with drawn and replaced with fresh solvent inequal volumes at pre-determined time intervals during the study. Quantification of MTX was carried out employing UV spectrophotometry. Drug release data were subsequently fitted into various different kinetic models to arrive at the possible mechanism(s) of MTX release from the prepared NPs.
3.6In VitroCell Culture Studies
Cells were harvested and grown in a tissue culture flask (25 cm2) (BD, Falcon, New Jersey,USA), according to the requisite guidelines [45]. Later, the cells were passaged to an alternate flask, as per the standard protocol for in vitro cell culture studies [46].
3.6.1 MCF-7 Cell culture investigations
3.6.1.1MTT cellularviability studies
Cell viability assay using free MTX and the prepared zein NPs was conducted employing an MTT assay on the MCF-7 cells, according to the procedure already reported by us [32, 47, 48]. Harvested cells (1*105cells/well) were seeded in a 96-well plateand were kept for adherence to the flask surface. Free MTX and zein NPs containing equivalent amount drug amounts were added in serial concentrations, and kept aside for incubationat 37 ± 1°C under CO2 (5%) environment. Subsequently, excess of zein NPs, free MTX and medium were removed, and washed with buffer (PBS; pH 7.4). Optical density (OD) was measured at 550 nm employing an ELISA plate reader (MultiscanTM FC microplate, Thermo Fisher ScientificTM, New Jersey, USA), and percentage of cell viability was quantified.
3.6.1.2 Apoptosisassay
Apoptosis assay using MCF-7 cells was carried out using Annexin V-FITC/PI kit [49]. Cells (2x105) were collected, incubated with MTX and zein NPs, and processed according to the standard protocol [50]. Cells were further treated with Annexin V-FITC:PI and kept aside in dark for incubation, followed by addition of binding buffer prior to its analysis using a flowcytometer (BD AccuriTMC6, California, USA).
3.6.1.3 Quantitative cell uptake analysis: Flow cytometry
A flow cytometer (BD AccuriTM C6, California, USA) was employed to study the quantitative cellular uptake of rhodamine-labelled NPs by MCF-7 cells [51]. Cells (1*105cells/well) were seeded in a 6-well plate, along with complete growth medium, and were set aside for an overnight. The cells were separately incubated with free rhodamine and zein NPs-rhodamine, and kept aside for 4 h, followed by their quantification using the flow cytometer.
3.6.2 Caco-2 cells culture
3.6.2.1 Qualitative uptake using confocal microscopy
Caco-2 cells (2*105cells/well) were placed in a 6-well plate (BD, Falcon, New Jersey,USA), and were setaside for 24 h for attachment of cells over the flask surface[51]. Additionally, the cells were separately incubated with free dye and dye-loaded zein NPs for 4 h. Cells were subsequently rinsed with the PBS (pH 7.4) to wipe out the excess of medium carefully and were fixed with a solution of glutaraldehyde inethanol (2.5% v/v). After proper treatment, microphotographs of the nanocarriers entrapped within the cell(s) were captured with a confocal laser scanning microscope (Nikon C2+, Tokyo, Japan).
3.7 In VivoAnimal Studies
Animal studies were conducted as per institutional ethical committee guidelines of the Panjab University, India, after attaining the essential permission, vide number, PU/IAEC/S/15/31.
3.7.1 In vivo animal pharmacokinetic studies
Pharmacokinetic investigations were conductedon free MTX and zein NPs using Sprague Dawley female rats (weights: 180to225 g), supplied by the Central Animal House Facility of the Panjab University. Prior to studies, the animals were housed in regulated conditions (25±2°C/60±5% RH) and fasted overnight, but were allowed ad libitum access to water. Further, 3 rats were randomly placed into two different groups, and were administered with free MTX (10 mg) and equivalent amount of zein NPs, employing oral gavage.
Blood samples, measuring approximately 150 µL each, were there after collected from rat retro-orbital plexus under mild anaesthesia at the scheduled time-points of 0.25, 0.50, 1, 2, 6, 8, 12, 24, and 48 h in HiAnticlot® vials (Himedia, Mumbai,India). Blood samples, thus collected, were centrifuged at 12,000 rpm (16,128 g) for 10 minutes, and supernatant plasma samples were estimated for MTX by an HPLC method, reported earlier by the authors [52]. The pharmacokinetic data analysis and modelling were conducted employing an add-in PK Solver® MS-Excel spread-sheet [53], adopting Wagner-Nelson technique. Diverse pharmacokinetic parameters like area-under-curve till 48 hours (AUC48h), maximal plasma drug concentration (Cmax), time to attain Cmax (tmax), biological half-life (t½), and mean residence time (MRT) were computed, interpreted critically, and compared to those obtained with pure drug suspension.
3.8 In vitro/in vivo correlation (IVIVC)
Attempts were made to establish point-to-point linear level A IVIVC between percentages of in vitro drug released data with that of in vivo drug absorbed at the corresponding time points for MTX as well as its zein NPs, and the significance of the correlations was statistically deciphered according to the standard Fisher’s ratio criterion at the appropriate degrees of freedom. The magnitudes of the total drug absorbed were calculated using Modified Wagner–Nelson technique, as MTX was found to obey one-compartment pharmacokinetic model[32].
3.9 Stability Studies
The lyophilized zein NPs were investigated for stability studies in order to predict their quality and integrity during different storage conditions over the course of time, employing an environmental chamber (Newtronic Lifecare, Mumbai, India). MTX being a photosensitive drug, amber-coloured glass vials were kept for a time period of 180 days at varied conditions of temperature and relative humidity (RH), viz., 5±3ºC and 25±2ºC (both at 60 ± 5 % RH); and 40±2ºC and 75±5% RH (n=3) [2, 54]. The formulations were periodically evaluated on intervals of 1, 2, 3 and 6 months for the values of their identified CQAs and compared to data obtained at the start of studies [55].