Novel Methotrexate-Loaded Zein Nanoparticles With Improved and Extended Biopharmaceutical Performance: QbD-Steered Development, and Extensive In Vitro and In Vivo Evaluation

The current work entails QbD-enabled preparation of methotrexate-loaded nanoparticles (NPs) using zein as the release-controlling natural polymer. Initially, quality risk estimationand factor screening studies using Taguchi design were undertaken to delineate “vital few” process and material attributes among “plausible so many”. Further, formulation optimization using central composite design and validation using correlation plots and percent predictive bias was carried out. Optimized NPs exhibited mean size of 159 nm, zeta potential of 14.85 mV and entrapment of 50.23%. In vitro dissolution kinetic modelling unearthed non-Fickian drug release extension mechanism from the proposed zein NPs. In vitro MTT and apoptosis assay using MCF-7 cells and cellular uptake studies using Caco-2 cells indicate remarkably superior anticancer potential of zein NPs over pure methotrexate, ascribable to their nanometric size and cationic nature. In vivo pharmacokinetic studies in rat construed signicant enhancement by 2.15-fold in AUC 48h (p<0.001), 1.30-fold in C max (p<0.05), 3.67-fold in T max (p<0.001), and 1.38-fold in T 1/2 (p<0.01), along with notably reduced variability in biopharmaceutical performance. Establishment of signicant point-to-point level A in vitro/in vivo correlations (IVIVC) and kinetic modeling construed the robustness and prognostic ability of drug release studies. Robustness of the nanoformulation was ratied under refrigerated storage through six months’stability studies. Overall, the studies unequivocally indicate development of a stable nanoparticulate formulation with signicantly enhanced extent, extension and consistency of biopharmaceutical performance, along with improved anticancer potential of methotrexate.


Introduction
Cancer, a proliferative syndrome, has proved to be serious threat to human health and life, since decades [1]. Breast cancer, in particular, has been the primary cause of deaths in women, with approximately 2.1 million new cases reported every yearacross the globe [2,3]. Methotrexate (MTX), in this regard, plays a vital role in the therapeutic management of cancers [4]. Exhibiting poor water solubility (0.01 mg/mL) couple with poor permeability (cLog P of 0.53), it can safely be categorizedas a Class IV drugas per Biopharmaceutical Classi cation System (BCS) [5]. Reported to follow saturable absorption through proximal intestine,mediated through speci c transporters, MTX shows dose-dependent oral bioavailability [6,7]. Accordingly, lower doses of MTX have been observed to demonstrate relatively higher oral bioavailability [8,9]. Besides, owing to its poor solubility and permeability, coupled withsaturation of transporters, MTX is documented to exhibit inconsistent pharmacokinetic behaviour and erratic bioavailability [10,11]. As MTX is also known to observe a narrow therapeutic index, it is highly recommended to maintain and sustain plasma levels of drug at the optimal levels [12,13].
Myriad types of nanostructured delivery systems of MTX, in this context, have been explored not only for improving its extent of bioavailability, but also in reducing the intensity and frequency of undesirable side effects like nephrotoxicity, bone marrow suppression, chronic interstitial obstructive pulmonary disease and hepatotoxicity [4,14]. Sustained drug release characteristics has been another distinct advantage of employing such nanocarriers in drug delivery [15]. Drug delivery systems, like liposomes [16], microcapsules [17], synthetic polymer-based NPs [4,18], lipid-based non-vesicular drug delivery systems like solid lipid nanoparticles (SLNs) [19,20], and lipid-polymer hybrid NPs [21], have been explored to improve the biopharmaceutical potential of MTX and/or controlled release characteristics, but with limited fruition. Many of such nanocarrier systems also tend to pose serious problems o nept robustness and stability, and rapid degradation in the gastric milieu [22]. Systematic drug delivery system development to provide the required quality traits, while optimizing different possible process and material parameters, is a formidable task. This could be achieved using Quality by Design (QbD) paradigms, involving the cardinal principles of Quality Risk Management (QRM) and Design of Experiments (DoE) [32,33]. Thus, the present work envisages organized development, characterization and evaluation of MTX-encapsulated NPs for attaining improved, sustained and consistent biopharmaceutical attributes of the drug. Zein was selected to formulate such MTX-loaded NPs for e cient and cost-effective controlled drug delivery to potentially facilitate effective drug transportation to the target cells [34].

Materials
MTX was obtained from IPCA Laboratories, Mumbai, India, as a gift drug sample. Zeinwasprocured fromAcros organics, Thermo Fisher Scienti c, New Jersey, USA,while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V-FITC apoptosis kit were procured from Sigma Aldrich, Missouri, USA,and BD Bioscience, New Jersey, USA, respectively. MCF-7 breast cancer and Caco-2 intestinal epithelial barrier model cells were arranged from National Centre of Cell Science, Pune, India. Different other laboratory excipients, reagents and solvent of pure analytical grade were used without any further alteration throughout the study.

3.1Preparation of Polymeric Nanoparticles
Zein-based NPs encapsulating MTX were formulated using an already reported evaporation method, after minor modi cations [4,32,35]. Brie y, 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.

QTPP and CQAs
As per QbD approach, the quality target product pro le (QTPP) for the proposed polymeric nanocarriers was rstly 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 e ciency (EE), were identi ed, and plausible cause-effect relationships among the chosen CQAs and probable method or material parameters were delineated using an Ishikawa sh-bone diagram.
Consequently, an initial risk estimation matrix (REM) exercise involving all the potential factors in uencing the QAs (PS, ZP and % EE) was conducted by assigning these to low, medium and high levels of risk [37][38][39].

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 in uential method and material parameters. Only the high-and medium-risk factors, previously identi ed 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].

Optimization Studies on Zein NPs
The CMPs, i.e., amount of polymer (X 1 ) and percentage of surfactant (X 2 ), 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.

Modelling data analysis, validation and optimum search
Subsequently, in order to t 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 coe cient (R) and lack-of-t analysis. The 2Dcontour 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 con rmatory 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.

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, xed at an angle of 90º.

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.

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 magni cation(s).

Encapsulation e ciency
The values of % EE of the prepared zein NPs were obtained by adopting a reported method [41,42]. Brie y, 2 mL aliquots of the optimized prepared formulation were centrifuged (Sorvall TM legend TM XTR, Thermo Fisher Scienti c, Massachusetts, USA) at 10,000 rpm (11,200g) for10 minutes, and free drug in the supernatant was estimated at a λ max of 257 nm using a UV-Vis spectrophotometer (UV 3000 + ,Labindia, Mumbai, India).

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. Quanti cation of MTX was carried out employing UV spectrophotometry. Drug release data were subsequently tted 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 ask ( Apoptosis assay using MCF-7 cells was carried out using Annexin V-FITC/PI kit [49]. Cells (2x10 5 ) 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 owcytometer (BD AccuriTMC6, California, USA).

Quantitative cell uptake analysis: Flow cytometry
A ow cytometer (BD Accuri TM C6, California, USA) was employed to study the quantitative cellular uptake of rhodaminelabelled NPs by MCF-7 cells [51]. Cells (1*10 5 cells/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 quanti cation using the ow cytometer.
3.6.2 Caco-2 cells culture 3.6.2.1 Qualitative uptake using confocal microscopy Caco-2 cells (2*10 5 cells/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 ask 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 xed 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).

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.

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 (AUC 48h ), maximal plasma drug concentration (C max ), time to attain C max (t max ), biological half-life (t ½ ), and mean residence time (MRT) were computed, interpreted critically, and compared to those obtained with pure drug suspension.

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 signi cance 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 Modi ed Wagner-Nelson technique, as MTX was found to obey one-compartment pharmacokinetic model [32].

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 identi ed CQAs and compared to data obtained at the start of studies [55].

Optimization studies and Response Surface Mapping (RSM)
A second-order CCD was applied to carry out the optimization studies, followed by data analysis using Eq2, accounting for mainand interaction effects between the selected CMPs. Y= β 0 + β 1 X 1 + β 2 X 2 + β 3 X 1 X 2 + β 4 + β 5 …Eq. 2 where, β 0 is the intercept term, β 1 and β 2 represent the coe cients of factors, and respectively, β 3 represents the interaction term coe cient between X 1 and X 2, and β 4 and β 5 are the corresponding quadratic coe cients of .

Search for Optimized Formulation and Validation Studies
The most suitabledrug delivery formulation was selected by balancing variegated CQAs to obtain the preferred targets, i.e., minimal value of PS (which aids in drug absorption) and cumulative drug release till 48 h (ensures extended release pro le), and maximal values of ZP (imperative for stability indication) and EE (ensures loading capacity). Following the aforesaid criteria, the optimum search was accomplished using numerical optimisation, aiming for the desirability values close to unity. Figure 5 shows the overlay plot delineating the constitution of the optimalzein NPs as per the ag, as polymer (78 mg) and surfactant (0.35 %), attaining mean PS of 159.30 nm,ZP of 14.85 mV, EE of 50.23% and cumulative drug release of 33.90 %.
Table S 1 illustrates the composition of the different formulations, along with the observed and the anticipated values of CQAs and % PPE between these. The absolute bias or % PPE ranged between -4.55 and 4.86% with low magnitude of overall prognostic error (i.e., 0.11% ± 3.06), construinga high level of predictive capability of the adopted QbD methodology.  Figure 7 (a and b)show the TEM and FESEM microphotographs of the optimised formulation, respectively, construing segregated and round-shaped particles with smooth surface and size under 200 nm. Such interpretation of size ranges was found to be in agreement with those obtained earlier during particle size measurement (Section 4.4.1).

Drug Release Kinetics Modelling
Studies on cumulative MTX release pro les on zein NPs revealed a sustained release pattern of MTX from the polymeric NPs, when compared to naïve drug suspension (Figure 8). It is quite evident that more than half fraction of pure MTX got released within the rst 30 minutes, while it took over 8 h to release the same extent of drug from the optimized NPs. Table 4 enlists the outcomes of tting of release data into various mathematical models. Statistical signi cance of regression (i.e., F ratio) construed Korsmeyer-Peppas mathematical model (p <0.001) as the most suitable strategy to unravel the mechanism of MTX release kinetics [57]. The values of diffusional release exponent (n) indicated Fickian release mechanism of MTX (n=0.181), and non-Fickian or anomalous release behaviour from the optimized NPs (n=0.5681) [55]. Weibull model tting on drug release data revealed the values of dissolution curve shape parameter (β)to be ranging between 0.444 and 0.723 (i.e., <1) for MTX as well zein NPs, thus representing an initial steep slope, followed by an exponential-type of curvature. On the other hand, the magnitude of Weibull time-scale parameter (α) for zein NPs (i.e., 4.854) were found to be superior to those of pure drug (i.e., 0.876), indicating discrete drug release extension from the NPs [58].
The values of location parameter (Ti) obtained with both pure MTX as well as with the optimized NPs indicated that the onset of dissolution process was quite instantaneous [36]. Attempts to t the data in Baker-Lonsdale model for NPs too indicated high degree of tness (R=0.9840; p<0.001) construing regulated drug release characteristics from the spherically shaped NPs [59]. On the other hand, pure drug data failed to yield statistically signi cant results.
4.4 Cell culture study 4.4.1.MTT assay Figure 9 depicts concentration-dependent cell cytotoxicity of MCF-7 cells on interacting with plain MTX suspension and zein NPs, evidently indicating much higher cytotoxicity of the latter vis-à-vis the former. The IC 50 values were found to be approximately 37.78 and 12.77 µg/mL for pure drug and optimized NPs, respectively, thus justifying almost 3-folds improvement in cytotoxicity and e cacy of the latter. The enhanced cytotoxicity of the optimized formulation could be attributed to its cationic nature and minuscule particle size, which may enhance cellular internalization across the MCF-7 cells, followed by subsequent release of MTX from zein NPs [60]. Figure 10 shows the quantitative estimation of programmed cell death caused by MTX and its zein NPs in the stoichiometrically equivalent concentraions (i.e., 12.77µg/mL). Almost 12.9 % of cells were observed to be in the initial apoptotic phase on treatment with zein NPs, to that of 2.1 and 0.5 % cells for MTX pure and reference control of untreated cells, respectively. Nevertheless, the count of cells in the late apoptotic phase increased to 0.9 % (reference control) and 1.1% (MTX) to 3.4 % (zein NPs), on treatment. Superior apoptosis was observed with zein NPs, which can be ascribed to the enhanced permeation of the carrier system due to their positive charge and the subsequent internalization across the cell membrane due to its minuscule size [56]. Figure 11 (a-d) illustrates the cellular uptake of C 6 by MCF-7 cells, when exposed to free MTX and its optimized zein NPs. The quantum of percent C 6 values, measured using FACS analysiswas found to be approximately 141-folds with the latter vis-à-vis the control (i.e., untreated cells). Improved uorescent intensity of C 6 was found with zein NPs, which can again be attributed to the integrated effect of nanometric size and cationic nature of the polymer [61]. Figure 12 (a-d) depicts the qualitative cellular uptake of C 6 -loaded zein NPs (C 6 -zein NPs) and pure C 6 in Caco-2 cell lines.

Cellular uptake studies
Negligible uorescent characteristics were noticed, as free C 6 was not freely internalised by the cell lines [62]. Besides, high uorescent intensity was noticed withC 6 -zein NPs (Figure 12 (d)), assignable again the positive charge and diminutive size of the NPs thus facilitating their endocytosis across the cellular membrane [63, 64]. Figure 13 displays 48-h plasma drug level (mean ± SD) pro les of pure MTX suspension and zein NPs. The respective inset represents percentage alteration in the values of pharmacokinetic metrics following intake of MTX NPs compared pure MTX. The results showed quite enhanced biopharmaceutical attributes of zein NPs vis-à-vis naïve drug, with with nearly 2.15-folds improvement in AUC 48h (p<0.001), 1.30-folds in C max (p<0.05), 3.67-folds in T max (p<0.001), 1.38-folds in T 1/2 (p<0.01), and 1.64folds in MRT (p<0.01). Also, the values of percent coe cient of variation (% CV) noticed with zein NPs (i.e., ranging between 5.8 and 59.8%; mean CV: 23.5%) indicated superior consistency in plasma level data over that of pure MTX suspension (i.e., ranging between 12.3 % and 81.8 %, mean CV: 41.9 %). Signi cant improvement in various absorption parameters and consistency in plasma level data were noticeable with NPs, than pure MTX. Overall, distinctive improvement in the magnitude of biopharmaceutical attributes demonstrates signi cant augmentation in the extent of biovailbility, as well as extension and consistency in of plasma MTX levels, on its incorporation in the zein NPs. Figure 14 portrays the linear curves for level A IVIVC for MTX and its zein NPs. High values of coe cient of correlation (R) were observed for MTX zein NPs (i.e., R=0.9716, p<0.01) as well as for pure MTX (i.e., R= 0.9426, p<0.05) with linearmodel tted between cumulative percentages of dissolved drug in vitro with those of absorbed drug in vivo [32,36]. Embarking upon of statistically valid IVIVC's, accordingly, corroborate the robustness of in vitro drug dissolution evaluation methodology, including that of drug release medium composition, dissolution apparatus type, sampling schedule and stirring conditions, as quite prognostic and simulative of in vivo biopharmaceutical performance of MTX. Successful establishment of such IVIVC's have already been reported in the literature for various BCS class II and IV drugs, and their consequent drug delivery systems [32,36,47,65].

Stability studies
The results of stability studies are summarized in Table S2. Relatively mild to notable rise in mean size of particles may be ascribed to the reduced viscosity of the delivery system, during different storage conditions. This eventually led to the high kinetic energy and number of collisions throughout the particles ultimately resulting in aggregation of NPs at room temperature The current studies were planned to explore the plausible enhancement and extension in biopharmaceutical performance of MTX, when formulated as zein NPs. Implementation of QbD strategy aided in holistic know-how of the nanoparticulate formulation development while achievingthe QTPP requirements. NPs demonstrated signi cantly higher cytotoxicity and improved uptake potential than pure MTX during in vitro evaluation, ostensibly ascribable to their nanometric size and cationic surface charge. Notable extension in pharmacokinetic pro le, augmentation in the extent of bioavailability, and diminution in data variability were observed with zein NPs. Stability studies rati ed the robustness of zein NPs under refrigerated conditions when stored for three months. Concisely, the ndings of the current research work substantiate superior formulation robustness, and extended, regulated and consistent biopharmaceutical performance of MTX NPs, resulting in its high therapeutic potential in breast tumour(s). Successful outcomes observed with polymeric NPs, employing zein as a natural, biodegradable and economical polymer, can be rationally explored and extrapolated for other biopharmaceutucally enigmatic drug molecules too.        Figure 1 An Ishikawa-sh bone diagram depicting various factors affecting the formulation development process of zein nanoparticles   3D-response surfaces and 2D-contour plots for different CQAs, viz., particle size (a-b), zeta potential (c-d), % EE (e-f), and cumulative % drug release (g-h)

Figure 5
An overlay plot depicting the design space of the optimized formulation Photo-images of (a) particle size (b) zeta potential of the optimized zein nanoparticles In vitro cell cytotoxicity pro les of MTX (•) and zein nanoparticles (♦), performed on MCF-7 cells lines. Data are expressed as Mean ± SD; n=3 SupplementaryZeinBSB2.docx