Triethanolamine

Ti₃AlC₂ powder (400 mesh) was purchased from Nanochemazone (Leduc, Alberta, Canada). Hydrofluoric acid (HF, 48–51%) and dimethyl sulfoxide (DMSO, ACS reagent, 99.9%) were supplied by Thermo Scientific (Fair Lawn, NJ, USA). Methanol (≥99.9%), ammonium persulfate (APS, ACS reagent, ≥98.0%), isopropyl alcohol (IPA), triethanolamine (TEA, reagent grade, 98%), thiophene (≥99%), methylene blue (MB), orange G (OG), and rhodamine B (RhB) were all obtained from Sigma-Aldrich Korea (Seoul, Republic of Korea). Cetyltrimethylammonium bromide (CTAB, 99%) was purchased from Daejung Chem (Siheung, Republic of Korea).

Leishmania infantum (MHOM/ES/92/LLM-320; isoenzyme typed MON-1) was cultured in M199 medium supplemented with 20% fetal bovine serum (Gemini Bio-Products), 100 μ/mL penicillin and 100 μg/mL streptomycin (Gibco), 2 mM L-glutamine (Gibco), 40 mM HEPEs (Gibco), 0,1 mM adenine (Sigma) in 50 mM HEPEs, 5 mg/mL hemin (Sigma) in 50% triethanolamine (Sigma), and 1 mg/mL 6-biotin (Sigma). Cultures were maintained at 26 °C. For mouse infections, metacyclic promastigotes were purified from stationary-phase cultures by Ficoll (Sigma) density gradient (8% and 20%), as adapted from Spath and Beverley^{65 (link)}, and centrifuged at 500 × g for 10 min, at 25 °C. Mice were intravenously inoculated with 3 × 10⁶ metacyclic promastigotes in 100 μl of 1 × PBS.

Vancomycin hydrochloride was obtained from Jaberabne Hyan Pharmaceutical Company, Iran. Edragit RS 100 was obtained from RÖhm Pharma GMBh (RÖhm Pharma GMBh, Weiterstadt, Germany), Poly vinyl alcohol (PVA) with molecular weight of MW 95000 was obtained from Acros Organics (Acros Organics, Geel, Belgium) and dichloromethane, methanol, glacial acetic acid, triethanolamine, hydrochloric acid, potassium chloride, sodium chloride, sodium hydrogen phosphate (dibasic), and potassium dihydrogen phosphate were obtained from Merck (Merck, Darmstadt, Germany). Silastic membrane (#10,000 Da) was provided by Biogene (Mashhad, Iran). All other materials used were of analytical or HPLC grade.
Experimental designThe experimental design was a modified Box-Behnken design for five variables. This design was suitable for exploring quadratic response surfaces and constructing second-order polynomial models. Four independent formulation variables analyzed during the study including the amounts of emulsifier (X1), volume of organic solvent (X2), and the amount of dispersing medium (X3), time of stirring (X4) and rate of stirring (X5). The investigated dependent variables were the drug content (DC, Y1), loading efficiency (LE, Y2), particle size (PS, Y3), and production yield (PY, Y4). The complete design consisted of 27 experimental points, which are included three replications. The 81 experiments were carried out in random order. Data were analyzed to fit the polynomial equation to Y (9 ).
Preparation of nanoparticlesVCM-loaded Eudragit RS100 nanoparticles were prepared by W1/O/W2 solvent evaporation method using different ratios of drug to polymer (1:1, 1: 2 and 1: 3). Briefly, 5 mL of aqueous internal phase (containing 100 mg VCM) was emulsified for 15 sec in 20 mL of methylene chloride (containing 100, 200 and 300 mg Eudragit RS100) using homogenizer (22000 rpm). This primary emulsion was poured into 25 mL of a 0.2% PVA aqueous solution while stirring using a homogenizer for 3 min, immersed in an ice water bath, to create the water in oil-in-water emulsion. Three to four mL of NP suspension was obtained after the solvent evaporation under reduced pressure (Evaporator, Heidolph, USA). Nanoparticles were separated from the bulk suspension by centrifugation (Hettich universal 320R, USA) at 22,000 g for 20 min. The supernatant was kept for drug assay as described later and the sediment nanoparticles were collected and washed with three portions of 30 mL water and were redispersed in 5 mL of purified water before freeze-drying. Blank nanoparticles (without drug) were prepared under the same conditions (11 (link), 12 ).
Micromeritic propertiesA laser light scattering particle size analyzer (SALD-2101, Shimadzu, Japan) was used to determine the particle size of the drug, polymer and nanoparticulate formulations. Samples were suspended in distilled water (nanoparticles and polymer) or acetone (drug) in a 1 cm cuvette and stirred continuously during the particle size analysis.
Zeta potential measurementZeta (ζ) potential measurements of diluted samples were made with a ZetaSizer (Malvern Instruments Ltd., Malvern, UK). Zeta potential values obtained from ZetaSizer were average values from twenty measurements made on the same sample. Initial measurements on several samples of the same kind showed that this number is sufficient to give a representative average value. VCM nanoparticles were diluted with deionized water before the measurement.
Loading efficiency and production yield (%) determinationThe drug concentration in polymeric particles was determined spectrophotometrically (UV-160, Shimadzu, Japan) at 280.2 nm by measuring the amount of non-entrapped VCM in the external aqueous solution (indirect method) before freeze-drying. In the case of nanoparticles, the external aqueous solution was obtained after the centrifugation of colloidal suspension for 20 min at 22,000 g.
The loading efficiency (%) was calculated according to the following equation:
Loading efficiency(%) = (actual drug content in nanoparticles/theoretical drug content) × 100
The production yield of the nanoparticles was determined by accurately calculating the initial weight of the raw materials and the last weight of the polymeric particles obtained. All of the experiments were performed in triplicate (Table 1).
VCM dissolution patterns from freeze-dried nanoparticles were obtained under sinking conditions. Dissolution studies were carried out using a dialysis bag rotating method. A set amount of nanoparticles (20 mg of drug) was added to 200 mL dissolution medium (phosphate buffered saline, pH = 7.4), preheated and maintained at 37 ± 1°C in a water bath, then stirred at 100 rpm. Then, 3 mL of solution was withdrawn at appropriate intervals (0.5, 1, 2, 3, 4, 5, 6, 8, 12 and 24 h). The filtrate (VCM) was replaced by 3 mL of fresh buffer. The amount of VCM in the release medium was determined by UV at 279.8 nm (12 , 13 ).
In order to have a better comparison between different formulations dissolution efficiency (DE), t50% (dissolution time for 50% fraction of drug) and difference factor, f1 (used to compare multipoint dissolution profiles) were calculated and the results are listed in Table 2.
DE is defined as the area under the dissolution curve up to a certain time (t), expressed as a percentage of the area of the rectangle arising from 100% dissolution in the same time. The areas under the curve (AUC) were calculated for each dissolution profile by the trapezoidal rule (14 (link)). DE can be calculated by the following:
DE = t dt 100 ∫ y
Here, y is the drug percentage dissolved at time t. All dissolution efficiencies were obtained with t equal to 1440 min. The in-vitro release profiles of different nanoparticle formulations were compared with physical mixture formulation using difference factor (f1), as defined by:
f1= (Σ t = 1n |Rt - Tt|) / (Σ t = 1n Rt) × 100
Here, n is the number of time points at which %dissolved was determined. Rt is the %dissolved of one formulation at a given time point and Tt is the %dissolved of the formulation to be compared at the same time point. The difference factor fits the result between 0 and 15, when the test and reference profiles are identical and approaches above 15 as the dissimilarity increases.
Data obtained from in-vitro release studies were fitted to various kinetic equations to find out the mechanism of drug release from the Eudragit RS100 nanoparticles. The kinetic models used were:
Qt = k0t (zero-order equation)
ln Qt = ln Q0 – k1.t (first-order equation)
Qt = K. S. t0.5= kH. t0.5
(Higuchi equation based on Fickian diffusion)
Here, Q is the amount of drug release in time t, Q0 is the initial amount of drug in the nanoparticles, S is the surface area of the nanoparticle and k0, k1 and kH are rate constants of zero order, first order and Higuchi equation, respectively. In addition to these basic release models, the release data was fitted to the Peppas and Korsmeyer equation (power law):
Mt/M∞ = k.tn
Here, Mt is the amount of drug release at time t and M∞ is the amount release at time t = ∞, thus Mt/M∞ is the fraction of drug released at time t, k is the kinetic constant, and n is the diffusion exponent which can be used to characterize the mechanism of drug release (14 (link), 15 (link)).
Optimization of the VCM nanoparticlesResponse surface methodology (RSM) is a very useful statistical technique for the optimization of VCM formulations. In this design, 5 factors were evaluated, each at 4 levels, and experimental trials were performed at all 27 possible combinations. The amounts of emulsifier (X1), volume of organic solvent (X2) and the amount of dispersing medium (X3), were selected as independent variables. The drug content (DC), loading efficiency (LE), particle size (PS), and percentage production yield (PY) were dependent variables (Table 3).
Various batches of the selected formulation (F2) were made, but the stirring rate was the only parameter that was varied between 22000, 24000 and 26000 rpm. In addition, while keeping the other parameter constant, time of homogenizer stirring was changed (1.5, 3 and 4.5 min). After drying, the weighed batch of nanoparticles was subjected to drug content, loading efficiency, particle size and drug release experiments.
The influence of process variables on nanoparticle formation, micromeritics and drug release characteristics, was investigated. These variables included the emulsifier concentration (0.1, 0.2 and 0.4%) and volume of organic solvent (15, 20 and 25 mL) and dispersing medium (15, 25 and 35 mL).
Regression analysisThe targeted response parameters were statistically analyzed by applying one-way ANOVA at 0.05 levels. Individual response parameters were evaluated using the F-test and quadratic models of the form given below were generated for each response parameter using the multiple linear regression analysis (17 (link)).
Y = b0 + b1X1+ b2 X2 + b3 X3 + b4 X4 + b5X5 + b11 X12 + b22 X22 + b33 X32 + b44 X42 + b55 X52 + b12 X1 X2 + b13 X1 X3 + b14 X1 X4 + b15 X1 X5 + b23 X2 X3 + b24 X2 X4 + b25 X2 X5 + b34 X3 X4 + b35 X3 X5 + b45 X4 X5
In this equation, Y is the predicted response, X1, X2, X3, X4 and X5 are independent variables, b0 is the intercept, b1, b2, b3, b4 and b5 are linear effects, b12, b13, b14, b15, b23, b24, b25, b34 and b45 are interaction terms. The main effects (X1, X2, X3, X4 and X5) represent the average result of changing one factor at a time from its low to high value. The interaction terms (X1X2, X1X3, X1X4, and X1X5) show how the response changes when five factors are simultaneously changed. The polynomial terms (X1X1, X2X2, X3X3, X4X4 and X5X5) are included to investigate nonlinearity. Three-dimensional surface (3D) plots were drawn to illustrate the main and interactive effects of the independent variables on production yield, drug content, loading efficiency and particle size. The optimum values of the selected variables were obtained from the software and also from the response surface plots.
Numerical optimization using the desirability approach was employed to locate the optimal settings of the formulation variables to obtain the desired response (17 (link)). An optimized formulation was developed by setting the constraints on the dependent and independent variables. The formulation developed was evaluated for the responses and the experimental values obtained were compared with those predicted by the mathematical models generated.

Tissue samples for immunoblotting were placed in 10 ml ice-cold isolation solution, containing 250 mM sucrose, 10 mM triethanolamine (Sigma-Aldrich, St. Louis, MO, USA), 1 mg/ml leupeptin (Sigma-Aldrich) and 0.1 mg/ml phenylmethylsulfonyl fluoride (Sigma-Aldrich) titrated to pH 7.6, and the mixture was homogenized at 13,600 × g with three strokes for 15 sec using a tissue homogenizer (PowerGun 125; Thermo Fisher Scientific, Pittsburgh, PA, USA). Following homogenization, the total protein concentration was measured using a bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific, Rockford, IL, USA), which was adjusted to 2 mg/ml with isolation solution. Equal amounts of protein and sample buffer were separated using 12% gradient SDS-PAGE, stained with Coomassie Brilliant Blue and transferred to a polyvinylidene fluoride membrane. The blotted membrane was blocked with Tris-buffered saline containing 5% milk, and incubated with HIF-1α, VEGF or CD34 rabbit polyclonal antibodies (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), followed by incubation with a HRP-coupled secondary antibody (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA). The proteins were detected using enhanced chemiluminescence (Thermo Fisher Scientific). All immunoblots were representative of at least three independent experiments.