*2.1. Materials*

*Trans*-resveratrol was supplied by Ningbo Liwah Pharmaceutical Co., Ltd. (Zhejiang, China), and micronized to a mean particle size of 2.6 μm and purity of 99.1% by using an air jet mill. Polyvinylpyrrolidone K 12/25/30/90 (PVP K12/K25/K30/K90), polyvinylpyrrolidone vinyl acetate 64 (PVP VA 64), polyvinyl caprolactampolyvinyl acetate-polyethylene glycol graft copolymer (Soluplus ®), macrogol (15)-hydroxystearate (KolliphorTM HS 15), <sup>d</sup>-<sup>α</sup>-Tocopherol polyethylene glycol 1000 succinate (TPGS), and poly (ethylene glycol)-block-poly (propylene glycol)-block-poly(ethylene glycol) (Poloxamer 188/407) were obtained from BASF (Ludwigshafen, Germany). Hydroxylpropylmethyl cellulose (HPMC 3 cp/4.5 cp/6 cp) and low viscosity hydroxylpropyl cellulose (HPC-SSL) were provided by Shin-Etsu chemical Co., Ltd. (Tokyo, Japan), and Nippon Soda Co., Ltd. (Japan), respectively. Polyethylene glycol 6000, sodium carboxymethylcellulose (CMC), and sodium lauryl sulfate (SLS) were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Sucrose laurate (RyotoTM Ester L-1695, Mitsubishi-Kagaku Foods Co., Tokyo, Japan) was gifted by Namyung commercial Co., Ltd. (Seoul, Korea). Sorbitan monolaurate (Span® 20), sorbitan monostearate (Span® 60), sorbitan monooleate (Span® 80), and polysorbate 20/60/80 (Tween® 20/60/80) were purchased from Daejung Chemicals and Metals Co., Ltd. (Siheung-si, Korea). Propylene glycol dicaprolate/dicaprate (LabrafacTM PG, Gattefossè, Saint-Priest, France), medium chain triglycerides (LabrafacTM Lipophile WL1349, Gattefossè, Saint-Priest, France), oleoyl polyoxyl-6 glycerides (Labrafil® M 1944CS, Gattefossè, Saint-Priest, France), linoleoyl polyoxyl-6 glycerides (Labrafil® M 2125CS, Gattefossè, Saint-Priest, France), lauroyl polyoxyl-6 glycerides (Labrafil® M 2130CS, Gattefossè, Saint-Priest, France), caprylocaproyl polyoxyl-8 glycerides (Labrasol®, Gattefossè, Saint-Priest, France), propylene glycol monolaurate type II (LauroglycolTM 90, Gattefossè, Saint-Priest, France), and polyglyceryl-3 dioleate (Plurol® Oleique CC 497, Gattefossè, Saint-Priest, France) were kindly provided by Masung Chemicals (Seoul, Korea). All organic solvents and reagents used were either of high-performance liquid chromatography (HPLC) grade or analytical grade and were purchased from Honeywell Burdick and Jackson (Muskegon, MI, USA) or Daejung Chemicals and Metals Co., Ltd. (Siheung-si, Korea), respectively.

#### *2.2. Solubility Studies of Trans-Resveratrol in Aqueous Solutions Containing Various Additives*

The solubility of *trans*-resveratrol in aqueous solutions was measured to test different polymers and surfactants. First, aqueous solutions containing 1% polymer or 1% surfactant (w/v) were prepared and excess resveratrol was added to an amber vial containing 10 mL of each solution. Samples were sonicated for 1 h then incubated in a shaking water bath at 37 ◦C for 72 h to reach equilibrium. Next, samples were centrifuged at 12,000× *g* for 15 min and filtered through a 0.2 μm glass fiber syringe filter. Then, 1 mL of filtrate was taken into an amber volumetric flask and diluted with methanol. The concentration of resveratrol was then determined using a Shimadzu HPLC system (Shimadzu, Tokyo, Japan) consisting of an SPD-20A ultraviolet–visible (UV/VIS) detector, CBM-20A communications bus module, SIL-20AC autosampler, LC-20AT liquid chromatograph, DGU-20A 5R degassing unit, and a C18 analytical column (4.6 × 150 mm, 5 μm, Shiseido, Tokyo, Japan). HPLC analysis conditions were as follows: a 40% acetonitrile in water mobile phase, 0.8 mL/min flow rate, 30 ◦C column temperature, 10 μL injection volume, and UV detector wavelength of 303 nm.

#### *2.3. Nanoparticle Preparation Using an SAS Process*

For preparation of *trans*-resveratrol-loaded composite nanoparticles, an SAS process was applied using the equipment as previously described [26,27]. CO2 gas was liquefied using a cooler, raised to the required temperature using a heat exchanger, and was pumped through an ISCOTM pump (Model 260D, Teledyne Technologies Inc., Thousand Oaks, CA, USA) into a high-pressure precipitation vessel. Alongside an SAS process, drug solutions were prepared by dissolving resveratrol/polymer (HPMC 6 cp)/surfactant (gelucire 44/14, TPGS, or poloxamer 407)) in a mixture of methanol and dichloromethane (1:1, w/w) in an amber vial. When the system reached a certain temperature and pressure (40 ◦C and 12 MPa), the drug solution was injected through the nozzle into a high-pressure vessel at a constant rate (1 g/min), along with supercritical carbon dioxide (40 g/min). The temperature of the precipitation vessel was maintained by circulating water with a temperature-controlled bath circulator. After injection of the drug solution was complete, additional carbon dioxide was applied to remove residual solvent dissolved in supercritical carbon dioxide. The pressure in the precipitation vessel was then slowly reduced to atmospheric pressure using the back-pressure regulator and the precipitated particles inside the vessel were collected.

#### *2.4. Trans-Resveratrol Content Analysis*

Levels of *trans*-resveratrol within the composite particles were determined using HPLC analysis of sample solutions. Composite particles were dissolved in a mixture of methanol and dichloromethane (1:1, w/w). After dilution with methanol, the concentration of *trans*-resveratrol was determined using a Shimadzu Prominence HPLC system (Shimadzu, Tokyo, Japan). The encapsulation e fficiency (%) was calculated by dividing the measured concentration by the theoretical concentration and then multiplying this by 100.

#### *2.5. Scanning Electron Microscopy (SEM)*

The morphology of the samples was examined using a scanning electron microscope (SUPRA 25 or 40, Zeiss, Oberkochen, Germany) operating at a voltage of 5 kV. Before observation, samples were fixed to aluminum stubs with double-sided adhesive carbon tape and then gold coated at a pressure of 8–10 Pa for 1 min to increase electrical conductivity of the sample.

#### *2.6. Particle Size Measurements*

The average particle size of the samples was determined with dynamic light scattering (ELSZ-1000, Otsuka Electronics, Tokyo, Japan). Samples were su fficiently dispersed in mineral oil and sonicated for 10 min, then size measurements performed at least four times.

#### *2.7. Specific Surface Area Measurements*

The specific surface area of composite nanoparticles was measured with a Micromeritics TriStar II 3020 instrument (Micromeritics, Norcross, GA, USA), using the adsorption of nitrogen at the temperature of liquid nitrogen.

#### *2.8. Di*ff*erential Scanning Calorimetry (DSC)*

DSC analysis was performed using a thermal analyzer (DSC25, TA instruments, Inc., New Castle, DE, USA). Prior to each analysis, temperature and heat capacity calibration were performed using high purity indium and aluminum oxide sapphire, respectively, with a temperature range of 0 to 390 ◦C, a modulation rate of 0.6 ◦C/min every 40 s, and a scan speed of 5 ◦C/min. Next, 2–4 mg of sample was weighed and placed in a pre-weighed aluminum hermetic pan, then sealed with an aluminum cover. Closed, empty aluminum pans were used as reference samples. Analysis was carried out by heating samples from 0 to 350 ◦C at a heating rate of 10 ◦C/min under a nitrogen purge of 400 mL/min.

#### *2.9. Powder X-ray Di*ff*raction (PXRD)*

Powder X-ray di ffraction analysis for samples was performed from 5◦ to 60◦ using an X-ray Di ffractometer (Xpert 3, Panalytical, Almelo, Netherlands) with Ni-filtered Cu-K α radiation. Data were collected at a scanning speed of 3◦/min and a step size of 0.01.

#### *2.10. Kinetic Solubility Study*

A sample of 100 mg *trans*-resveratrol was placed in a water-jacked beaker containing 50 mL of distilled water maintained at 37 ◦C with magnetic stirring at 300 rpm. At predetermined time intervals, 3 mL of sample was withdrawn from the medium and centrifuged at 12,000× *g* for 15 min, then filtered using a 0.2 μm glass fiber syringe filter to remove insoluble material. After dilution with methanol, the concentration of *trans*-resveratrol was quantified using HPLC analysis. All sample measurements were repeated six times.

#### *2.11. Flux Measurements via In Vitro Dissolution and Permeation Studies*

To compare *trans*-resveratrol flux between di fferent composite nanoparticles prepared by an SAS process and to evaluate the correlation between in vitro flux data and in vivo pharmacokinetic

data for *trans*-resveratrol, a flux measurement study of *trans*-resveratrol was carried out using a miniaturized dissolution–permeation apparatus (μFLUXTM apparatus, Pion Inc., Billerica, MA, USA) [28]. This apparatus contains a horizontal diffusion cell composed of a donor cell, membrane, and receiver cell. The membrane (diffusion area of 1.54 cm2) used to separate the donor and receiver cells was prepared by impregnating support material (polyvinylidenfluoride, 0.45 μm pore size, 70% porous, 120 μm thickness) with 50 μL of GITTM lipid solution consisting of 20% phospholipid in a dodecane lipid solution (Pion Inc., Billerica, MA, USA). The donor cell was filled with 16 mL of simulated intestinal fluid (pH 6.8, with pancreatin), while the receiver cell was filled with 16 mL of acceptor sink buffer (ASB, Pion Inc., Billerica, MA, USA), consisting of a hydroxyethyl piperazine ethane sulfonicacid (HEPES)-based pH 7.4 buffer containing surfactant micelles to ensure the sink condition of *trans*-resveratrol. The temperature of the diffusion cell was maintained at 37 ◦C by circulating water through the heating block with a temperature bath circulator. A 32 mg sample of *trans*-resveratrol was placed in the donor cell with magnetic stirring at 150 rpm. The concentration of drug in both the donor cell and receiver cell was quantified using UV fiber optic probes (2 mm path length for the donor cell and 20 mm path length for the receiver cell) connected with Pion Rainbow spectrometers, using a wavelength of 306 nm. Data were collected every 1 min for the first 30 min, then every 5 min for the next 240 min. The calibration curve for *trans*-resveratrol levels in simulated intestinal fluid (pH 6.8, with pancreatin) in the donor cell was generated by diluting a stock solution of *trans*-resveratrol in a mixture of ethanol and simulated intestinal fluid at pH 6.8 (with pancreatin). The calibration curve for *trans*-resveratrol levels in the receiver cell was generated using serial dilutions of a stock solution of *trans*-resveratrol in acceptor sink buffer. All sample tests were repeated four times. Flux (*J*), the mass transfer through the membrane, is calculated using Equation (1):

$$J = \frac{dm}{Sdt} = \frac{V}{S} \cdot \frac{dc}{dt} \tag{1}$$

where *dc*/*dt* is the slope of the concentration of *trans*-resveratrol vs. the time profile in the range with linear slope (except for lag time), *V* is the volume (mL) of medium in the donor cell, and *S* is the permeation area (cm2).

#### *2.12. Pharmacokinetic Study of Oral Delivery in Rats*

The animal study protocol is in compliance with institutional guidelines for the care and use of laboratory animals and was approved by the ethics committee of Kyungsung University (No. 17-004A). To investigate the oral bioavailability of *trans*-resveratrol composite nanoparticles, the in vivo pharmacokinetics of*trans*-resveratrol in male Sprague–Dawley (SD) rats were evaluated. Thirty-six male SD rats (200 ± 10 g; Hyochang Science, Daegu, Korea) were divided into six treatment groups of six rats each. The six experimental groups received either micronized *trans*-resveratrol, *trans*-resveratrol/HPMC composite nanoparticles (1:4 or 1:5), or *trans*-resveratrol/HPMC/surfactant composite nanoparticles (1:4:1) at *trans*-resveratrol doses of 20 mg/kg by oral administration. Samples were dispersed in 1 mL of water immediately prior to oral dosing. Blood samples (approximately 0.25 mL each) were collected in heparinized tubes from the jugular vein of the treated rats at 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, and 12 h after dosing. Blood samples were centrifuged at 12,000 × *g* for 10 min at 4 ◦C. The amount of *trans*-resveratrol in plasma was determined using HPLC, following a previously reported analytical method [29]. Data were then used to determine the maximum plasma concentration of *trans*-resveratrol (*C*max), and the time required to reach *C*max (*T*max) and the area under the plasma concentration versus time curve (*AUC*0→<sup>12</sup> h) were calculated using the linear trapezoidal method.

#### *2.13. Ex Vivo Skin Permeation Study of Skin Delivery*

For application of *trans*-resveratrol-loaded composite nanoparticles in skin delivery, ex vivo skin permeation studies were carried out for 24 h at 32 ◦C with vertical static-type Franz diffusion cells, using the skin of SD rats as the diffusion membrane. Firstly, after anesthesia, rat abdominal

skin was shaved using electric and hand razors, then removed surgically. Skin samples were then cleaned of adherent subcutaneous fat and immersed in cold normal saline solution (pH 7.4) for 2 h. Powdered samples of *trans*-resveratrol-loaded composite nanoparticles were placed on the skin membrane surfaces, with effective diffusion areas of 1.86 cm2. Skin membrane surfaces and sampling ports were later covered with parafilm and aluminum foil to minimize the influx of the external compounds and the degradation of *trans*-resveratrol from light. The loading dose of *trans*-resveratrol was 0.5 mg/cm<sup>2</sup> [30] The receptor cell was filled with 11.5 mL of a 60:40 mixture of phosphate-buffered saline (pH 7.4) and ethanol and stirred with a magnetic bar at 150 rpm to ensure uniform mixing. At predetermined time intervals, 0.2 mL of receptor medium was taken from the receptor compartment and replaced by an equal volume of fresh medium (32 ◦C). Samples were then centrifuged for 15 min at 12,000× *g*, and a 10 μL aliquot of the supernatant was injected into an HPLC system, as described above. The amount of *trans*-resveratrol permeated per unit area of the skin was then calculated. All sample tests were repeated six times.
