| Ropivacaine is a new, long-acting aminoamide local anesthetic, which is the first enantiomerically pure local anesthetic and exists as the S-enantiomer. Ropivacaine was proved less prone to produce central nervous system toxicity and cardiovascular changes than its dextroisomer after intravenous infusion. The property of the great degree of differential block between nerve fibers responsible for transmission of pain and those that control motor function was considered to offer considerable clinical advantages in proving analgesia with minimal motor block. Actually, ropivacaine showed great separation of motor and sensory block in clinical trials. Ropivacaine has been introduced into clinic in the form of ropivacaine hydrochloride or mesylate, however, the intravenous infusion is offering poor patient convenience since it is found painful and risky. Thus, aims to develop novel external preparation to alter traditional intravenous infusion, the present study designed ropivacaine-loaded lipid nanocapsules (RPV-LNCs) and characterized its potential for transdermal delivery. The main studies and results were shown as follows:1. Preparation and physicochemical characterization of RPV-LNCsThe preparation of RPV-LNCs is based on phase-inversion phenomenon of the emulsion. MCT was selected as oil phase, Solutol HS15 as surfactant, lecithin as cosurfactant and NaCl as the phase-inversion temperature regulator. The influence of each factor was analyzed by simplex lattice design with entrapment efficiency (EE%) and drug loading (DL%) of RPV as indexes. Based on the high DL% and EE% within the feasibility domain of RPV-LNCs, the suggested composition of optimized formulation was established as follows:20% of MCT (200 mg),30% of Solutol HS15 (300 mg) and 50% (500 mg) of water. The obtained RPV-LNCs were an optically slightly bluish dispersion. Under TEM, the RPV-LNCs presented a homogeneous structure without aggregation. The mean particle size of the optimal RPV-LNCs was 62.1±1.7 nm with the polydispersity index of 0.137. The zeta potential values of the optimal RPV-LNCs were-11.81 mV. The results of differential scanning calorimeter (DSC) analysis and X-ray diffraction analysis (XRD) confirmed that the RPV-LNCs were prepared successfully. The results of in vitro release study showed that the prepared RPV-LNCs represented a remarked controlled-release profile.2. In vitro permeation studyThe in vitro permeation study of RPV-LNCs was evaluated by using full thickness dorsal skin excised from Kunming mice (male,25±2 g), and conducted by Franz cell. It was revealed that the flux rate of RPV from LNCs was significantly higher than that from the propylene glycol solution. The cumulative amounts of RPV from LNCs at 24 h was 349.0 ± 11.5 μg·cm-2, which was 2.17 times than that from propylene glycol solution. It indicated that the permeation followed zero order release kinetics and the steady-state flux (Js) of RPV-LNCs was 9.058 μg·cm-2·h-1 whereas that of RPV propylene glycol was 2.163 μg·cm-2·h-1.3. In vivo permeation studyThe in vivo permeation study was conducted based on the results of in vitro permeation study. The concentration of ropivacaine in plasma, epidermis and dermis was determined respectively. The results showed that RPV-LNCs yielded higher plasma concentrations (5.743 μg/mL) compared to the RPV propylene glycol solution (0.81 ug/mL). The plasma AUC (0-12) of RPV-LNCs was 23.789 mgh/mL, which was significantly larger than that of the RPV propylene glycol solution (5.695 mgh/mL) (P< 0.05). For epidermis, RPV level for the propylene glycol group increased drastically and higher than that for LNCs group at 6,9,12 h. For dermis, the retained amount of RPV was 27.9±5.2 μg/mL for LNCs, obviously remarkable than that of the propylene glycol group (15.59±3.9 μg/mL). Even though achieved high level in epidermis, the relatively low level in dermis for RPV propylene glycol solution proved the limitation in transporting RPV into deep skin, let alone blood circulation.4. Investigation on the mechanisms of penetration enhancementThe results of in vitro and in vivo permeation study showed that lipid nanocapsules could promote the permeability of ropivacaine. The mechanism of penetration enhancement was evaluated by the DSC analysis, histopathology study and scanning electron microscope (SEM) of skin. The untreated skin showed significant endothermic peaks at 208.22℃, 283.96 ℃, and 324.63 ℃ whereas that of skin treated by RPV-LNCs showed significantly changed endothermic peaks. The enthalpy value for the endothermic transitions at 283.96 ℃ shifts to 5.02 J·g-1 compared to 20.37 J·g-1 for that of the blank skin. Enthalpy variation reflects the changes in structures indirectly, thus the results of DSC illustrated the structure of skin changed much after treated by RPV-LNCs. The results of histopathology study and scanning electron microscope (SEM) of skin both showed that the untreated skin had a compact stratum corneum, with the adjacent corneocyte layer conjugated tightly. Application of RPV propyleneglycol could slightly change the tight junction of the stratum corneum. The skin treated by RPV-LNCs appeared swollen, with total stratum corneum thickness increased. In a conclusion, RPV-LNCs could weaken the barrier function of stratum corneum and promote drug permeation.5. Pharmacodynamic experimentIn order to evaluate the antinociceptive effect of RPV-LNCs formulation, the pharmacodynamics study was conducted by writhing test. As a result, the blank 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 blank 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 and reducing the writhing response in mice. |
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