Poly(lactic acid) (PLA) and PLA-poly(ethylene glycol) (PLA-PEG) nanoparticles containing resveratrol (RVT) were developed, and their antioxidant activity was evaluated. An analytical method using high performance liquid chromatography (HPLC)/photodiode array (PDA) detection was also developed and validated for RVT determination in nanoparticles. The mobile phase consisted of methanol : water (51 : 49, v/v) flowed at 0.9 mL/min, and the PDA detector was set at wavelength of 306 nm. The mean diameter of the nanoparticles varied between 180 and 220 nm, and the encapsulation efficiency of RVT ranged from 60% to 88%. The nanoparticles containing RVT were evaluated for their ability to scavenge the radical (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (ABTS•+). The profile obtained from the PLA nanoparticles containing RVT demonstrated that after 24 h, there was almost no increase in antioxidant activity, which was lower than that of the free RVT and RVT-loaded PLA-PEG nanoparticles. For PLA-PEG nanoparticles, the radical-scavenging activity of RVT was shown to increase with time, and after 48 h, it was similar to that observed with free RVT.
The human body constantly produces reactive oxygen species that are generated as by-products of biological reactions or by exogenous factors derived from the metabolism of oxygen [
The use of exogenous antioxidant compounds to compensate for this imbalance has received great attention, mainly in natural product-based compounds. Among these, transresveratrol (3,4′,5-trihydroxystilbene, RVT), a polyphenolic compound found mainly in grapes, peanuts, and herbs, is rich in pharmacological activities. Studies demonstrate high antioxidant activity [
Although there is therapeutic potential for this molecule, RVT presents pharmacokinetic drawbacks; for example, it is extensively metabolized after oral administration, resulting in low oral bioavailability. Additionally, a large portion of the dose is converted to conjugate sulfates, which is the limiting step in the systemic bioavailability of RVT [
The use of colloidal drug carriers as polymeric nanoparticles is a strategy to combat these disadvantages. The physicochemical characteristics of nanoparticles influence the pharmacokinetics of the drug, affecting its bioavailability and biodistribution. Additionally, it promotes controlled and prolonged drug release to help reduce toxicity [
Obtaining nanoparticles requires extensive characterization and determination of the drug content within the nanoparticles. This parameter must be properly verified because the drug must be efficiently loaded into the nanoparticles to reach its therapeutic goal. Therefore, a suitable and validated quantification method is required. Several analytical methods have been developed to quantify RVT in samples, such as plasma, urine, wine, and butter; however, few analytical methods have been reported for the determination of RVT in nanoparticles. UV-Vis spectroscopy [
Trans-RVT was obtained from Pharmanostra (Brazil). PEG (10 kDa), PLA (85,000–160,000 Da), and polyvinyl alcohol (PVA, 31 KDa, and 88% hydrolyzed) were purchased from Sigma-Aldrich (USA). Ethyl acetate (P.A) and dimethyl sulfoxide (P.A, DMSO) were purchased from Biotec (Brazil), and dichloromethane was purchased from FMaia (Brazil). HPLC-grade methanol was purchased from J.T. Baker (USA). Water was purified using a Milli-Q Plus system (Millipore) with a conductivity of 18 MΩ. ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and potassium persulfate (dipotassium peroxydisulfate) both were obtained from Sigma-Aldrich (USA).
The HPLC system consisted of a Waters 2695 Alliance (Milford, MA, USA) combined with a photodiode array wavelength detector (PDA) (Waters 2998). This system was equipped with a quaternary pump, an autosampler, an online degasser, and a column compartment with temperature control. Data acquisition, analysis, and reporting were performed using the Empower chromatography software (Milford, MA, USA). The analysis was conducted using a reverse phase C18 column (Xterra Waters) with a 5
Chromatographic analyses were performed in the isocratic mode with a mobile phase consisting of a methanol and water mixture (51 : 49, v/v) pumped at a flow rate of 0.9 mL/min. The sample injection volume was 20
An RVT stock standard of 1 mg/mL was prepared in a methanol : water mixture (50 : 50, v/v), and subsequent dilutions were carried out to obtain six standard solutions (10, 20, 25, 30, 40, and 50
Prior to injection, the standards and samples were filtered through a 0.22
The HPLC method was validated in terms of specificity, linearity, precision (intra- and interday), accuracy, robustness, LOD, and LOQ according to the International Conference on Harmonization (ICH) guidelines [
The specificity was evaluated by comparing the representative chromatograms of samples containing possible interfering substances (excipients used in nanoparticle composition) and samples containing RVT. Additionally, specificity was demonstrated by performing stress studies (i.e., light stability, pH, and temperature variation).
The linearity was determined by calculating a regression line from the plot of the peak area versus concentration for the six standard solutions in a 50 : 50 (v/v) methanol : water mixture (10, 20, 25, 30, 40, and 50
Precision was assessed at two levels: repeatability and interday variability. The repeatability of the measurements was assessed by testing three different standard solutions (10, 25 and 50
The accuracy was determined by calculating the percent recovery of the RVT at three concentration levels and then determining the RSD. The mean concentration value obtained for each level was compared to the theoretical value, which was considered to be 100%.
The robustness was evaluated by deliberately varying the temperature of the analytical column (20 or 30°C), while using a standard C18 column (5
The LOD and LOQ were determined from the specific calibration curve obtained using six standard solutions (1, 2, 4, 6, 8, and 10
The RVT-loaded nanoparticles were obtained by a single-emulsion solvent evaporation technique and subsequently lyophilized and stored in a light-protecting container. PLA was dissolved in dichloromethane either with or without PEG at room temperature, and the RVT were then added to the solution. This solution was poured rapidly into a PVA aqueous solution (1%, w/v) and emulsified by means of sonication for 10 min (80–100% of 500 W, Unique Ultrasonic Mixing, mod. DES 500, equipped with a 4 mm probe, Unique Group, Brazil), which resulted in an oil-in-water (O/A) emulsion. Next, the organic solvent was rapidly removed by evaporation under vacuum at 37°C (20 min). The particles were then recovered by ultracentrifugation (19,975 g, 30 min, 4°C, Cientec CT-15000R centrifuge, Brazil) and washed twice with water to remove the surfactant. The nanoparticles were dispersed in the cryoprotectant sucrose (5%, w/v), and the resulting nanosuspension was subsequently cooled to −18°C and freeze-dried (Terroni, Brazil).
The mean particle size, size distribution, and polydispersity index were determined by dynamic light scattering (BIC 90 plus, Brookhaven Instruments Corp.). The analyses were performed at a scattering angle of 90° and a temperature of 25°C. For each sample, the mean particle diameter, polydispersity, and standard deviation for ten determinations were calculated.
The amount of RVT in the nanoparticles was determined indirectly. The supernatant obtained from the ultracentrifugation process was diluted in the mobile phase (1 : 1000), filtered through a 0.22
The cation radical ABTS●+ was employed to measure the antioxidant activity of free RVT, RVT-loaded nanoparticles (PLA and PLA-PEG nanoparticles), and blank nanoparticles.
A mixture of ABTS (7 mM) and potassium persulfate (2.45 mM) was prepared and allowed to stand at room temperature [
The concentration of RVT that resulted in 50% of inhibition of ABTS●+ (IC50) was calculated by the linear regression of the RVT concentration versus percentage of ABTS●+ inhibition curves.
Initial runs were performed using a mobile phase mixture of methanol and acetonitrile based on existing methods for RVT quantification in plasma [
Representative HPLC chromatograms of RVT standard (40
Linearity was evaluated at six concentration levels (10–50
The validity of the assay was confirmed by an analysis of variance, which showed that the linear regression was significant and that the deviation from linearity was not significant (
Accuracy was assessed by calculating the percent recovery and the RSD of the mean concentration of the analyte at three different concentrations. Three standard solutions (10, 30, and 50
Accuracy results for the RVT concentrations in standard solutions (
RVT standard solution ( |
Recovery (%) | RSD (%) |
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10 | 96.49 | 0.09 |
30 | 100.69 | 1.25 |
50 | 100.73 | 0.09 |
RSD: relative standard deviation.
The precision is a measure of the relative errors of the method, expressed as the RSD for repeatability and intermediate precision. Three concentrations of RVT (10, 30, and 50
Precision results for the different levels of RVT in standard solutions.
RVT standard solution (µg/mL) | Measured concentration ± SDa (µg/mL) | RSDb (%) |
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Analysis repeatability ( |
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10 |
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0.32 |
30 |
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0.16 |
50 |
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0.12 |
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Intermediate precision ( |
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Day 1 | ||
10 |
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0.43 |
30 |
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1.25 |
50 |
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0.09 |
Day 2 | ||
10 |
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0.17 |
30 |
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0.35 |
50 |
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0.29 |
Day 3 | ||
10 |
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1.51 |
30 |
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0.37 |
50 |
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0.18 |
Robustness is a measure of the influence of small changes to the analytical procedures/parameters on the response. The robustness was evaluated based on the percent recovery and RSD values obtained using different parameters for column temperature and commercial mark (Table
Percentage of recovery and RSD obtained in the analysis of robustness after changes in original temperature of the method (25°C) and column mark (
Changes to original method | Percentage of recovery ± RSDa | |||
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10 µg/mL | 30 µg/mL | 50 µg/mL | Mean | |
Temperature 30°C |
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Temperature 20°C |
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Similar column |
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In the present study, the lowest concentration at which an analyte can be detected (LOD) or quantified (LOQ) with acceptable precision and accuracy was calculated from the SD of the response and the slope obtained from linear regression of a specific calibration curve (1–10
The specificity of the method was evaluated by comparing the chromatograms of RVT standards and samples to those with potential interfering formulation components. For this purpose, blank nanoparticles (drug-unloaded nanoparticles) were prepared as described in Section
Representative HPLC chromatograms of RVT sample in supernatant from nanoparticles (a) and supernatant from blank nanoparticles (b). Conditions: mobile phase, methanol : water (51 : 49, v/v); flow rate, 0.9 mL/min; PDA detection wavelength, 306 nm; column temperature, 25°C; injection volume, 20
Tests were also performed under stress conditions (i.e., temperature, visible light, and pH variation) to detect the occurrence of possible interfering peaks at 306 nm resulting from the degradation of RVT. The results showed no alterations in the RVT retention time when the sample was exposed to temperature, visible light, and acid medium. However, a decrease in RVT recovery caused by photodegradation was observed. Exposure to the alkaline conditions resulted in sample degradation, making RVT peak identification impossible. Additionally, in all stress conditions evaluated, no peaks for RVT metabolites were observed. This method can be considered highly specific because no potential interfering peak was observed.
The proposed method was applied to the analysis of RVT in PLA and PLA-PEG nanoparticles and serves as a tool for the determination of the encapsulation efficiency without any interference, as demonstrated in the specificity assay.
The single-emulsion solvent evaporation method was successfully developed for obtaining PLA and PLA-PEG nanoparticles containing RVT. The mean diameter of the PLA and PLA-PEG nanoparticles was approximately
The nanoparticles containing RVT were evaluated for their ability to scavenge the radical ABTS●+. The results of radical inhibition percentage obtained from the 1, 5, 10, 20, and 25
Percentage of inhibition of the radical ABTS●+ from free and nanoencapsulated RVT in concentrations of 1, 5, 10, 20, and 25 µM for 0, 24, 48, and 72 h.
RVT concentration (µM) | Inhibition of ABTS●+ (%) | ||
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RVT-loaded PLA nanoparticles | RVT-loaded PLA-PEG nanoparticles | Free RVT | |
0 h | |||
1 | 6.84 (±1.04)a | 8.95 (±1.70)a | 13.29 (±1.25)b |
5 | 28.56 (±9.27)a,b | 15.38 (±0.76)a | 46.33 (±2.66)b |
10 | 35.50 (±1.58)a | 33.23 (±5.18)a | 69.21 (±1.58)b |
20 | 53.35 (±3.88)a | 56.40 (±2.63)a | 95.48 (±4.61)b |
25 | 65.54 (±7.30)a | 64.00 (±5.42)a | 99.70 (±0.29)b |
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24 h | |||
1 | 5.67 (±0.45)a | 2.92 (±2.79)a | 7.35 (±2.27)a |
5 | 10.89 (±0.58)a | 14.97 (±3.87)a | 28.68 (±3.29)b |
10 | 22.75 (±1.87)a | 24.09 (±1.57)a | 49.35 (±5.10)b |
20 | 50.49 (±4.17)a | 55.66 (±1.65)a | 95.46 (±5.20)b |
25 | 65.75 (±3.14)a | 75.87 (±5.86)b | 97.88 (±1.91)c |
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48 h | |||
1 | 2.90 (±2.33)a,c | 7.50 (±0.95)b | 2.21 (±1.98)c |
5 | 7.87 (±1.07)a | 21.76 (±2.17)a | 25.47 (±12.53)a |
10 | 20.82 (±2.30)a | 35.01 (±4.68)b | 47.74 (±4.56)c |
20 | 45.76 (±5.92)a | 62.14 (±320)b | 88.40 (±0.88)c |
25 | 69.07 (±12.22)a | 85.13 (±2.52)a,b | 98.34 (±0.68)b |
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72 h | |||
1 | 12.13 (±0.65)a | 14.01 (±1.07)a | 3.83 (±0.76)b |
5 | 13.30 (±2.19)a | 25.22 (±1.67)b | 28.30 (±1.96)b |
10 | 24.90 (±4.45)a | 40.58 (±4.30)b | 51.43 (±3.25)c |
20 | 57.26 (±13.78)a | 70.45 (±2.39)b | 92.17 (±4.86)c |
25 | 76.43 (±2.49)a | 90.99 (±3.45)b | 95.52 (±1.58)b |
At 0 h, it can be observed that the two nanoparticle formulations exhibit the same ability to scavenge ABTS●+ at all the concentrations tested (
The IC50 of RVT scavenging ABTS●+ as a function of time was obtained, and the results are shown in Table
IC50 of RVT (free or nanoencapsulated) over the capture of the ABTS●+ cation radical in sodium phosphate buffer (50 mM, pH 7.4) and in the absence of light at room temperature (
Time (h) | IC50 | ||
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RVT-loaded PLA nanoparticles ± RSD* | RVT-loaded PLA-PEG nanoparticles ± RSD* | Free RVT ± RSD* | |
0 |
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24 |
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48 |
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72 |
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The polyphenol RVT is extensively used for pharmaceutical applications and has received great attention in recent years due to its prophylactic and therapeutic abilities against reactive oxygen species. However, its low aqueous solubility and high metabolism significantly decrease its bioavailability. Polymeric nanoparticles have been proven to increase the therapeutic benefits of drugs, decrease the toxic effects of drugs, and deliver the drug to a specific site of action. The physicochemical parameters of nanoparticles influence the pharmacokinetics of the drug, affecting its bioavailability and biodistribution [
The main objective of this study was to develop an analytical method coupled with a PDA detector to quantify RVT loaded in PLA and PLA-PEG nanoparticles by the indirect method. This quantification method is extensively used [
Several analytical methods are described in the literature with the purpose of quantifying RVT in samples, such as wines [
The literature mainly describes spectrophotometric methods for RVT quantification in nanoformulations [
The HPLC method developed and validated in this work represents an alternative to these other methodologies and satisfies the requirement for detailed data in the literature for analyzing RVT in nanoparticles via HPLC-PDA detection. The short retention time of RVT allows for the analysis of a large number of samples in a short period of time and reduces costs because of the solvents used and the absence of buffer in the composition of the mobile phase.
The proposed method was applied to the analysis of RVT in PLA and PLA-PEG nanoparticles. The nanoparticles were successfully obtained by the single-emulsion solvent evaporation method, which is ideal for a hydrophobic drug such as RVT. The particles presented nanometric sizes and high encapsulation efficiency. The antioxidant activity of RVT in nanoparticles was evaluated by the ABTS●+ assay. In the analysis of the IC50 values, we observed that better results for RVT-loaded PLA-PEG nanoparticles were obtained with time, probably due to the prolonged drug release characteristics promoted by the nanoparticles, but this profile was not observed with PLA nanoparticles, since the IC50 at 0 h was the same as that in 72 h. At 48 and 72 h, the free RVT and RVT-loaded PLA-PEG nanoparticles presented the same efficacy. Comparing the two nanoparticles, the difference between the results obtained by PLA and PLA-PEG formulations can be explained by the presence of PEG. PEG is able to modify the amount of drug released from the polymeric matrix. In recent work, we observed that the drug release profile from PLA-PEG nanoparticles was faster than that observed from PLA nanoparticles. This difference can be attributed to the enhancement of water permeation and drug diffusion through the polymeric matrix because of the hydrophilicity of PEG. The presence of PEG causes the polymeric matrix to become more amphiphilic than the PLA matrix, while the wettability of the nanoparticle surface also increases. These characteristics contribute to an increase in the drug release profile [
These results indicate that PLA and PLA-PEG nanoparticles are potential carriers for RVT. Despite the fact that RVT-loaded PLA nanoparticles demonstrated inferior antioxidant ability compared to PLA-PEG nanoparticles and free RVT, and the RVT from PLA-PEG nanoparticles exhibited the same antioxidant activity as free RVT only after 48 h; we must consider the advantages of drug-loaded nanoparticles over free drug, such as improved pharmacokinetics, prolonged and controlled drug release. The
The reverse-phase HPLC method using PDA detection was developed and validated according to the guidelines of ICH and was shown to be fast, simple, and reliable during the determination of the encapsulation efficiency of RVT in PLA and PLA-PEG nanoparticles. The RVT-loaded nanoparticles, especially in PLA-PEG nanoparticles, were very effective as a scavenger of the ABTS radical, suggesting that the polymeric nanoparticles could be used as RVT carriers for applications in prophylaxis or the treatment of diseases involving oxidative stress. More studies are necessary to test this hypothesis.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors would like to thank CAPES for scholarships.