Preparation and in vitro – in vivo characterization of trans-resveratrol nanosuspensions

Abstract

Nanosuspensions technique is an important tool to enhance the saturation solubility and dissolution velocity of poorly soluble drugs. Trans-resveratrol (t-Res) with extensive pharmacological effects was severely restricted by poor solubility and short biological half-life. In this study, anti-solvent precipitation was employed to development trans-resveratrol nanosuspensions (t-Res NS) with PVPK30 as stabilizer. The physicochemical properties, in vitro release and in vivo pharmacokinetics of t-Res NS were investigated. The mean particle size, zeta potential, encapsulation efficiency and drug loading of t-Res NS prepared by the optimal prescription were 96.9 nm, −20.4mV, 78% and 28.1%, respectively. The morphology of t-Res nanoparticles was spherical indicated by SEM with amorphous phase verified by XRD and DSC. The t-Res NS present a good physical stability as well as enhanced chemical stability. Compared to crude drug, the in vitro dissolution rate of t-Res NS was increased with fitting Higuchi equation ( $Q = 0.3215 t^{1 / 2} + 0.0070$ ). The in vivo pharmacokinetic test in rats showed that the ${AUC}_{0 \sim t}$ of t-Res NS (559.4 μg/mL·min) was about 3.6-fold higher than that of t-Res solution. Meanwhile, the MRT of t-Res nanosuspensions was longer than that of t-Res solution. These results suggested that NS may be a potentially nanocarrier for clinical delivery of t-Res.

Keywords

Nanosuspensions trans-resveratrol anti-solvent precipitation

Table content used only: Resveratrol belonged to stilbene family is abundant in many natural foods, such as grapes, peanuts, berries and others. Resveratrol is synthesized in these plants in response to pathogen attack and classified as phytoalexin [1]. Chemically, resveratrol has trans- and cis-geometrical isomers, and trans-resveratrol (t-Res, shown in Fig. 1) is more stable and active [2].

Many studies have proved that t-Res exerts a lot of in vitro and in vivo pharmacological activities, including anti-inflammatory, antioxidant, anticancer, cardiovascular-protective effects and so on [3–5]. Previous reports indicated that t-Res mediates its effects through the activation of the sirtuin class of nicotinamide adenine dinucleotide (NAD)+-dependent histone deacetylases [6–8]. Recently, a pioneering research on t-Res reported that PARP1-(auto-poly-ADP ribosylation of poly (ADP-ribose) polymerase1) and NAD+-dependent dimension are the physiological mechanism of t-Res nullifying the catalytic activity and redirecting tyrosyl transfer-RNA (tRNA) synthetase (TyrRS) to a nuclear function [9]. No matter what kind of mechanism t-Res does, many researchers have been attracted by this drug for its chemopreventive activity and little side effects [10–12].

Fig. 1.

Chemical structure of trans-resveratrol.

However, the application of t-Res is limited by its poor physicochemical property, especially the poor water solubility (3 mg/100 mL) and instability by isomerized and oxidized [13,14]. In addition, t-Res is fast and extensively converted to glucuronic acid and sulfate metabolites after administration resulted low in vivo bioavailability [15,16]. To overcome the above defects of t-Res, many drug delivery systems including liposomes, nanoparticles, polymeric micelles, and self-nanoemulsion have been utilized [17–20]. However, a large amount of excipients and surfactants used in these drug delivery systems causes many side effects such as toxicity and hemolysis.

Nanosuspensions (NS), a promising strategy for delivery of hydrophobic drugs with small amount of surfactants, have developed into a mature drug delivery system and achieved great progress in clinical application [21,22]. It was reported that several drug NS, such as Rapamune, Emend and Megace, had been approved by the FDA and now in the pharmaceutical market [23]. The advantages of NS are as follows: (i) simple and feasible preparation, (ii) improvement of drug’s solubility and dissolution rate by nanosizing, (iii) low drug’s toxicity due to the lack of co-solvents and high amounts of surfactants, (iv) changes in drug’s natural distribution with passive targeting in solid tumors by enhanced permeability and retention effect, and (v) easily to scale-up and transform into more convenient dosage forms, such as tablets, pellets, and capsules [24–27]. The preparation methods of NS can be divided into two categories: top-down and bottom-up approaches [28]. In top-down processes, large drug particles are reduced into smaller particles by means of attrition forces, such as high-pressure homogenization and medium grinding. And in bottom-up processes, nanoparticles grew from individual molecules by rapidly supersaturating such as anti-solvent precipitation and microfluidisation method [29–31].

Based on the advantages of NS and the medicinal value of t-Res, trans-resveratrol nanosuspensions (t-Res NS) were prepared by anti-solvent precipitation method. The aim of this study was to set up optimized formulation of t-Res NS and evaluate the improvement of the water solubility, the rate of dissolution, AUC and MRT. After optimizing the prescription, the physicochemical properties of t-Res NS, including particle size, Zeta potential, entrapment efficiency, drug loading, stability and in vitro drug release kinetics were systematically investigated. Finally, we conducted animal experiment to validate the in vivo behavior and the results demonstrate significant improvement of t-Res NS compared to t-Res solution.

1. Results and discussion

During the process of t-Res NS prepared by anti-solvent precipitation method, nanoparticles formed by rapidly supersaturation. Stabilizers, determined by the specific nature of drug, greatly influence on the system stability [32,33]. We have tried to use different stabilizers, such as PVPK30, SDS, P188, Lecithin and so on. Compared with PVPK30, other stabilizers couldn’t effectively stabilize the NS. PVPK30, as a non-ionic surfactant, was chosen as the proper stabilizer. As shown in Table 1, the low ratio of PVPK30 to t-Res couldn’t stabilize the system. As the ratio increased, the system became stable with the particle size increased as well. To get a smaller particle size and a more stable system, the ratio of 3:2 was selected. The effect of PVPK30 on particle size and stability of the NS could ascribe to the coverage area of PVPK30 on the particle surface. As the ratio of PVPK30 increasd to 3:2, it was sufficient for PVPK30 to cover the particle surface and provide enough steric repulsion.

The drug content in NS was increased with the concentration of t-Res increasing from 2 mg/mL to 8 mg/mL when the volume of organic phase was fixed. However, the particle size raised from 127.1 nm to 387.4 nm and the system became less stable (Table 2). This phenomenon may be explained by the enhanced interaction between nanoparticles with the higher concentration of t-Res. In view of the particle size of nanoparticles, the stability of the colliod system and the drug content in NS, it was rational to fix the concentration of t-Res at 4 mg/mL.

Table 1
Influence of the ratio of PVPK30 to t-Res on the storage stability and particle size of nanoparticles ( $n = 3$ )

The ratio of PVPK30 to t-Res Storage stability Particle size (nm)

1:2 Stable for 3 days 95.5 ± 2.8

1:1 Stable for a week 149.7 ± 2.7

3:2 Stable for 2 weeks 186.3 ± 1.9

2:1 Stable for over 3 weeks 239.4 ± 2.2

The ratio of PVPK30 to t-Res	Storage stability	Particle size (nm)
1:2	Stable for 3 days	95.5 ± 2.8
1:1	Stable for a week	149.7 ± 2.7
3:2	Stable for 2 weeks	186.3 ± 1.9
2:1	Stable for over 3 weeks	239.4 ± 2.2

Table 2

Influence of the concentration of t-Res on the storage stability and particle size of nanoparticles ( $n = 3$ )

Concentration of t-Res (mg/mL)	Storage stability	Particle size (nm)
2	Stable for over 2 weeks	127.1 ± 2.5
4	Stable for a week	150.2 ± 2.2
6	Stable for 2 days	218.6 ± 3.1
8	Stable for 10 h	387.3 ± 2.9

Preliminary experiments revealed injection speed and nucleation temperature are main factors influenced the particle size in the process of preparing t-Res NS. As shown in Fig. 2(a), quicker injection speed resulted smaller particle size, which was in accordance with the study of Bajaj et al. [34]. As the injection speed increased from 1 mL/min to 24 mL/min, the particle size reduced from 250.5 nm to 146.9 nm. Figure 2(b) indicated the mixing temperature dropped to 0–4°C (in ice water), the particle size decreased from 129 nm to 96.9 nm. Quicker injection speed and lower precipitation temperature generated a higher degree of supersaturation and resulted ultrafine crystals in the instantaneous formation [35]. Based on above results, 24 mL/min and 0–4°C were selected as the optimal injection speed and precipitation temperature.

Fig. 2.

Effect of the injection speed (a) and temperature (b) on particle size of NS.

Fig. 3.

Appearance of t-Res suspension (a1) and t-Res NS (a2) with its schematic diagram (b).

The appearance of t-Res NS prepared with the ratio of PVPK30 to t-Res as 3:2, the concentration of t-Res as 4 mg/mL and injection speed as 24 mL/min under 0–4°C was displayed in Fig. 3, attached with its schematic diagram. As we can see, t-Res NS was a transparent uniform colloidal dispersion with blue opalescence, whereas the t-Res suspension without stabilizers was a turbid dispersed system with visible white particles.

Figure 4(a) showed that the PS of t-Res NS as an intensity mean size was about 96.9 nm, affiliated with particle size distribution (PDI) as 0.052. The lower the PDI value is, the more monodisperse the particles are. The narrow PDI of t-Res NS suggested that the particles in the NS were quite uniform. ZP is a parameter to represent the electric charge at the surface of the particles which related to the stability of nanoparticles [36]. And ZP value of t-Res NS was measured about −20.4 mV as shown in Fig. 4(b). In this colloid system, PVPK30 as nonionic surfactant with not too high charge repulsion and steric hindrance could maintain the stability of t-Res nanoparticles in aqueous dispersions. The EE% and DL% of t-Res NS was 78% and 28.1%, meaning that the most of drug were in the form of nanoparticles with small amount of surfactants and the high drug loading.

Fig. 4.

Particle size distribution (a) and zeta potential (b) of t-Res NS.

Fig. 5.

SEM images of nanocrystals in t-Res NS (a) and raw t-Res powder (b).

SEM image of t-Res nanocrystals in t-Res NS was shown in Fig. 5(a). Differences are clearly found between the t-Res nanocrystals in t-Res NS and the raw crystals. The particles in t-Res NS were spherical in the nano-scale range, whereas the raw crystals were needle-like with a micrometer-scale size. The particle size in SEM was slightly smaller than that of determined by dynamic laser scattering (DLS). This may be explained by surface water of hydration and the fact that light scattering is more sensitive to an admixture of larger particles in the sample by DLS [37].

The XRD spectra of t-Res crude drug, PVPK30 and t-Res NS are shown in Fig. 6. Crude t-Res drug exhibited obvious crystallization peaks and PVPK30 showed week diffraction peak. Compared to the typical crystalline peaks of crude t-Res powder, t-Res NS almost has no diffraction peaks with intensity from 0 to 25,000 a.u. (Fig. 6(c)), just shown a broadly weak peak with intensity of 15,000–15,170 a.u. Based on the results of XRD analysis, we speculated the crystallinity of t-Res in NS has changed greatly almost to an amorphous state. The inherent crystal of t-Res may be hindered by PVPK30 which strongly adsorbed on particles and slowed down the surface integration kinetics of t-Res [38,39].

DSC analysis was performed to obtain the thermograms for t-Res NS formulation along with its individual components. Figure 7 showed the obtained different shape of the DSC curves. The raw material of t-Res revealed a sharp peak at 271.97°C (curve a) and PVPK30 showed a relatively weak peak at 162.71°C (curve b). The melting point of the physical mixture is at 265.86°C and 162.35°C, respectively (curve c), which is lower than the melting point of pure t-Res and PVPK30. For t-Res NS, the signal peak of t-Res disappeared and a displacement peak of PVPK30 appeared at 172.01°C (curve d). This suggested that no crystallization of t-Res occurred during the preparation of NS. The crystallinity may be changed because of the interaction between matrix and surfactant, which agreed with the result of XRD.

Fig. 6.

XRD patterns of crude t-Res (a), PVPK30 (b) and freeze-dried powder of t-Res NS (c). Inset is the XRD of freeze-dried t-Res NS of (c) with intensity of 15,000–15,170 a.u.

Fig. 7.

DSC of crude t-Res drug (a), PVPK30 (b), physical mixture (c) and freeze-dried t-Res NS (d).

Fig. 8.

Physical stability of t-Res NS (a) and chemical stability of t-Res NS vs. t-Res solution (b).

In order to investigate the particle growth through agglomeration and Ostwald ripening as well as the chemical degradation by oxidation and isomerization, the physical and chemical stability of t-Res NS was monitored over a period of time compared to t-Res solution. The NS here could be placed for over a month at room temperature without precipitation, and particle size increased slightly from 96.6 nm to 112 nm (Fig. 8(a)) which indicated the physical stability of t-Res NS. As shown in Fig. 8(b), the degradation of t-Res in NS was much less than t-Res in solution which means that the chemical stability of t-Res was greatly enhanced by nanosizing and the protection of stabilizer.

It is important to investigate drug release behavior, based on which we can predict the in vivo performance. The release behavior of t-Res NS compared to t-Res crude drug and t-Res solution was displayed in Fig. 9. The results revealed that during 720 min, t-Res NS showed the accumulative release of 83.1%, while the crude drug was only released 49.7%. The improved dissolution rate of t-Res NS was due to the reduced particle size and the corresponding increased water solubility. On the other hand, compared to quickly release of t-Res solution within 120 min, t-Res NS could sustain the drug release for a longer period of time. Release of t-Res in NS follows Higuchi equation unitarily $Q = 0.3215 t^{1 / 2} + 0.0070$ ( $r = 0.9948$ , Q means cumulative released quantity). And there was a quick release about 30% at the initial 60 min followed by a prolonged release with the rest of time. The quick release at the initial time could attribute to free drugs that weren’t encapsulated into nanoparticles.

Fig. 9.

In vitro release profile of t-Res solution, crude t-Res drug and t-Res NS.

In this study, we investigated the in vivo pharmacokinetics of t-Res NS by intravenous administration with t-Res solution as control. As shown in Fig. 10, the rats treated with t-Res NS showed higher drug concentrations at every time point in comparison with t-Res solution group. 3P97 software was used to calculate the pharmacokinetic parameters. The main pharmacokinetic parameters of t-Res NS and the reference formulation are listed in Table 3. The ${AUC}_{0 \sim t}$ value of t-Res NS (559.4 μg/mL·min) were approximately 3.63-fold greater than that of reference preparation (154.2 μg/mL·min) ( $p < 0.05$ ). The MRT of the nanoparticle (175.3 min) was significantly extended than that of the solution (95.8 min) ( $p < 0.05$ ). To summarize, administration of t-Res NS resulted in the prolonged residence time of t-Res in system blood circulation which is confirmed to our slow-release design.

Fig. 10.

Mean concentration-time profiles of t-Res NS and t-Res solution after intravenous administration of a single dose of 3.2 mg/kg in rats ( $n = 6$ ).

Table 3

The main PK parameters of t-Res NS and the reference formulation in SD rats resulted from the intravenous administration (means ± S.D. $n = 6$ )

Parameters	t-Res NS	t-Res solution
$t_{1 / 2}$ (min)	160.81 ± 29.74	104.6 ± 11.62
${AUC}_{0 \sim t}$ (μg/mL·min)	559.4 ± 108.61	154.2 ± 46.3
MRT (min)	175.3 ± 34.85	95.8 ± 20.11
Cl (mg/kg)/(μg/mL)/min	0.00572 ± 0.0092	0.0208 ± 0.005

2. Conclusions

In summary, t-Res NS was successfully prepared by simple anti-solvent precipitation under mild conditions. The system was homogeneous and stable with a mean particle size of 96.9 nm. In comparison to t-Res solution, the chemical stability of t-Res NS was greatly enhanced. The dissolution rate of t-Res NS was improved with sustained release. After intravenous injection to rats, the AUC_0-t of t-Res NS was enhanced 3.63-fold and the MRT of t-Res NS was significantly extended in comparison to t-Res solution. Moreover, the small amount of adopted stabilizer is biocompatible, non-cytotoxic and non-hemolytic which guaranteed the safety of this preparation. Thus NS may be a potentially nanocarrier for the delivery of t-Res.

3. Materials and method

T-Res (99.4%, purity) was purchased from Chengdu LanBei Plant & Chemical Technology (China), and PVPK30 was obtained from Jiangsu Chemical Company (China). Methanol and acetonitrile were of HPLC grade. The water used in the study was doubly distilled and deionized. All the other reagents were of analytical purity grade.

Preparation of t-Res NS. Briefly, a quantity of t-Res was dissolved in ethanol to prepare the organic phase. Meanwhile, the aqueous phase as anti-solvent was prepared by dispersing certain amount of PVPK30 in distilled water. At a fixed temperature, 4 mL of organic solution was quickly injected into 25 mL of aqueous phase under mechanical stirring. The nanoparticles were immediately formed in water by supersaturating. After rotary evaporated under reduced pressure for 1 h at 40°C to remove the ethanol, t-Res NS were obtained.

Experimental design. T-Res NS was prepared by anti-solvent precipitation with water as anti-solvent to rapidly supersaturate t-Res to grow its nanoparticles. And ethanol with low toxic, water-soluble and high soluble for t-Res was chosen as solvent in this bottom-up process. To optimize the formulation, we systematically investigated the concentration of t-Res in ethanol solution (2, 4, 6 and 8 mg/mL), the polymer to drug ratio (1:2, 1:1, 3:2 and 2:1), the injection speed (1, 2, 4, 12, 24 mL/min), the stirring rate (100, 300, 500 and 1000 r/min), and the temperature (0–4°C and 25°C) by single-factor analysis with other variables fixed. Overall consideration the particle size of nanoparticles, the stability of the colloidal dispersion system and the drug content in NS, we finally determined the optimal prescription. Experiments were performed on triplicate samples and the results were presented as the means ± S.D. values.

Particle size (PS) and zeta potential (ZP) measurement. The PS and ZP of the t-Res NS were analyzed by Dynamic Laser Scattering (DLS) method with Malvern Zetasizer 3000HS (Malvern Instrument, Malvern, UK). Prior to the measurement, the samples were diluted with distilled water to a suitable concentration range. The scattering angle was set at 90° and samples were maintained at 25.0°C during the experiments. The PS was measured with 20 runs and the ZP was determined 100 times for each sample. And ionic strength was 0.5 mmol/l at ZP measurement.

HPLC analysis of t-Res. The concentration of t-Res was analyzed by HPLC method. HPLC was equipped with Shimadzu LC-15C (Shimadzu, Kyoto, Japan) connected to a SPD-15 C UV detector. Sample separation was carried out with a COSMOSIL C18 column (5 μm, 4.6 mm × 250 mm; Nacalai Inc, Kyoto, Japan) at the column temperature of 30°C. The mobile phase, comprising of acetonitrile and water (35:65, v/v) was used at a flow rate of 1.0 mL/min. UV wave length were quantified at 306 nm and the injection volume was 20 μL. All the samples need to be filtered with millipore filtration before injected. Drug concentrations were quantified by the standard curve of t-Res.

Entrapment efficiency (EE%) and drug loading (DL%) analysis. An aliquot of NS was dissolved thoroughly by methanol and analyzed by HPLC to calculate the actual drug concentration. EE% and DL% were calculated using equation (1) and (2) by the ultracentrifugation method. Briefly, 2 mL of sample was put in the filter tubes and then centrifuged at 3000 rpm at 4°C for 15 min. The outer tube fluid was immediately analyzed as the concentration of free drug ( $C_{2}$ ) $\begin{array}{l} (1) & EE % = (1 - C_{2} / C_{1}) \times 100 %, \\ (2) & DL % = M_{0} * % EE / (M_{1} + M_{0} * % EE) \times 100 % . \end{array}$ Here, $C_{1}$ and $C_{2}$ represent the drug concentration and the free drug concentration of t-Res NS, while $M_{0}$ and $M_{1}$ mean the weights of drug and stabilizers in NS.

SEM observation. The morphology of t-Res NS and crude t-Res drug were conducted by field emission scanning electron microscope (FESEM, JEOL-6700F, Japan). A drop of NS and crude drug were added to the clean copper. After the solvent was air-dried, the copper was fixed on aluminum stubs using double-sided adhesive tape, and then sputter-coated with Au to enhance the conductivity of t-Res. Images of the samples were obtained at an accelerating voltage of 5–10 kV.

X-ray powder diffraction (XRD) analysis. XRD was measured on silica substrate by Philips X’Pert Pro Super Diffractometer Cu Kα radiation ( $λ = 1.541874 Å$ ), and the operating voltage and current were maintained at 40 kV and 40 mA, respectively. The scanning speed was 10°/min over an angular range of 10–80°of 2θ, with a step size of 0.02°.

Differential scanning calorimetry (DSC) analysis. DSC analysis was performed by DSC (Q200 F3 NETZSCH Germany). For DSC measurement, about 10 mg of each sample was weighed into an aluminum pan and sealed hermetically, and the thermal behavior was determined in the range of 25–300°C at a scan rate of 10°C/min. An empty aluminum pan was used as reference to calibrate the temperature and energy scale of the DSC apparatus.

Physical and chemical stability investigation. Physical stability was determined by the precipitation phenomenon and the change of particle size of the NS during storage. For chemical stability, HPLC was used to assess possible chemical degradation during the storage at 25°C exposing to light. Any decrease in the area of the drug peak of the stored sample compared to the pre-storage sample was considered as degradation. The samples were conducted at regular time intervals over the one month period (1, 8, 15, 22, 30 days) and the values were reported as the means ± S.D. values.

Drug release analysis. Dissolution behavior of t-Res NS was analyzed in comparison with different controls (t-Res crude drug and t-Res solution) by paddle method. The paddle speed and bath temperature were set at 100 rpm and 37 ± 0.5°C, respectively. 4 mL t-Res NS and the equivalent amount of controls were introduced into dialysis bag (⩽12,000 Da, China). To maintain sink conditions, 500 mL deionized water were used as the dissolution media. At predetermined time intervals (30, 60, 120, 180, 240, 360, 480, 720 min for t-Res NS and t-Res crude drug, and 10, 20, 30, 40, 60, 80, 100, 120 min for t-Res solution), 1 mL of dissolution medium was withdrawn and filtered through 0.22 μm filters and the same volume of fresh medium was added. The initial filtrate was removed to ensure that the filters were saturated as much as possible. The concentration of t-Res was analyzed with HPLC method. Each experiment was performed in triplicate and values were reported as the mean ± S.D. values.

Pharmacokinetic study. Twelve healthy SD rats (200 ± 20 g) were supplied by Experimental animal center of Anhui province (2011-002, Hefei, Anhui, China). All animal procedures were conducted in accordance with all appropriate regulatory standards according to the protocol approved by the Institutional Animal Care and Use Committee of the Anhui Medical University Experimental Animal Center. Animals were randomly divided into two groups with half male and half female for control and experimental group. Animals were fasted overnight but allowed to free access to water before experiment. The experimental group received t-Res NS while the control group received t-Res solution. Each animal was intravenously injected the single dose of 3.2 mg/kg of t-Res. Blood samples were collected by retroorbital venous plexus puncture at the specific schedule of 5, 15, 30, 60, 90, 120, 180, 240, 360, 480, 600 and 720 min, respectively. The heparinized blood was centrifuged for 15 min at 3000 rpm to separate plasma from the whole blood. Then, adding 100 μL HCl of 0.05 mol/l together with 200 μL acetonitrile to the plasma, vortex for 2 min, water bath at 50°C for 30 min, centrifugation at 12000 rpm for 10 min and finally get the supernatant for HPLC determination. Avoid light as far as possible during the process of biological samples. The software program 3P97 (Chinese Pharmacological Society, Chain) was employed to estimate the pharmacokinetic parameters. Student’s t tests and ANOVA were performed to determine the significance of any differences. All results were presented as means ± S.D. values.

Footnotes

Acknowledgements

We acknowledge the funding support from the Ministry of Science and Technology of China and the National Basic Research Program of China (Grants 2012BAD32B05-4, 2010CB934700, 2013CB931800), the National Natural Science Foundation of China (Grants 91022032, 91227103, 21001099, 21061160492, 51303006, 81171829), the Chinese Academy of Science (Grant KJZD-EW-M01-1) and the Provincial Natural Science Foundation (1408085MH196, KJ2012ZD09) of Anhui Province.

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

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