RETRACTED ARTICLE: Preparation and characterization of novel lipid nanocapsules of ropivacaine for transdermal delivery

Abstract

Ropivacaine, a novel long-acting local anesthetic, has been proved to own superior advantage. However, the application form used in clinic, ropivacaine hydrochloride (Naropin® Injection), which should be administed intravenously, is causing poor patient convenience. The purpose of this study was to formulate ropivacaine (RPV) in lipid nanocapsules (LNCs) and character the potential of LNCs in delivering RPV transdermally to exploit novel external preparation. The RPV-LNCs were successfully prepared by phase inversion technique and the formulation was characterized in terms of size, zeta potential, ex vivo permeation study, and pharmacodynamics. The prepared RPV-LNCs displayed a typical core-shell structure with a narrow size distribution of 62.1 ± 1.7 nm and drug loading of 1.35 ± 0.20%. The results of differential scanning calorimetry (DSC) analysis and X-ray diffraction showed that RPV was in amorphous crystalline state when encapsulated into LNCs. Furthermore, the results of ex vivo permeation study displayed that RPV-LNCs had an improved permeability (349.0 ± 11.5 μg cm⁻² versus 161.0 ± 1.3 μg cm⁻²) compared with free RPV. The results of histopathology study showed that interaction between LNCs and skin could break the close conjugation of corneocyte layers. In the mice writhing test, RPV-LNCs exhibited obvious analgesic effect by both prolonging pain latency and reducing the writhing response with an inhibition rate of 91.3% compared to the control group. In conclusion, RPV-LNCs could be a promising delivery system to encapsulate RPV and deliver RPV for transdermal administration.

• Lipid nanocapsules
• Ropivacaine
• transdermal delivery
• Writhing test

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Introduction

Ropivacaine is a relatively new long-acting local anesthetic introduced into the clinic in the last few years to offer a safer alternative to bupivacaine, after the reports of its severe toxicity like seizure and cardiac arrest (Albright, 1979). This agent belongs to the family of the n-alkylsubstituted pipecoloxylidide (McClure, 1996), which contains a chiral center on the piperidine ring, resulting in optically active stereoisomers (dextrorotatory (R+) or levorotatory (S−) stereoisomer). Ropivacaine is pure levorotatory (S−) stereoisomer and seems to have less-toxic effects on the central nervous system and cardiovascular system because S−enantiomer has been demonstrated to have different affinity for ion channels of sodium, potassium, and calcium when compared to that of the R−enantiomer (Åberg, 1972). A recent study (Groban, 2003) showed a high convulsive dose of ropivacaine (4.5 mg/kg) in a rat model and confirmed a reduced neurotoxic profile. Another study pointed out a higher recovery of unassisted ambulation for ropivacaine (Senardet al., 2004). Ropivacaine also showed relevant advantage of a great separation of motor and sensory block, which was specifically beneficial when early mobilization was important to accelerate post-operative recovery (Chaykovska et al., 2014). Until now, some new drug deliveries for ropivacaine have been studied, such as large unilamellar vesicles (Araujo et al., 2008) and Liposome (Shen et al., 2011).

Although RPV is currently state-of-the-art, a major drawback from the formulation point of view is that the application form used in clinic, ropivacaine hydrochloride (Naropin® Injection), which should be administered intravenously, is causing poor patient convenience (Simpson et al., 2005). With the improvement of better patient convenience, the needs for smart and innovative preparation forms are accelerating since intravenous infusion is found inconvenient and painful (Segeren et al., 1999). In addition, adverse reactions of RPV like arrhythmia may occur as a consequence of unwanted excessive dose, which is induced by the too fast rate of intravascular injection and the rapidity with which a certain plasma level is achieved (Leone et al., 2008). Thus, develop novel preparation forms for ropivacaine may provide a feasible convenient alternative to the traditional intravascular injection form.

External preparations applied onto skin for transdermal delivery can be taken into account due to the advantage of transdermal delivery as compared with the traditional administration route. Generally, the principal advantages are the controlled drug release thus avoids major fluctuations of plasma levels (Mostafa et al., 2014) and confers steady blood concentration (Raza et al., 2013). Furthermore, it is easy of application and of practical management, promoting patient convenience (Langley & Heel, 1988). End points, patients can achieve autonomous administration rather than infused by specialized nurses. Particularly, approximately total 16 active ingredients and their transdermal products have been authorized for use globally till now, such as CATAPRES-TTS®, IONTOCAINE® and TESTODERM TTS®. Ointment, cream or others have been exploited as the carriers of external preparations (Yamaguchi et al., 2014). However, the poor permeability made it incapable in transporting drug into deep skin, let along blood circulation.

Lipid nanocapsules (LNCs), kinds of new generation of lipid based nanocarriers, were recently proposed by Benoit et al. (Huynh et al., 2009). High quantities of surfactants and cosurfactants, Like Solutol HS15 which is a mixture of free polyethylene glycol 660 and polyethylene glycol 660 hydroxystearate (Li et al., 2011), would favor high-permeability since polyethylene glycol can act as permeation enhancers (Megrab et al., 1995). Specially, the lecithin in the formulation may also play a key role because it can interact with the skin corneum, joining lipid bilayer and loosening intercellular regions between corneocytes (Sinico et al., 2005). The subsequent increment of lipid fluidization could facilitate drug permeation through the skin barrier. Moreover, as a colloidal system, LNCs are supposed to form an oppressed film covered on the skin surface after application (Tan et al., 2011), which makes drug carrier instantly accessible to the skin. The long-term hydration of skin induced by the formed occlusive film on skin surface may open up the tight junctions of stratum corneum, facilitating maximum penetration of drug into blood through the skin (Kawadkar et al., 2013). Specially, although some other nanocarriers were proposed to deliver ropivacaine transdermally, such as multivesicular liposomes (Shen et al., 2011), the potential of LNCs to deliver ropivacaine had not yet been reported.

Thus, taking advantages of transdermal administration and the novelty of design into account, the aim of the present study was to develop lipid nanocapsules formulation of ropivacaine (RPV-LNCs) intended for transdermal delivery. The RPV-LNCs were prepared by a phase inversion method and the formulations were characterized in terms of morphology, size, zeta potential, differential scanning calorimetry (DSC) analysis, and X-ray diffraction study. Moreover, the ex vivo permeation study of RPV-LNCs was conducted to evaluate their potential of delivering RPV transdermally. In addition, the histopathology study of skin was performed to investigate the permeation mechanism and the pharmacodynamics of the RPV-LNCs was also investigated by Writhing test.

Materials and methods

Materials

Ropivacaine (RPV) was purchased from Jinan Dexinjia Pharmaceutical Co. Ltd. (Jinan, China). Lipoid (soybean lecithin at 69% of phosphatidylcholine) was purchased from Lipoid Gmbh (Ludwigshafen, Germany). Solutol HS15 (mixture of free polyethylene glycol 660 and polyethylene glycol 660 hydroxystearate) were acquired from BASF (Ludwigshafen, Germany). Labrafac (medium chain triglyceride, MCT) was provided by Beiya reagent corporation (Tieling, China).

Preparation of RPV-LNCs

The preparation of LNCs is based on phase-inversion phenomenon of the emulsion (Figure 1), which totally avoids using of organic solvents (Ding et al., 2013). At room temperature, all the components (RPV, Solutol HS 15, MCT, and distilled water) were mixed at different proportions. Then the mixture was heated to 85 °C and then cooled to 60 °C at the rate of 4 °C /min to reach the inversion process. In order to obtain a stable oil/water system, another two temperature cycles (85 °C –60 °C –85 °C) were applied to repeat the inversion process. An irreversible shock was induced to the system by adding cold distilled water (0 °C) when the last temperature cycles cooled at 72 °C. The formulation was then stirred for 5 minutes to form stable nanocapsule suspension.

Figure 1. Preparation process of lipid nanocapsules by phase inversion procedure. Step one is a phase inversion between a W/O to an O/W emulsion. Step two leads to the formation of stable nanocapsules by fast irreversible shock dilution induced by cold water (0–2 °C).

Preparation of RPV-LNCs freeze-dried powder

In order to facilitate DSC and X-ray diffraction research, the powder of RPV-LNCs was prepared by lyophilization. Mannitol (5.0%, w/v) was adopted in the freeze-drying process as cryoprotectant. The samples were frozen at −20 °C, and then continue to pre-freezed at −80 °C for 24 h using an ultracold freezer (MDF-382E, SANYO, Japan). Lyophilization was carried out using a lyophilizer (LGJ0.5, Beijing Sihuan Instrument Company, China) at pressure of 0.1 mbar and temperature of −60 °C for 48 h. The obtained RPV-LNCs powders were collected and kept in desiccators for further analysis.

Optimization of the formulation

The choice of formula dosage is very important in the preparation of LNCs. The LNCs formation highly depends on the ratio of the various excipients (Solutol HS15, Labrafac, Lipoid, NaCl and Water) rather than specific amount (Heurtault et al., 2003a) . Based on previous studies, the proportions of NaCl and Lipoid were fixed at 1.75% and 1.5%, respectively (Barras et al., 2009). The influence of other factors was analyzed by simplex lattice design with entrapment efficiency (EE%) and drug loading (DL%) of RPV as indexes. For the determination of EE% and DL%, the amount of entrapped RPV in RPV-LNCs should be firstly determined. A Sephadex G-50 minicolumn centrifugation technique was adapted to separate the RPV-LNCs from free RPV. Specifically, the RPV-LNCs dispersion was placed onto a minicolumn and then centrifuged for 60 s at 500 rpm, followed by washed with 3 mL of distilled water (six times, 0.5 mL per time). Then the RPV-LNCs nanoparticles were achieved within the eluates. The eluates were then mixed with methanol and vortexed for 3 minutes to break down the structure of LNCs. Thereafter, the amount of entrapped RPV was detected using HPLC. The EE% and DL% of RPV-LNCs were calculated from the following formulas:

Where W_loaded represents the amount of RPV entrapped in LNCs, whereas W_added represents the amount of RPV in the formulation. W_total stands for the total weight of all components in the formulation.

Study on physicochemical properties of RPV-LNCs

The physicochemical properties of RPV-LNCs were evaluated in terms of morphology, particle size and zeta potential.

The morphology of RPV-LNCs was analyzed by transmission electron microscopy (Zheng et al., 2013) (JEM-1200EX; JEOL, Tokyo, Japan). Briefly, one drop of diluted RPV-LNCs was deposited on a 200-mesh film-coated copper grid and negatively stained with 2% (w/v) aqueous solution of phosphotungstic acid. The samples were then allowed to dry naturally for contrast enhancement and finally examined by transmission electron microscopy. All batches were analyzed in triplicate.

The average particle diameter, polydispersity index (PI) and the zeta potential of RPV-LNCs was determined by dynamic light scattering (DLS), using a Delsa™ Nano C Particle Analyzer (Beckman Coulter. Inc. Brea, CA) at a fixed angle of 90^o. The RPV-LNCs dispersion was diluted by deionized water at 25 °C in order to assure convenient scatter intensity. For each sample, the measurement was carried out in triplicate and the average value was recorded.

Differential scanning calorimeter analysis

The thermal behavior of RPV-LNCs was analyzed using differential scanning calorimeter (DSC, 1/1600HT, METTLER, Switzerland). Firstly, blank porcelain crucible was used as the standard reference material to calibrate the temperature and energy scale of the instrument. Approximately 5 mg of samples was accurately weighed and placed into porcelain crucible and then heated from 30 to 400 °C at a scanning rate of 10 °C /min. The DSC analysis was carried out under the protection of nitrogenin.

X-ray diffraction analysis

For further determining of the crystalline state of LNCs, wide-angle X-ray scattering (WAXS) was investigated by an X-ray diffractometer (D8, Advance, Bruker, Germany) with a Cu Kα radiation source. The Cu Kα radiation source was fixed at 40 kV and 100 mA and the wavelength was set at 1.5405Å (0.154 nm). The XRD was performed from the initial 2θ = 10 ° to the final 2θ = 70 ° with a scanning rate of 1 °/s.

Ex vivo permeation study

Aims to evaluate the potential of LNCs in delivering RPV transdermally, the ex vivo permeation study was performed by using Franz cells. Full thickness dorsal skin excised from Kunming mice (male, 20 ± 2 g) was employed in this study. Prior to the study, excised skin was prepared. The hair on the dorsal skin was shaved with a razor and the residual fur was removed by using 8% Na₂S solution, without scratching the skin surface. Then the mice were sacrificed and the dorsal skin was harvested. Subcutaneous connective tissues and fat were carefully removed (Chen et al., 2013).

In the experiments, the skin samples were mounted on Franz diffusion cells with the stratum corneum side facing upward into the donor compartment. The receptor compartment was filled with physiological saline (20 mL) contained 1% Tween 80 to meet the sink condition. The receptor fluid was maintained at (32 ± 0.5) °C by using a water bath. RPV-LNCs or control solution were uniformly applied on the stratum corneum side of the skin surface. At regular intervals (0.5, 1, 2, 3, 6, 9, 12 and 24 h), approximately 0.5 mL of the acceptor fluid was withdrawn and replaced by an equal volume of fresh medium.

The sample was mixed with 1 mL of acetonitrile to precipitate protein and vortexed for 5 minutes, and then centrifuged at 12 000 rpm for 10 minutes. Finally, the supernatant was collected and analyzed by HPLC.

Histopathology study of skin

To visualize the topical effect of RPV-LNCs on skin, histopathology study of skin was conducted. Briefly, RPV-LNCs dispersion and RPV propylene glycol were applied to the dorsal skin of hair-removed mice, respectively. After 12 h, the mice were sacrificed and the treated skin pieces were harvested. Then the skin samples were washed twice with physiological saline and collected into formalin for tissue fixation for 24 h. Later the specimen was embedded in paraffin wax, followed by subjected to hematoxylin/eosin staining. Finally, paraffin tissue sections were cut and examined by using microscope.

Writhing test

In order to evaluate the analgesia effect of RPV-LNCs, the writhing test (Tjølsen et al., 1997) was carried out. The writhing test, consisting of a syndrome called ‘writhing’ induced by intraperitoneal (i.p.) injection of an irritant, is a well-known animal model of visceral pain and normally used to evaluate peripheral antinociceptive activity. In this study, Kunming mice (20 ± 2 g) were used to study the antinociceptive characterization of RPV. All mice were randomly divided into 3 groups (12 animals per group) either as control group, RPV propylene glycol group, or RPV-LNCs group. The experimental group was administered either RPV propylene glycol or RPV-LNCs transdermally, while the control mice did not receive the treatment. After a certain time, the mice were injected i.p. a 0.6% acetic acid solution in the left lower quadrant of the abdomen (0.1 mL/10 g), and then observed for 15 min by recording the latent period and counting the number of writhes. Latent period refer to the time delay between stimulation induced by 0.6% acetic acid and the beginning of first writhe. A writhe is indicated by abdominal constriction, arching of the back and extension of the hind limbs (Abdollahi et al., 2003). The reduction% was determined for each experimental group as follows:

Where N_con represents the number of writhing in control group, N_ex represents number of writhing in experimental groups.

HPLC analysis of ropivacaine

A HPLC method is developed for determination of ropivacaine with reversed chromatographic column (C18, 4.6 × 250 mm, 5 µm) and UV absorbance detector (Agilent G1314–60100). A degassed mixture of acetonitrile−phosphate buffered solution at a ratio of 60:40 (v/v) containing 0.05% triethylamine was selected as the mobile phase, which was pumped at a flow rate of 1.0 mL/min. The wavelength of the UV detector was set at 225 nm. The column temperature was maintained at room temperature.

Statistical analysis

One-way analysis of variance (ANOVA) was used to determine the statistical significance. Multiple comparisons between the groups were performed by Tukey's test. A value of p < 0.05 was established as acceptable level of significant difference.

Results and discussion

Method validation

The HPLC analysis method was evaluated through specificity, linearity, intra- and inter-day precision and accuracy. Under optimized HPLC conditions, RPV were eluted within 10 min. The calibration curves of RPV were linear over a range of 0.1−40 μg/mL, with correlation coefficients r > 0.99. The precision and accuracy of this method indicated that all coefficients of variation were below 15‰.

Optimization of the formulation

Simplex lattice design, which requires fewer experimental runs and provides savings on time and resources, was adopted to optimize the formulation variables. In this design, the total amount of the oil (MCT), surfactant (Solutol HS15), and water was set as 100%; the weight percentages of the three components were evaluated by changing their percentages simultaneously but keeping the total amount constant (1 g). The factor values and observed results of simplex lattice design was listed in Table 1 and analyzed via response surface plots by using the software Origin 8.5 and the results were shown in Figure 2. In order to find the optimum results, the response surface contour plots were designed. In Figure 2(c), the curves represent EE%, while in Figure 2(d) the curves stand for DL%. The common part of the contour plot (Figure 2e) between DL% and EE% was the best chosen area for the proportion of MCT and water. Taking all the factors including higher DL%, EE% into account, the optimum results were as follows: 20% of Labrafac, 30% of Solutol HS15 and 50% of water. Since the total amount of formulation variables was fixed at 1 g, the suggested composition of the optimized formulation was 17.5 mg of NaCl (1.75%), 15 mg of Lipoid (1.5%), 200 mg of Labrafac (20%), 300 mg of Solutol HS15 (30%) and 500 mg of water (50%).

Figure 2. Predicted response surface plots: (a) EE%; (b) DL%; and Response surface contour plots: (c) EE%; (d) DL%; (e) represents the overlay chart of (c) and (d).

Table 1. The factor values and observed results of simplex lattice design.

Formulation	Formulation Components
Numbers	Water/%	MCT/%	HS15/%	EE%	DL%
1	80	10	10	84.9 ± 4.75	1.29 ± 0.05
2	40	50	10	85.1 ± 2.75	1.13 ± 3.5
3	40	10	50	91.4 ± 2.2	1.16 ± 1.0
4	60	30	10	93.6 ± 2.05	1.24 ± 0.08
5	60	10	30	72.3 ± 1.17	1.25 ± 0.16
6	53.3	23.3	23.3	80.7 ± 0.3	0.98 ± 0.05
7	40	30	30	87.1 ± 0.05	0.69 ± 2.0

At optimum results, the EE% and DL% of the RPV-LNCs were 92.6 ± 1.3% and 1.35 ± 0.20%, respectively. Based on the consideration of high-hydrophobicity, RPV should mainly disperse or dissolve in the oily core of LNCs, while a small portion of RPV still adsorb within the shell layer. The reason why the DL% of RPV-LNCs was low might be that the solubility of RPV in the selected lipid materials was poor, since the encapsulation of drug was governed and restricted by its solubility in the lipids. Therefore, selecting high compatible lipid materials or improving the compatibility of RPV with carrier materials might be a promising method to improve the DL%.

Morphology, particle size and zeta potential analysis

In appearance, the prepared RPV-LNCs dispersion was bluish and transparent (Figure 3a), whereas crude RPV dispersed in water at the same drug concentration was the suspension with some precipitation of drug (Figure 3b). It was indicating that an improvement of the dispersibility of RPV was achieved after formulated to RPV-LNCs dispersion. The morphology of RPV-LNCs was analyzed by transmission electron microscopy (TEM) and displayed in Figure 3(c). The results revealed that the RPV-LNCs were in typical core-shell structure and non-adherent among each other. The homogeneous core-shell structure could be explained by a schematic of LNCs. Lipid nanocapsules, consisted of an oily core which was corresponding to free Labrafac and a surfactant shell which was made up of the mixture of Lipoid anchored in the oily phase, and Solutol HS15 oriented towards the water phase. Since the fast cooling-dilution process, the forming temperature of the final RPV-LNCs dispersion was below the melting point of HS15, crystallization could occur, leading to a rigid shell coating the oily core (Heurtault et al., 2003b) . Moreover, the PEG chains of Solutol HS 15 may stretch within the outer water phase and intertwine like balls of loose twine, preventing aggregation and inducing a steric stabilization among particles.

Figure 3. Photographic images (a, b) and TEM micrograph (c) of the optimal RPV-LNCs formulations.

Particles size and polydispersity index are fundamental indexes, which plays an important role in the size and uniformity evaluation of nanoparticles. Polydispersity varies from 0 to 1, but closer to zero, higher the homology between the particles (Joshi & Patravale, 2008). The average particle size of the optimized RPV-LNCs dispersion was 62.1 ± 1 nm with a polydispersity index of 0.137, indicating narrow particle size distribution (Figure 4a). In addition, the encapsulation of RPV increases the size of the LNCs in 8 nm compared to that of the blank LNCs (54.3 ± 2.1 nm). The reason could be attributed to the encapsulation of RPV in the oily core and the adsorption of RPV in the shell of LNCs.

Figure 4. Particle size distribution (a) and the zeta potential (b) of the optimal RPV-LNCs.

Zeta potential is a valid factor to indicate the storage stability of nano-formulations. Higher electric charge on the surface of the colloid particles is able to prevent aggregation among the colloid particles because of the repellent forces between each other. The zeta potential values of blank LNCs and the optimal RPV-LNCs were −11.16 ± 0.20 mV (not shown) and −11.81 ± 0.40 mV (Figure 4b), respectively. Zeta potential may depend on chemical nature of the polymer, the stabilizing agent or pH of the medium. In this study, the negative zeta potential might be related to the amphipathic surfactant Solutol HS15 which was integrated at the capsule interface since hydrogen bond may occur among the hydroxyl groups in the PEG chains of Solutol HS 15 or between PEG chains and water molecules (Perrier et al., 2010).

Differential scanning calorimetry analysis

The Differential scanning calorimetry (DSC) analysis was performed to inspect the changes of nanoparticles crystalline structure compared with the raw materials (Jia et al., 2012). Figure 5 displayed the DSC thermograms of the raw materials, their physical mixture (RPV, mannitol, lecithin) and RPV-LNCs freeze-dried powder. It is evident from Figure 5 that each raw material exhibited particular endothermic peaks. For instance, the melting peak of pure RPV was at 149.47 °C (a), and that of mannitol was at 168.04 °C (b). The characteristic peaks of RPV were also present in the DSC curves of physical mixture (c), meaning that the initial crystalline form of the drug was retained. Nevertheless, the peak of RPV in physical mixture showed a slight shift (146.09 °C). This might be related to the interaction of the excipients of lecithin and mannitol with RPV. On the contrary, no melting peak corresponding to the fusion of RPV was observed in the curve of RPV-LNCs freeze-dried powder (d). It is indicating that the crystalline form of RPV was unchanged when physically mixed with the excipients, but transformed into amorphous state when encapsulated into LNCs. These results proved that RPV had been successfully incorporated into LNCs in amorphous structure. In addition, as proved in literature (Corrigan et al., 2003), the amorphous form was considered to have higher energy with increased surface area, subsequently higher solubility.

Figure 5. Differential scanning calorimetry thermograms: (a) ropivacaine; (b) mannitol; (c) the physical mixture of RPV-LNCs; (d) RPV-LNCs freeze-dried powder.

X-ray diffraction analysis

Further investigation on whether RPV undergo polymorphic transformation or not when formulated to LNCs was confirmed by X-ray diffraction (XRD) analysis (Luan et al., 2014). The XRD analysis of the raw materials, their physical mixtures (RPV, mannitol and lecithin) and RPV-LNCs freeze-dried powder was carried out and the diffractograms were displayed in Figure 6. It is evident that the pure RPV showed a strong absorption peak at 10.56 ° (a), while the cryoprotectant mannitol showed a characteristic absorption peak at 10.88 ° (b). The two peaks could also be present in the curve of the physical mixture (c), which is illustrative that the crystal form of RPV was the same with that of the pure RPV powder. On the contrary, the characteristic peak of RPV disappeared in the curve of RPV-LNCs freeze-dried powder (d), indicating a disordered crystalline state of RPV in LNCs. These results further proved that RPV had been incorporated into the lipid matrix of LNCs in amorphous or disordered structure.

Figure 6. X-ray diffraction patterns: (a) ropivacaine; (b) mannitol; (c) the physical mixture of RPV-LNCs; (d) RPV-LNCs freeze-dried powder.

Ex vivo permeation study

In order to evaluate the potential of RPV-LNCs in delivering RPV through skin, the ex vivo skin permeation study was carried out by using Franz diffusion cells. The permeability of RPV-LNCs was examined by analyzing the accumulative penetration amount (Q_n) and penetration rate of RPV in the receive cells and the results were displayed in Figure 7. It was revealed that the Q_n and penetration rate of RPV from RPV-LNCs were both significantly increased compared with that from the propylene glycol solution. The Q_n of RPV from RPV-LNCs at 24 h was 349.0 ± 11.5 μg cm⁻², almost 2.17 times than that from propylene glycol solution (161.0 ± 1.3 μg cm⁻²). Furthermore, the permeation reached steady state after 6 h and followed zero order release kinetics with a penetration rate of 9.06 μg cm⁻² for RPV-LNCs, also much higher than that of the RPV propylene glycol group (2.63 μg cm⁻²).

Figure 7. Ex vivo permeation profile through the excised mice skin of RPV-LNCs (▪) and RPV propylene glycol (▴) (mean ± S.D.; n = 5).

The reason that two experimental groups showed different permeability may be related to the function mode of the carriers. As reported by Barry (Barry et al., 1987), propylene glycol may act as penetration enhancer in the ex vivo permeation study, which could achieve accelerant ability by penetrating into the corneum and changing drug partition coefficient between stratum corneum and vehicle. Thus, RPV in the control group should permeate independently to yield fluxes. As for LNCs, the RPV-LNCs are supposed to form an appressed film covered on the skin surface, which makes LNCs instantly accessible to the skin. The close contact may facilitate RPV permeation across the stratum corneum into deep skin. Further, the small size of RPV-LNCs (62.1 nm) may also favor RPV penetrating into the viable skin, even into the systemic circulation since it was reported that fluorescence-labeled nanoparticles at a size of 70 nm passed the skin barrier and was detectable in deeper skin layers (Lademann et al., 2011).

Histopathology study of skin

Aims to evaluate the topical action of RPV-LNCs on skin, the histopathology study of untreated skin, skin treated with RPV propylene glycol solution and RPV-LNCs was investigated by hematoxylin/eosin staining and the results were visualized by light microscopy (Figure 8). It could be observed visually that the untreated skin had tight junctions within stratum corneum (Figure 8a), while the skin treated with RPV propylene glycol solution revealed a loose structure (Figure 8b). Particularly, the skin stratum corneum treated by RPV-LNCs was more scattered and loose compared with that of treated by propylene glycol solution, meanwhile appeared more swollen (Figure 8c). In conclusion, RPV-LNCs could weaken the barrier function of stratum corneum and thus promote drug permeation.

Figure 8. Histopathology photomicrographs (400×) of untreated skin (a), skin treated with RPV propylene glycol solution (b) and RPV-LNCs dispersion (c).

The reason for interaction occurred between LNCs dispersion and skin may be related to the colloidal properties of LNCs and can be illustrated in Figure 9. Firstly, a colloidal film was supposed to form onto the skin surface since water evaporation after the application of RPV-LNCs, making the drug carrier instantly accessible to the skin. The covered film might significantly reduce or prevent skin dehydration, providing better wetting function to the skin and loosening the barrier properties (Tan et al., 2011). Moreover, some components of LNCs, particularly lecithin, may fuse or mix with skin lipids by disturbing the lamellar arrangement of the lipids within stratum corneum or even achieve lipid exchange (Sinico et al., 2005). All these factors combined to make the structure of stratum corneum loose, correspondingly promote drug permeation.

Figure 9. Permeation mechanism of LNCs. LNCs can promote adhesiveness and increase skin hydration, finally lead to loose structure, fluidization and even lipid exchange within the intercellular lipid domain.

Writhing test

Only when RPV permeate through skin into systemic circulation can the analgesia effect works. In order to evaluate the antinociceptive effect of RPV-LNCs formulation, the pharmacodynamics study was conducted by writhing test with 12 Kunming mice per group. All mice received either RPV-LNCs formulations or RPV propylene glycol transdermally for 6 h. The antinociceptive responses were determined by measuring the pain latency and writhing numbers within 15 min after injected i.p. a 0.6% acetic acid solution. As a result (Table 2), the control group showed a latent period of 4.02 ± 0.79 min, while the RPV-LNCs and RPV propylene glycol showed a latent period of 13.90 ± 1.12 min and 10.50 ± 0.33 min, respectively. Moreover, compared to the control group, both experimental groups induced significant reduction in writhing response as follow: RPV propylene glycol (74.7%, p < 0.05), RPV-LNCs (91.3%, p < 0.05). In addition, the RPV-LNCs formulations also showed significant reduction compared to RPV propylene glycol group (p < 0.05). Therefore, we can draw a conclusion that the RPV-LNCs formulations provide obvious analgesic effect by both prolonging pain latency in mice and reducing the writhing response within 15 min.

Table 2. Antinociception of RPV-LNCs on writhing test of Kunming mice induced by intraperitoneal injection (i.p.) of 0.6% acetic acid (0.1 mL/10 g).

Groups	N.	Latent Period/min (Mean ± S.D.)	Number of writhing (Mean ± S.D.)	Reduction in writhing response/%	p value
Control	12	4.02 ± 0.79	41.5 ± 16.2	—
RPV propylene glycol	12	10.50 ± 0.33	10.5 ± 2.6	74.7	<0.05 versus control
RPV-LNCs	12	13.90 ± 1.12	3.6 ± 2.1	91.3	<0.05 versus control <0.05 versus RPV propylene glycol

N: Number of Kunming mice per group. Reduction% was obtained in comparison to control group.

Among the several models of visceral pain, the writhing test has been mostly used as a standard screening method specially in evaluating antinociceptive properties (Golshani et al., 2004). The acetic acid-induced writhing test is widely applied for the evaluation of peripheral antinociceptive activity (Gené et al., 1998), which is sensitive in detecting antinociceptive effects of drug that may be inactive in other methods such like tail-flick test (Bentley et al., 1981). The entire analgesia effect of RPV could be due to the systemic uptake of the drug with subsequent wide spread actions. Therefore, from the results of pharmacodynamics study, we may draw an inference that RPV-LNCs could deliver RPV into blood circulation successfully. Further, corresponding to the results of ex vivo permeation study, the cumulative amount of RPV from LNCs penetrating through the skins was 2.17 times (349.0 ± 11.5 μg·cm⁻² versus 161.0 ± 1.3 μg cm⁻²) than that from propylene glycol solution. Thus, the RPV-LNCs formulations showed significant reduction compared to RPV propylene glycol group (p < 0.05) since the higher RPV transdermal permeation.

Conclusion

Ropivacaine, a novel long-acting local anesthetic, is regarded as a promising agent for anesthesia and analgesia. In this study, aims to exploit innovative administration route for RPV, RPV-LNCs were prepared by phase inversion process and investigated as transdermal carriers for RPV. As a result, the novel lipid nanocapsules (LNCs) composed of 20% of Labrafac, 30% of Solutol HS15 and 50% of water, exhibited high potential in delivering ropivacaine (RPV) transdermally. The optimized RPV-LNCs represented typical core-shell structure with the mean diameter of 62.1 ± 1.7 nm. The EE% and DL% were 92.6 ± 1.3% and 1.35 ± 0.20%, respectively. DSC and XRD analysis revealed the amorphous state of RPV in LNCs. In the ex vivo permeation study, the cumulative amount of RPV penetrated through excised skin from LNCs formulations was 2.17-folds than that of the propylene glycol solution. The histopathology study proved the interaction between RPV-LNCs and skin could loosen the structure of stratum corneum barrier. Moreover, the RPV-LNCs formulation showed significant antinociceptive effect in the writhing test. In conclusion, our studies provided evidences in detail that LNCs as a lipid-based colloid system were advantageous to enhance the transdermal penetration of ropivacaine, which laid foundation of developing novel external preparation for ropivacaine.

Declaration of interest

The authors confirm that this article content has no conflicts of interest. This work is supported by grants from Shanghai Municipality Science and Technology Commission (12nm0500700, 11DZ1971400) and the National Nature Science Foundation (No.81171766, No.81373896) and the Natural Science Foundation of Shandong Province, China (No.ZR2011HM026).