BSA (fraction V) was purchased from Merck, Darmstadt, Germany. ACV micronized was obtained from Zhejiang Wuyi Pharmaceutical Factory, Jinhua, China. Glacial acetic acid, ethanol, and acetonitrile were obtained from Labscan, Gliwice, Poland. Sodium hydroxide was from Carlo Erba, Val-de-Reuil, France. Trehalose was from Sigma-Aldrich, St. Louis, MO, USA. All chemicals were of analytical or reagent grade.

ACV-loaded BSA nanoparticles were prepared by desolvation method as previously described (Suwannoi et al. 2017). BSA for 100 mg and 0, 6.5, or 12.5 mg of ACV were dissolved together in 5 mL of sterile water for injection (SWI) and allowed for 20 min incubation. Subsequently, 30 mL of ethanol was submergely pumped at the rate of 15 mL/min (Model 505S, Watson Marlow, Wilmington, MA, USA) through a 0.5-mm spinal needle into the ACV-BSA bulk solution with continuous stirring at 500 rpm. The mixture was then stirred for 10 min and subject to hardening process at 60 °C for 2 h with continuous stirring. Finally, the obtained nanoparticles were purified by ultrafiltration technique using centrifugal filter unit (Vivaspin, 100,000 MWCO, GE Healthcare, Little Chalfont, Buckinghamshire, UK), and then resuspended in various vehicle media, i.e. SWI, phosphate buffer saline (PBS) pH 7.4, or isotonic trehalose solution. The purified formulations were kept at 4 °C until use.

Particle size and polydispersity index (PdI) of ACV-loaded BSA nanoparticles were determined by dynamic light scattering (DLS) technique (Zetasizer, Nanoseries, Malvern, Worcestershire, UK). The samples were measured at 25 °C with a scattering angle of 173 °. The nanoparticles were directly loaded into a cuvette of the particle electrophoresis instrument (Zetasizer, Nanoseries, Malvern, Worcestershire, UK), the zeta potential of which was determined by measuring the direction and velocity of the particle movement in the applied voltage of 150 V at 25 °C.

The contents of ACV entrapped in BSA nanoparticles were directly determined by subtracting the initial amount of drug added into the system by the amount of extracted drug from ACV-loaded BSA nanoparticle formulations. ACV extraction was done by treating the formulation with 1.0 N NaOH at 1:1 ratio. After continuous stirring for 30 min with magnetic stirrer, the mixture was centrifuged at 2,000 rpm for 30 min (Biosan, Riga, Latvia). The supernatant was then collected and the contents of ACV were analyzed by HPLC method. The percentage loading efficiency and loading capacity were calculated, respectively, as follows:

$\%\text{ Loading efficiency }= \frac{\text{ Amount of drug recovered }}{\text{ Initial amount of drug added }}\times 100$ (1)

$\%\text{ Loading capacity }=\frac{\text{ Amount of drug recovered }}{\text{ Amount of carrier BSA }}\times 100$ (2)

Analysis of ACV contents were done by HPLC system equipped with a high-precision pump (LC-20AD, Shimadzu), UV-vis detector (SPD-20A, Shimadzu), and system controller (LC-20AD, Shimadzu, Tokyo, Japan) as previously described (Suwannoi et al. 2017). A C₁₈ reverse-phase column (Mightysil RP-18 Aqua 250–4.6 (5 µm), Kanto Kagaku Co., Tokyo, Japan) was used at ambient temperature. The mobile phase consisted of 95% of 0.1% glacial acetic acid in water and 5% of acetonitrile, which was eluted at a flow rate of 1.0 mL/min. The sample injection volume was 40 µL and detection wavelength was 254 nm. The retention time was about 2.5 min. Calibration curve with the coefficient of determination (r²) for linear regression of at least 0.999 was acceptable. The accuracy and precision of the HPLC method were validated.

It was shown in Fig. 1 that all formulations of ACV-loaded BSA nanoparticles with small and monodisperse sizes around 120–200 nm could be obtained by this preparation process for those resuspended in SWI and isotonic trehalose solution. In contrast, those resuspended in PBS were found to exhibit much larger sizes with high polydispersity. This might be attributed to the effect of ionic strength of different vehicle media. The solution with high concentration of ions could neutralize the surface charge and shift the zeta potential to nearly zero, which was followed by aggregation of the particles. Adsorption of neutral molecules on the charged surface should cause the movement of the slip plane away from the surface causing a decrease in the absolute value of zeta potential. The high absolute value of zeta potential generates a repulsive electrostatic force between particles, which is a key property of an agglomeration resistant suspension (Kaasalainen et al. 2012). With low absolute value of zeta potential, agglomeration of the particles was generated and could be determined by an increase in particles sizes. Such explanation could be evident by the results of zeta potentials shown in Fig. 2. The surfaces of BSA nanoparticles were negatively charged with zeta potentials of around -7 to -50 mV due to higher pH of vehicle medium than the pI of BSA. The highly negative charge reduced the extent of protein-protein interactions to form larger particles. SWI and trehalose solution as vehicle medium could still maintain the electrostatic stability of the colloidal system, while PBS imposed the strong ionic strength effect resulting in aggregation of particles as mentioned above. This indicated that physical stability of nanoparticles was not only affected by the intrinsic electrical properties of the nanoparticles formulations, but also influenced by microenvironments (Jun et al. 2011; Kaasalainen et al. 2012).

Figure 1. 

Particle size (upper) and PdI (lower) of BSA nanoparticles with various ACV contents in different vehicle media. (Mean ± SD, n = 3).

Figure 2. 

Zeta potential of BSA nanoparticles with various ACV contents in different vehicle media. (Mean ± SD, n = 3).

It can be seen in Table 1 that substantially loading efficiency of around 60% of drug could be achieved in ACV-loaded BSA nanoparticles prepared by this preparation process. Besides, the results indicated that a 2-fold increase in ACV amount added to the system could significantly improve the drug loading capacity by almost the same ratio with similar percentage loading efficiency. This may be due to molecular mixing behavior between the bulk aqueous solution containing ACV and BSA, and flow of submerged circular jet of ethanol pumped into it as illustrated in Fig. 3. The volume of liquid entrained at the nominal boundary of the jet per unit time, causing the mixing in action, is proportional to the volume of liquid leaving jet nozzle per unit time as described by the following equation (McCabe et al. 1993)

Figure 3. 

Molecular mixing behavior between the bulk aqueous solution containing ACV and BSA, and flow of submerged circular jet of ethanol (upper), and calculated flow (mL/min) of bulk aqueous liquid containing ACV and BSA entrained at the nominal boundary of the ethanol jet submersibly pumped at 15 mL/min through 0.5-mm spinal needle as a function of distance from needle tip (lower).

q_e = [(x/4.3D_j) - 1]q_o (3)

where D_j is the diameter of submerged circular jet, q_e is the volume of liquid entrained per unit time at distance x from nozzle, and q_o is the volume of liquid leaving jet nozzle per unit time. In this study, the diameter of submerged circular jet of ethanol (D_j) is equal to that of 0.5-mm (0.05 cm) spinal needle used and leaving jet nozzle at a flow rate (q_o) of 15 mL/min. Substituting these values into Eq. (3) would yield Eq. (4)

q_e = 69.77x - 15 (4)

The calculated volume flow rates (mL/min) of bulk aqueous ACV-BSA solution entrained at the nominal boundary of the ethanol jet pumped through needle at various distances from needle tip are shown in Fig. 3. It was shown that the volume flow rate of ACV-BSA solution entrained increases drastically as a function of distance compared to that of ethanol jet. Therefore, ACV in solution could be efficiently adsorbed and embedded at a higher extent onto the simultaneously precipitating BSA nanoparticles. In addition, from Eq. (4) the mass flow rate (mg/min) of ACV in entrained solution that mixed with ethanol at distance x from nozzle is

Q_e = (69.77x – 15) C_ACV (5)

where C_ACV is the ACV concentration in solution. Eq. (5) implies that increase in ACV concentration would result in an increase in mass mixing rate, and hence drug loading capacity, by almost the same increasing ratio of ACV concentration.