*2.14. Data Analysis*

Data are expressed as mean ± standard deviation (*n* = 4 or 6). To evaluate the statistical significance of differences between groups, one-way ANOVA was carried out, followed by least significant difference (LSD) and Student-Newman-Keuls (SNK) tests using SPSS 25.0 software (IBM SPSS Statistics, IBM Corporation, Armonk, NY, USA).

#### **3. Results and Discussions**

*Trans*-resveratrol-loaded composite nanoparticles were prepared using an SAS process with hydrophilic polymers and surfactants. For a preliminary study of various additives, the equilibrium solubility of *trans*-resveratrol in aqueous solutions containing 1% additive were determined at 37 ◦C and are presented in Figure 1. The solubility of *trans*-resveratrol was 55.3 μg/mL in water, 52.3 μg/mL in pH 1.2 buffer solution, 53.7 μg/mL in pH 4.0 buffer solution, and 51.1 μg/mL in pH 6.8 buffer solution. These results indicate that the solubility of *trans*-resveratrol is very poor in aqueous solution and is similar to previously reported data [31]. Among the hydrophilic polymers, *trans*-resveratrol showed the highest solubility in HPMC (6 cp). Interestingly, the solubility of *trans*-resveratrol increased with increasing viscosity of HPMC, while *trans*-resveratrol solubility showed the opposite trend in PVP. Surfactants dramatically increased *trans*-resveratrol solubility via micelle formation. The most effective surfactant tested was poloxamer 407, followed by TPGS, tween, and gelucire 44/14. The solubility of *trans*-resveratrol with poloxamer 407 was approximately 20-fold the solubility of *trans*-resveratrol alone. Based on the solubility tests, HPMC (6 cp) was selected as the polymer for nanoparticle construction and poloxamer 407, TPGS, and gelucire 44/14 were evaluated as surfactants for preparation of *trans*-resveratrol-loaded composite nanoparticles using an SAS process.

**Figure 1.** Solubility of *trans*-resveratrol in aqueous solutions containing 1% of various additives at 37 ◦C. Note: PVP = polyvinylpyrrolidone; HPMC = hydroxylpropylmethyl cellulose; SLS = sodium lauryl sulfate; CMC = sodium carboxymethylcellulose; HPC-SSL = hydroxylpropyl cellulose; TPGS = D-α-Tocopherol polyethylene glycol 1000 succinate; PEG = polyethylene glycol.

#### *3.1. Preparation and Characterization of Trans-Resveratrol Composite Nanoparticles*

In this study, we prepared composite nanoparticles containing 1:4 and 1:5 ratios of *trans*-resveratrol/HPMC using a SAS process and evaluated the molecular dispersion of *trans*-resveratrol within composite nanoparticles based on previously reported formulations [32]. In addition, the effects of surfactants (poloxamer 407, TPGS, and gelucire 44/14) on the physicochemical properties and in vivo performance of *trans*-resveratrol-loaded composite nanoparticles was evaluated at a 1:4:1 ratio of *trans*-resveratrol/HPMC/surfactants. In our previous work, nanoparticle agglomeration was observed in composite nanoparticles with high levels of low melting point surfactants (poloxamer 407, TPGS, and gelucire 44/14) prepared using an SAS process [33–35]. As shown in Figure 2 and Table 1, micronized *trans*-resveratrol morphology includes needle-shaped particles with mean particle sizes of 2.6 μm, while *trans*-resveratrol-HPMC nanoparticles are spherical particles with sizes of 180–190 nm and specific surface areas of 60–64 m<sup>2</sup>/g. No significant differences were observed between *trans*-resveratrol-HPMC nanoparticles composed of 1:4 and 1:5 ratios of *trans*-resveratrol/HPMC. However, surfactant addition to nanoparticles increased mean particle size and reduced specific surface areas. In particular, *trans*-resveratrol/HPMC/TPGS nanoparticles with mean particle sizes of 293.4 nm exhibited fusion and aggregations of nanoparticles with specific surface areas of 36.4 m<sup>2</sup>/g due to the low melting temperature of TPGS (37 ◦C). Nevertheless, mean particle sizes of all the composite nanoparticles produced were less than 300 nm. *Trans*-resveratrol was successfully incorporated into composite nanoparticles and the encapsulation efficiency exceeded 97% for all formulations, indicating that *trans*-resveratrol was not degraded during the SAS process (Table 1). In addition, SAS process yields were above 80% for all formulations.

**Figure 2.** SEM images of *trans*-resveratrol composite nanoparticles.

**Table 1.** Composition, encapsulation efficiency, mean particle size, and specific surface area of *trans*-resveratrol composite nanoparticles.


Data are expressed as the mean ± standard deviation (*n* = 4).

The crystallinity and dispersion of *trans*-resveratrol in composite nanoparticles was analyzed using modulated DSC and PXRD. In DSC thermograms (Figure 3A), the melting temperature and fusion enthalpy of raw *trans*-resveratrol are 268.96 ◦C and 270.96 J/g, respectively, in good agreemen<sup>t</sup> with previously reported data [36]. The complete disappearance of a single, sharp melting endotherm for *trans*-resveratrol was observed for composite nanoparticles, indicating that *trans*-resveratrol exists in an amorphous or molecularly dispersed state within composite nanoparticles. Furthermore, we also analyzed the glass transition temperature (*T*g) using modulated DSC measurements. If small molecule drugs act as a plasticizer, the *T*g value of the polymer should decrease with increasing drug content in polymer–drug blends. As shown in reversing heat flow versus temperature thermograms (Figure 3B), the *T*g value of the composite nanoparticles decreased with increasing *trans*-resveratrol level, and all composite nanoparticles exhibited one *T*g value. From these results, *trans*-resveratrol appears dispersed at the molecular level within the composite nanoparticles. In addition, samples of micronized *trans*-resveratrol and composite nanoparticles were characterized using PXRD to assess the preparation of amorphous composites of *trans*-resveratrol (Figure 3C). Micronized *trans*-resveratrol exhibited characteristic peaks at 2θ, similar to previously reported results [37] However, characteristic peak patterns for *trans*-resveratrol were not observed for all composite nanoparticle preparations, indicating that *trans*-resveratrol is molecularly dispersed within composite nanoparticles.

**Figure 3.** Differential scanning calorimetry thermograms of (**A**) heat flow versus temperature and (**B**) reversing heat flow versus temperature, and powder X-ray diffraction patterns (**C**) of *trans*-resveratrol composite nanoparticles.

The kinetic solubility of*trans*-resveratrol composite nanoparticles was determined in distilled water at 37 ◦C. As shown in Figure 4, the degree of solubility of *trans*-resveratrol in composite nanoparticles was dramatically increased compared to that of micronized *trans*-resveratrol. In particular, the solubility of *trans*-resveratrol/HPMC/poloxamer 407 (1:4:1) nanoparticles at 24 h was significantly higher (~7.2x) than that of micronized *trans*-resveratrol. The solubility values of *trans*-resveratrol-loaded composite nanoparticles at 24 h as ranked by the SNK test were as follows: drug/HPMC/poloxamer 407 (1:4:1) > drug/HPMC/TPGS (1:4:1) > drug/HPMC/gelucire 44/14 (1:4:1) > drug/HPMC (1:5) = drug/HPMC (1:4) > micronized *trans*-resveratrol. The maximum solubility of *trans*-resveratrol from composite nanoparticles was rapidly reached and maintained for at least 24 h through high inhibition of *trans*-resveratrol crystallization by HPMC [32,38].

**Figure 4.** Kinetic solubility profiles of *trans*-resveratrol composite nanoparticles in distilled water at 37 ◦C.

#### *3.2. Use of Trans-Resveratrol Composite Nanoparticles for Oral Delivery*

To compare *trans*-resveratrol flux of different composite nanoparticles prepared by the SAS process and establish correlations between in vitro flux data and in vivo pharmacokinetic data of *trans*-resveratrol, in vitro flux measurements and in vivo pharmacokinetic experiments of *trans*-resveratrol in SD rats were carried out using the oral delivery of *trans*-resveratrol composite nanoparticles. As shown in Figure 5A, the concentration of *trans*-resveratrol from composite nanoparticles in the donor cell increased with increasing solubility of composite nanoparticles. Higher concentrations of *trans*-resveratrol in the receiver cell were observed by increasing the driving force of *trans*-resveratrol through the membrane. In addition, the concentration of *trans*-resveratrol over time in the donor cell indicated maximum dissolution of *trans*-resveratrol from the composite nanoparticles within 2 min, indicating fast dissolution from multiple HPMC/surfactant combinations. Flux (*J*) was obtained from the slope of the concentration of *trans*-resveratrol vs. time profile in the range of 30 to 240 min in Figure 5B, and is presented in Table 2. The system reached steady state within 30 min after dissolution–permeation measurements. As shown in Table 2, *trans*-resveratrol/HPMC/poloxamer 407 (1:4:1) nanoparticles exhibited the highest flux of 0.792 μg/min/cm2, which was 3.0-fold higher than the flux of micronized *trans*-resveratrol. Flux ranks based on the SNK test for *trans*-resveratrol-loaded composite nanoparticles were as follows: drug/HPMC/poloxamer 407 (1:4:1) > drug/HPMC/TPGS (1:4:1) > drug/HPMC/gelucire 44/14 (1:4:1) > drug/HPMC (1:5) = drug/HPMC (1:4) > micronized *trans*-resveratrol. The trend of increased flux of composite nanoparticles is similar to the trend for kinetic solubility of *trans*-resveratrol. In addition, the very fast dissolution of *trans*-resveratrol from HPMC/surfactant nanoparticles was observed in the donor cell.

The increased solubility and flux of *trans*-resveratrol by composite nanoparticles increases the oral bioavailability of *trans*-resveratrol [39,40]. As shown in Figure 6, HPMC/surfactant composite nanoparticles have rapid absorption rates and significantly higher exposure 4 h after oral administration compared to micronized *trans*-resveratrol. *C*max ranks based on the SNK test for*trans*-resveratrol-loaded composite nanoparticles were: drug/HPMC/poloxamer 407 (1:4:1) = drug/HPMC/TPGS (1:4:1) > drug/HPMC/gelucire 44/14 (1:4:1)>drug/HPMC (1:5)=drug/HPMC (1:4)> micronized *trans*-resveratrol. The *AUC*0–12 h ranked by the SNK test for *trans*-resveratrol-loaded composite nanoparticles were as follows: drug/HPMC/poloxamer 407 (1:4:1) > drug/HPMC/TPGS (1:4:1) > drug/HPMC/gelucire 44/14 (1:4:1) > drug/HPMC (1:5) = drug/HPMC (1:4) > micronized *trans*-resveratrol. Composition ranks for flux data, thus, agreed with ranks for *AUC*0–12 h. Greater increases in *C*max and *AUC*0→<sup>12</sup> h

were observed for composite nanoparticles compared to micronized *trans*-resveratrol. In particular, *C*max and *AUC*0→<sup>12</sup> h of *trans*-resveratrol/HPMC/poloxamer 407 (1:4:1) nanoparticles were 9.7-fold and 3.0-fold higher, respectively, than those of micronized *trans*-resveratrol, which may be due to the rapid metabolism of *trans*-resveratrol [41].

**Figure 5.** In vitro dissolution (**A**) and permeation profiles (**B**) of *trans*-resveratrol composite nanoparticles. Data are expressed as the mean ± standard deviation (*n* = 4).

**Table 2.** In vitro flux data and in vivo pharmacokinetic data for *trans*-resveratrol composite nanoparticles.


Note: a *p* < 0.05 vs. micronized trans-resveratrol; b *p* < 0.05 vs. drug/HPMC (1:5); c *p* < 0.05 vs. drug/HPMC/Gelucire 44/14 (1:4:1); d *p* < 0.05 vs. drug/HPMC/TPGS (1:4:1). Data are expressed as the mean ± standard deviation (*n* = 4 or 6). *AUC*0→<sup>12</sup> h, the area under the plasma concentration versus time curve; *Cmax*, the maximum plasma concentration of trans-resveratrol; *T*max, the time required to reach *C*max.

To further investigate correlations between in vitro flux data and in vivo pharmacokinetic data for *trans*-resveratrol, linear regression analysis was used. A good correlation was observed between relative in vitro flux, relative in vivo *C*max, and in vivo *AUC*0→<sup>12</sup> h of composite nanoparticles and micronized *trans*-resveratrol (*R*<sup>2</sup> > 0.989). In fact, in vitro flux data reasonably represent in vivo pharmacokinetic data of *trans*-resveratrol, as previously reported for other poorly water-soluble compounds [33–35]. *Trans*-resveratrol/HPMC/poloxamer 407 (1:4:1) nanoparticles show 3.0-fold higher flux enhancement and 9.7-fold higher *C*max compared to micronized *trans*-resveratrol. However, the3.0-fold in vivo increase in *AUC*0→<sup>12</sup> h for *trans*-resveratrol/HPMC/poloxamer 407 (1:4:1) nanoparticles relative to micronized *trans*-resveratrol more closely follows the in vitro flux enhancement. Similar to a previously reported method [28], we performed regression analysis between the total amount of *trans*-resveratrol absorbed at 240 min in flux measurements and in in vivo *AUC*0–12 h. At 240 min, the amounts of permeated *trans*-resveratrol in the receiver cell were 89.6 ± 2.0 μg for micronized *trans*-resveratrol, 136.4 ± 2.2 μg for drug/HPMC (1:4), 139.1 ± 2.9 μg for drug/HPMC (1:5), 209.7 ± 4.2 μg for drug/HPMC/gelucire 44/14 (1:4:1), 260.9 ± 8.2 μg for drug/HPMC/TPGS (1:4:1), and 279.5 ± 5.3 μg for drug/HPMC/poloxamer 407 (1:4:1), with the same ranks observed for in vivo *AUC*0→<sup>12</sup> h. As shown in Figure 7C, there is good positive linear correlation between the total amount of *trans*-resveratrol absorbed at 240 min in flux measurements and in vivo *AUC*0→<sup>12</sup> h data obtained from plasma concentration–time profiles in rats (*R<sup>2</sup>* > 0.990). In fact, in vitro flux data can predict the in vivo pharmacokinetic data for *trans*-resveratrol. In addition, experimental conditions for flux measurements can be modified to establish proportional linear relationships between in vitro and in vivo data.

**Figure 6.** Plasma concentration versus time profiles of *trans*-resveratrol after oral administration of composite nanoparticles to Sprague–Dawley (SD) rats. Data are expressed as the mean ± standard deviation (*n* = 6).

Generally, the oral absorption of poorly water-soluble compounds can be accounted for by including the solubilization and dissolution processes as rate-limiting steps [42]. In this study, we demonstrated that a highly supersaturated solution generated using HPMC/surfactant nanoparticles is able to diffuse *trans*-resveratrol through membranes and enhance the flux of *trans*-resveratrol to receiver cells in in vitro flux measurement studies. Consequently, the enhanced flux of *trans*-resveratrol induces higher driving forces in the gastrointestinal epithelial membrane, resulting in enhanced oral delivery of *trans*-resveratrol in in vivo pharmacokinetic studies of rats [43,44]. Taken together, our results sugges<sup>t</sup> that *trans*-resveratrol-loaded composite nanoparticles prepared using an SAS process are useful for orally delivering *trans*-resveratrol in a manner that allows fast absorption in the initial phase, resulting in higher overall exposure.

**Figure 7.** Correlations between in vitro flux data and in vivo pharmacokinetic data of *trans*-resveratrol: (**A**) in vitro flux vs. in vivo *C*max of composite nanoparticles relative to micronized *trans*-resveratrol; (**B**) in vitro flux vs. in vivo *AUC*0–12 h of composite nanoparticles relative to micronized *trans*-resveratrol; (**C**) total absorbed *trans*-resveratrol at 240 min in flux measurements vs. in vivo *AUC*0→<sup>12</sup> h.

#### *3.3. Utilization of Trans-Resveratrol Composite Nanoparticles for Skin Delivery*

˧ To investigate the application of *trans*-resveratrol-loaded composite nanoparticles for skin delivery, we performed ex vivo permeation studies using skin from rats. The cumulative amount of permeated *trans*-resveratrol (*Q*) per unit area of skin (μg/cm2) was determined using HPLC analysis. To calculate flux, defined as the rate of diffusion of a substance through a permeable membrane, *Q* versus time profiles were generated (Figure 8). The steady state flux (*J*ss) of *trans*-resveratrol was calculated based on the slope of the linear portion of the *Q* versus time profiles. The permeation of *trans*-resveratrol-loaded composite nanoparticles was higher compared to permeation of micronized *trans*-resveratrol (Figure 8). In particular, the steady state flux (*J*ss) of *trans*-resveratrol/HPMC/poloxamer 407 (1:4:1) nanoparticles was significantly higher (~14.9-fold) than that of micronized *trans*-resveratrol. The steady state flux (*J*ss) ranks for *trans*-resveratrol-loaded composite nanoparticles based on the SNK test are: drug/HPMC/poloxamer 407 (1:4:1) > drug/HPMC/TPGS (1:4:1) > drug/HPMC/gelucire 44/14 (1:4:1) > drug/HPMC (1:5) = drug/HPMC (1:4) > micronized *trans*-resveratrol. Skin penetration of *trans*-resveratrol has been confirmed in multiple studies [7,10,11]. The enhancement of solubility and dissolution rates of *trans*-resveratrol by composite nanoparticles produced using the SAS process has been shown to enhance *trans*-resveratrol penetration. Interestingly, the steady state flux (*J*ss) of *trans*-resveratrol from composite nanoparticles containing poloxamer 407 and TPGS was much higher than that of composite nanoparticles containing gelucire 44/14, with approximately 2.2- and 1.9-fold increases, respectively, compared to micronized *trans*-resveratrol. This increase in skin permeation is likely a result of the penetration-enhancing properties of surfactants, such as poloxamer 407 and TPGS [45,46]. Surfactants can potentially solubilize stratum corneum lipids, and thus enhance penetration [47,48]. Surfactants have well-known effects on permeability characteristics of several biological membranes, including membranes in the skin, and thus can enhance the penetration of skin by other compounds present in formulations [49]. In addition, the enhanced permeation may be due to the permeation of solute-containing nanoparticles through shunt routes, such as hair follicles [50]. However, further mechanistic studies are needed of *trans*-resveratrol-loaded composite nanoparticles.

**Figure 8.** Cumulative ex vivo skin permeation profiles and flux (*J*ss) data for *trans*-resveratrol composite nanoparticles. Data are presented as means ± standard deviation (*n* = 6).
