Development of a Resveratrol Nanosuspension Using the Antisolvent Precipitation Method without Solvent Removal, Based on a Quality by Design (QbD) Approach

The purpose of this study was to develop a resveratrol nanosuspension with enhanced oral bioavailability, based on an understanding of the formulation and process parameters of nanosuspensions and using a quality by design (QbD) approach. Particularly, the antisolvent method, which requires no solvent removal and no heating, is newly applied to prepare resveratrol nanosuspension. To ensure the quality of the resveratrol nanosuspensions, a quality target product profile (QTPP) was defined. The particle size (z-average, d90), zeta potential, and drug content parameters affecting the QTPP were selected as critical quality attributes (CQAs). The optimum composition obtained using a 3-factor, 3-level Box–Behnken design was as follows: polyvinylpyrrolidone vinyl acetate (10 mg/mL), polyvinylpyrrolidone K12 (5 mg/mL), sodium lauryl sulfate (1 mg/mL), and diethylene glycol monoethyl ether (DEGEE, 5% v/v) at a resveratrol concentration of 5 mg/mL. The initial particle size (z-average) was 46.3 nm and the zeta potential was −38.02 mV. The robustness of the antisolvent process using the optimized composition conditions was ensured by a full factorial design. The dissolution rate of the optimized resveratrol nanosuspension was significantly greater than that of the resveratrol raw material. An in vivo pharmacokinetic study in rats showed that the area under the plasma concentration versus time curve (AUC0–12h) and the maximum plasma concentration (Cmax) respectively, than those of the resveratrol raw material. Therefore, the prepara values of the resveratrol nanosuspension were approximately 1.6- and 5.7-fold higher,tion of a resveratrol nanosuspension using the QbD approach may be an effective strategy for the development of a new dosage form of resveratrol, with enhanced oral bioavailability.

. Initial risk assessment of the resveratrol nanosuspension.

Resveratrol concentration
High If the concentration of resveratrol is excessively high, particles grow very quickly during the manufacturing process. Therefore, the risk level was high.
Stabilizer type High The ability to inhibit particle growth depends on the type of stabilizer. Therefore, the risk level was high. Stabilizer concentration High An appropriate concentration of stabilizer has an effective ability to inhibit particle growth. Therefore, the risk level was high.

Solvent type High
The solvent and anti-solvent should be sufficiently miscible, and the solvent should have a solubilization effect. Therefore, the risk level was high. Ratio of solvent/antisolvent High The solubility of resveratrol for the mixture solvents depends on the ratio of the solvent/anti-solvent. The solubility affects particle growth. Therefore, the risk level was high.
Mixing speed High Depending on the mixing speed, the mixing speed of the anti-solvent and solvent vary, and can affect particle growth rate. Therefore, the risk level was high. Mixing time High Mixing time can affect particle growth. Therefore, the risk level was high.
Injection rate (solvent) High Depending on the rate of injection of solvent, the mixing speed of the anti-solvent and solvent vary, which can affect the particle growth rate. Therefore, the risk level was high.

Temperature High
The solubility of resveratrol changes when the temperature of the solvent changes, which can affect particle growth. Therefore, the risk level was high.
Zeta Potential

Resveratrol concentration
Low The effect of changes in resveratrol concentration on the zeta potential is insignificant. Therefore, the risk level was low.

Stabilizer type High
Depending on the type of stabilizer, the surface charge of the particles differs. Therefore, the risk level was high. Stabilizer concentration High Depending on the concentration of stabilizer, the surface charge of the particles differs. Therefore, the risk level was high.

Low
The influence of the type of solvent on the surface charge of the particles was insignificant. Therefore, the risk level was low. Ratio of solvent/antisolvent Medium Depending on the solvent/anti-solvent ratio, the concentration of the stabilizer in the mixture solvent varies, which affects the surface charge of the particles. Therefore, the risk level was medium. Mixing speed Low The effect of mixing speed on the zeta potential is insignificant. Therefore, the risk level was low. Mixing time Low The effect of mixing time on the zeta potential is insignificant. Therefore, the risk level was low. Injection rate (solvent) Low The effect of injection rate on the zeta potential is insignificant. Therefore, the risk level was low. Temperature Low The effect of temperature on the zeta potential is insignificant. Therefore, the risk level was low.

Resveratrol concentration Low
Resveratrol is chemically stable when light is blocked. Nanosuspensions were prepared in a space where light was blocked, and the possibility of drug loss during the manufacturing process is insignificant. Therefore, the risk level was low.    Table S3. Particle size of resveratrol nanosuspensions prepared using a polymer/polymer combination.   Table S6. Updated risk assessment of resveratrol nanosuspension after preformulation and screening study.

Resveratrol concentration
Low In a preliminary experiment, the resveratrol concentration was fixed at 100 mg/mL to satisfy the define QTPP. Therefore, the risk level was reduced to low.

Stabilizer type Low
Based on preliminary experiments, PVP VA64, PVP K12, and SLS were selected as stabilizers. Therefore, the risk level was reduced to low.

Solvent type Low
Based on preliminary experiments, Transcutol ® HP was selected as a solvent. Therefore, the risk level was reduced to low.

Ratio of solvent/anti-solvent Low
Based on preliminary experiments, the solvent/anti-solvent ratio was fixed at 1/19 to satisfy the define QTPP. Therefore, the risk level was reduced to low.

Mixing speed Medium
In a preliminary experiment, a nanosuspension that satisfied the target values was prepared at a mixing speed of 750 rpm. However, the mixing speed can still affect particle size. Therefore, the risk level was reduced to medium.

Mixing time Low
In preliminary experiments, the effect of mixing time on the particle size distribution was insignificant. Therefore, the risk level was reduced to low.

Injection rate (solvent) Medium
In a preliminary experiment, a nanosuspension that satisfied the target values was prepared at an injection rate of 1.0 mL/min. However, the injection rate can still affect the particle size. Therefore, the risk level was reduced to medium.

Temperature Medium
In a preliminary experiment, a nanosuspension that satisfied the target values was prepared at 25°C. However, the temperature can still affect the particle size. Therefore, the risk level was reduced to medium.

Zeta Potential
Stabilizer type Low In preliminary experiments, PVP VA64, PVP K12, and SLS were selected as stabilizers. Therefore, the risk level was reduced to low.

Stabilizer concentration High
Depending on the concentration of stabilizer, the surface charge of the particles is different. Therefore, the risk level was high.

Ratio of solvent/anti-solvent Low
In preliminary experiments, the solvent/anti-solvent ratio was fixed at 1/19 to satisfy the define QTPP. Therefore, the risk level was reduced to low. R 2 , coefficient of determination; PRESS, predicted residual error sum of squares; CV, coefficient of variation.  Table S9. Updated risk assessment of resveratrol nanosuspension after optimization study.

Resveratrol concentration
Low Based on a preliminary experiment, the resveratrol concentration was fixed at 100 mg/mL. Therefore, the risk level was reduced to low.

Stabilizer type Low
In preliminary experiments, PVP VA64, PVP K12, and SLS were selected as stabilizers. Therefore, the risk level was reduced to low.

Stabilizer concentration Low
The particle size distribution in the optimized nanosuspension satisfies the set target range. Therefore, the risk level was reduced to low.

Solvent type Low
Based on preliminary experiments, Transcutol ® HP was selected as a solvent. Therefore, the risk level was reduced to low.

Ratio of solvent/anti-solvent Low
Based on preliminary experiments, the ratio of solvent/anti-solvent was fixed at 1/19. Therefore, the risk level was reduced to low.

Mixing speed Low
The particle size distribution of the nanosuspension prepared at 500 rpm-1000 rpm was within the set target range. Therefore, the risk level was reduced to low.

Mixing time Low
Based on preliminary experiments, the effect of mixing time on the particle size distribution was insignificant. Therefore, the risk level was reduced to low.
Injection rate (solvent) Low The particle size distribution of the nanosuspension prepared at an injection rate of 1.0 mL/min was within the set target range. Therefore, the risk level was reduced to low.

Low
The particle size distribution of the nanosuspension prepared at 20°C ~ 30°C was within the set target range. Therefore, the risk level was reduced to low.

Zeta Potential
Resveratrol concentration Low The effect of changes in the resveratrol concentration on the zeta potential is insignificant. Therefore, the risk level was low.
Stabilizer type Low Based on preliminary experiments, PVP VA64, PVP K12, and SLS were selected as stabilizers. Therefore, the risk level was reduced to low.

Stabilizer concentration Low
In the optimized nanosuspension, the zeta potential value was -32.9 mV to -39.6 mV and satisfied the target range. Therefore, the risk level was reduced to low.

Solvent type Low
The influence of the type of solvent on the surface charge of the particles was insignificant. Therefore, the risk was low.
Ratio of solvent/anti-solvent Low Based on preliminary experiments, the solvent/anti-solvent ratio was fixed at 1/19. Therefore, the risk was reduced to low.

Mixing speed Low
The effect of mixing speed on the zeta potential was insignificant. Therefore, the risk was low.

Mixing time Low
The effect of mixing time on the zeta potential was insignificant. Therefore, the risk was low.
Injection rate (solvent) Low The effect of injection rate on the zeta potential was insignificant. Therefore, the risk was low.

Temperature Low
The effect of temperature on the zeta potential was insignificant. Therefore, the risk was low.

Resveratrol concentration Low
Resveratrol is chemically stable when light is blocked. Nanosuspensions were prepared in a space where light is blocked, and the possibility of drug loss during the manufacturing process is insignificant. Therefore, the risk level was reduced to low.