Encapsulation of ropivacaine in a combined (donor-acceptor, ionic-gradient) liposomal system promotes extended anesthesia time

<div><p>Ropivacaine is a local anesthetic with similar potency but lower systemic toxicity than bupivacaine, the most commonly used spinal anesthetic. The present study concerns the development of a combined drug delivery system for ropivacaine, comprised of two types of liposomes: donor multivesicular vesicles containing 250 mM (NH₄)₂SO₄ plus the anesthetic, and acceptor large unilamellar vesicles with internal pH of 5.5. Both kinds of liposomes were composed of hydrogenated soy-phosphatidylcholine:cholesterol (2:1 mol%) and were prepared at pH 7.4. Dynamic light scattering, transmission electron microscopy and electron paramagnetic resonance techniques were used to characterize the average particle size, polydispersity, zeta potential, morphology and fluidity of the liposomes. <i>In vitro</i> dialysis experiments showed that the combined liposomal system provided significantly longer (72 h) release of ropivacaine, compared to conventional liposomes (~45 h), or plain ropivacaine (~4 h) (p <0.05). The pre-formulations tested were significantly less toxic to 3T3 cells, with toxicity increasing in the order: combined system < ropivacaine in donor or acceptor liposomes < ropivacaine in conventional liposomes < plain ropivacaine. The combined formulation, containing 2% ropivacaine, increased the anesthesia duration up to 9 h after subcutaneous infiltration in mice. In conclusion, a promising drug delivery system for ropivacaine was described, which can be loaded with large amounts of the anesthetic (2%), with reduced <i>in vitro</i> cytotoxicity and extended anesthesia time.</p></div

Analgesia (von Frey test) induced by ropivacaine in mice.

<p>The anesthetic was used in solution (0.75% and 2% plain RVC) or encapsulated in HSPC:cholesterol (2:1 mol %) liposomes: donor (RVC in LMVV 7.4_{in+ sulfate}), acceptor (RVC in LUV 5.5 _in) and combined formulation (RVC in LMVV 7.4_{in+ sulfate} + LUV 5.5_in).</p

EPR spectra of 5-SASL probe incorporated in liposomes, with or without 2% RVC: donor (LMVV 7.4_{in+ sulfate}) and acceptor (LUV 5.5_in) vesicles.

<p>EPR spectra of 5-SASL probe incorporated in liposomes, with or without 2% RVC: donor (LMVV 7.4_{in+ sulfate}) and acceptor (LUV 5.5_in) vesicles.</p

Cell viability (%) of 3T3 fibroblasts exposed to RVC, plain or encapsulated in liposomes: donors (LMVV 7.4_{in+ sulfate}), acceptors (LUV 5.5_in), and their combination (LMVV 7.4_{in+ sulfate} + LUV 5.5_in); N = 5.

<p>Cell viability (%) of 3T3 fibroblasts exposed to RVC, plain or encapsulated in liposomes: donors (LMVV 7.4_{in+ sulfate}), acceptors (LUV 5.5_in), and their combination (LMVV 7.4_{in+ sulfate} + LUV 5.5_in); N = 5.</p

Size, polydispersity index (PDI) and zeta potential of HSPC:cholesterol (2:1 mol %) liposomes.

<p>Size, polydispersity index (PDI) and zeta potential of HSPC:cholesterol (2:1 mol %) liposomes.</p

Transmission electron micrographs of HSPC:cholesterol (2:1 mol %) vesicles.

<p>A) donor—LMVV 7.4_{in+ sulfate} and B) acceptor—LUV 5.5_in, stained with 2% uranyl acetate (× 60,000 magnification).</p

<i>In vitro</i> release kinetics of RVC, plain or encapsulated in HSPC:cholesterol liposomes: donors (LMVV 7.4_{in+ sulfate}), acceptors (LUV 5.5_in), and their combination (LMVV 7.4_{in+ sulfate} + LUV 5.5_in); Ambient temperature (25°C), N = 3.

<p><i>In vitro</i> release kinetics of RVC, plain or encapsulated in HSPC:cholesterol liposomes: donors (LMVV 7.4_{in+ sulfate}), acceptors (LUV 5.5_in), and their combination (LMVV 7.4_{in+ sulfate} + LUV 5.5_in); Ambient temperature (25°C), N = 3.</p