Materials
The biodegradable polymer studied was PLGA (RESOMER® RG 504 molecular weight range is 38,000–54,000 and inherent viscosity is 0.45–0.60 dl/g) with a copolymer ratio of dl-lactide to glycolide of 50:50 gifted from Evonik Mumbai (India). The surfactant used in this process was polyvinyl alcohol (PVA) purchased from Sigma-Aldrich, Mumbai (India). Diazepam was received as gift sample from Windlas Biotech Ltd, Dehradun (India). Purified water of Milli-Q quality was used to prepare the solutions as well as the aqueous phases of the emulsions. All other reagents were of analytical grade.
Preparation of diazepam loaded nanoparticles
The diazepam loaded nanoparticles were prepared by an emulsion solvent evaporation method [10]. Typically, known amounts of mass of PLGA polymer and diazepam were added into ethyl-acetate, which was suitably stirred to ensure that all material was properly dissolved in solvent. Then, the solution of organic phase was slowly poured into the stirred aqueous solution of PVA. This mixture was sonicated using a microtip probe sonicator energy output of 55 W in a continuous mode (Soniweld Probe Sonicator, Imeco Ultrasonics, India) for a few minutes. The formed oil in water (O/W) emulsion was gently stirred at room temperature by a magnetic stirrer (Remi, India) for 5 hours to evaporate the organic solvent. The nanoparticles were recovered by centrifugation (22,000 rpm, 25 min; WX ultra 100 ultracentrifuge Thermofisher Scientific USA) and washed with distilled water 2–3 times to remove the surfactant. The purified nanoparticles were freeze-dried (YSI-250, Yorco Freeze Dryer (Lyophilizer), Yorco Sales Pvt. Ltd., India) to obtain the fine powder of nanoparticles, which was placed and kept in vacuum desiccators.
Nanoparticles characterization
The size (Z-average mean) and zeta potential of the nanoparticles were analyzed by photon correlation spectroscopy (PCS) or dynamic light scattering (DLS), respectively, in triplicate using a Zetasizer (Model- ZEN 3600, Malvern Instruments, U.K.). The dried powder samples were suspended in distilled water and slightly sonicated before analysis. The obtained homogeneous suspension was measured for the volume mean diameter and size distribution. Each measurement was done in triplicate. The shape, surface morphology and size analysis of the nanoparticles were analyzed by transmission electron microscopy (TECNAI 200 kV TEM (Fei, Electron Optics) Japan). A droplet of the nanoparticles was placed on a carbon-coated copper grid, forming a thin liquid film. The negative staining of samples was obtained with a 2 % (w/V) solution of phosphotungstate acid.
Entrapment efficiency
Nanoparticles were separated from dispersion by centrifugation at 22,000 rpm for 25 min. The supernatant obtained after centrifugation was suitably diluted and analyzed for free diazepam by UV–Visible spectrophotometer (Model No.-2201, UV–visible double beam spectrophotometer, Shimadzu, India) at 325 nm. The percentage entrapment efficiency was calculated as:
$$\% {\text{ Entrapment efficiency}} = \frac{{\left[ {Drug} \right]total - \left[ {Drug} \right]supernant }}{{\left[ {Drug} \right]total}}\varvec{ } \times 100$$
(1)
In-vitro drug release
The in-vitro drug release study of diazepam loaded PLGA nanoparticles formulations were studied by dialysis bag diffusion method [19]. Drug loaded nanoparticles (5 ml) were dispersed into dialysis bag and the dialysis bag was then kept in a beaker containing 100 ml of pH 7.4 phosphate buffer. The beaker was placed over a magnetic stirrer and the temperature of the assembly was maintained at 37 ± 1 °C throughout the experiment. During the experiment rpm was maintained at 100 rpm. Samples (2 ml) were withdrawn at a definite time intervals and replaced with equal amounts of fresh pH 7.4 phosphate buffer. After suitable dilutions the samples were analyzed using UV–Visible spectrophotometer at 325 nm.
To analyze the in vitro drug release data various kinetic models were used to describe the release kinetics. The zero order rate Eq. (2) explains the systems where the rate of drug release does not depend on its concentration [20]. The first order Eq. (3) explains the release from the system where rate of drug release is concentration dependent [21]. Higuchi [22] described the release of drugs from insoluble matrix as a square root of time dependent process based on Fickian diffusion Eq. (4). Korsmeyer et al. [23] derived a simple mathematical relationship which described the drug release from a polymeric system Eq. (5).
$$C \, = \, k_{o} t$$
(2)
where, C is the concentration of drug at time t, t is the time and k0 is zero-order rate constant expressed in units of concentration/time.
$$Log \, C_{0} {-} \, Log \, C \, = \, k_{1} t / 2.303$$
(3)
where, C0 is the initial concentration of drug and k1 is the first order rate constant.
$$C \, = \, K_{H} \sqrt t$$
(4)
where, KH is the constant reflecting the design variables of the system.
$$M_{t} / \, M_{\infty } = \, K_{KP} t^{n}$$
(5)
where Mt/M∞ is the fraction of drug released at time t, KKP is the rate constant and n is the release exponent.