This study reports an investigation of the pharmacological activity, cytotoxicity and local effects of a liposomal formulation of the novel local anaesthetic ropivacaine (RVC) compared with its plain solution. RVC was encapsulated into large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine, cholesterol and alpha-tocopherol (4:3:0.07, mole %). Particle size, partition coefficient determination and in-vitro release studies were used to characterize the encapsulation process. Cytotoxicity was evaluated by the tetrazolium reduction test using sciatic nerve Schwann cells in culture. Local anaesthetic activity was assessed by mouse sciatic and rat infraorbital nerve blockades. Histological analysis was performed to verify the myotoxic effects evoked by RVC formulations. Plain (RVC(PLAIN)) and liposomal RVC (RVC(LUV)) samples were tested at 0.125%, 0.25% and 0.5% concentrations. Vesicle size distribution showed liposomal populations of 370 and 130 nm (85 and 15%, respectively), without changes after RVC encapsulation. The partition coefficient value was 132 +/- 26 and in-vitro release assays revealed a decrease in RVC release rate (1.5 fold, P < 0.001) from liposomes. RVC(LUV) presented reduced cytotoxicity (P < 0.001) when compared with RVC(PLAIN). Treatment with RVC(LUV) increased the duration (P < 0.001) and intensity of the analgesic effects either on sciatic nerve blockade (1.4-1.6 fold) and infraorbital nerve blockade tests (1.5 fold), in relation to RVC(PLAIN). Regarding histological analysis, no morphological tissue changes were detected in the area of injection and sparse inflammatory cells were observed in only one of the animals treated with RVC(PLAIN) or RVC(luv) at 0.5%. Despite the differences between these preclinical studies and clinical conditions, we suggest RVC(LUV) as a potential new formulation, since RVC is a new and safe local anaesthetic agent.
The present study explored a new approach to the production of tubular conduits designed for peripheral nerve repair. Poly(L-lactic acid) (PLLA) and polycaprolactone (PCL) membranes were obtained after solvent evaporation and wrapped around a mandrel. The effectiveness of nerve regeneration was compared with that obtained with polyethylene and PCL extruded prostheses 30 and 60 days after surgery. The comparison between extruded and membrane-derived tubes clearly showed structural differences that were directly proportional to the hardness and transparency. An important factor to be considered is that the fiber count indicated that membrane-derived PCL tubes provided a significantly greater number of axons 30 days after repair. Sixty days after the procedure, the greatest regenerative performance was obtained with PCL, regardless of tube construction method. An intense imunolabeling of S100, type IV collagen, and laminin could be observed in the tissue obtained from membrane-derived PCL and PLLA groups, indicating that such constructs were able to positively stimulate Schwann cell responses. Overall, the results provided evidence that membrane-derived conduits are an alternative preparation method for tubular prostheses for peripheral nerve regeneration.
This study reports an investigation of the pharmacological activity, cytotoxicity and local effects of a liposomal formulation of the novel local anaesthetic ropivacaine (RVC) compared with its plain solution. RVC was encapsulated into large unilamellar vesicles (LUVs) composed of egg phosphatidylcholine, cholesterol and alpha-tocopherol (4:3:0.07, mole %). Particle size, partition coefficient determination and in-vitro release studies were used to characterize the encapsulation process. Cytotoxicity was evaluated by the tetrazolium reduction test using sciatic nerve Schwann cells in culture. Local anaesthetic activity was assessed by mouse sciatic and rat infraorbital nerve blockades. Histological analysis was performed to verify the myotoxic effects evoked by RVC formulations. Plain (RVC(PLAIN)) and liposomal RVC (RVC(LUV)) samples were tested at 0.125%, 0.25% and 0.5% concentrations. Vesicle size distribution showed liposomal populations of 370 and 130 nm (85 and 15%, respectively), without changes after RVC encapsulation. The partition coefficient value was 132 +/- 26 and in-vitro release assays revealed a decrease in RVC release rate (1.5 fold, P < 0.001) from liposomes. RVC(LUV) presented reduced cytotoxicity (P < 0.001) when compared with RVC(PLAIN). Treatment with RVC(LUV) increased the duration (P < 0.001) and intensity of the analgesic effects either on sciatic nerve blockade (1.4-1.6 fold) and infraorbital nerve blockade tests (1.5 fold), in relation to RVC(PLAIN). Regarding histological analysis, no morphological tissue changes were detected in the area of injection and sparse inflammatory cells were observed in only one of the animals treated with RVC(PLAIN) or RVC(luv) at 0.5%. Despite the differences between these preclinical studies and clinical conditions, we suggest RVC(LUV) as a potential new formulation, since RVC is a new and safe local anaesthetic agent.
We concluded that the new formulations presented a lower myotoxicity and a similar cytotoxic effect when compared to plain local anesthetic solutions.
The present in vitro study investigated the expression of basal lamina components by Schwann cells (SCs) cultivated on PCL and PLLA membranes prepared by solvent evaporation. Cultures of SCs were obtained from sciatic nerves from neonatal Sprague Dawley rats and seeded on 24 well culture plates containing the polymer membranes. The purity of the cultures was evaluated with a Schwann cell marker antibody (anti-S-100). After one week, the cultures were fixed and processed for immunocytochemistry by using antibodies against type IV collagen, laminin I and II. Positive labeling against the studied molecules was observed, indicating that such biomaterials positively stimulate Schwann cell adhesion and proliferation. Overall, the present results provide evidence that membrane-derived biodegradable polymers, particularly those derived from PLLA, are able to provide adequate substrate and stimulate SCs to produce ECM molecules, what may have in turn positive effects in vivo, influencing the peripheral nerve regeneration process.
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