DoE development of ionic gradient liposomes: A successful approach to improve encapsulation, prolong anesthesia and decrease the toxicity of etidocaine.

Etidocaine (EDC) is a long-acting local anesthetic of the aminoamide family whose use was discontinued in 2008 for alleged toxicity issues. Ionic gradient liposomes (IGL) are nanostructured carriers for which an inner/outer gradient of ions increases drug upload. This work describes IGLEDC, a formulation optimized by Design of Experiments, composed of hydrogenated soy phosphatidylcholine:cholesterol:EDC, and characterized by DLS, NTA, TEM/Cryo-TEM, DSC and 1H NMR. The optimized IGL showed significant encapsulation efficiency (41 %), good shelf stability (180 days) and evidence of EDC interaction with the lipid bilayer (as seen by DSC and 1H NMR results) that confirms its membrane permeation. In vitro (release kinetics and cytotoxicity) tests showed that the encapsulation of EDC into the IGL promoted sustained release for 24 h and decreased by 50 % the intrinsic toxicity of EDC to Schwann cells. In vivo IGLEDC decreased the toxicity of EDC to Caenorhabditis elegans by 25 % and extended its anesthetic effect by one hour, after infiltrative administration, at clinically used (0.5 %) concentration, in rats. Thus, this novel drug delivery system is a promise for the possible reintroduction of EDC in clinics, aiming at the control of operative and postoperative pain.

Inclusion Complex between Local Anesthetic/2-hydroxypropyl-ß-cyclodextrin in Stealth Liposome.

The drugs delivery system in the treatment of diseases has advantages such as reduced toxicity, increased availability of the drug, etc. Therefore, studies of the supramolecular interactions between local anesthetics (LAs) butamben (BTB) or ropivacaine (RVC) complexed with 2-hydroxypropyl-ß-cyclodextrin (HP-ßCD) and carried in Stealth liposomal (SL) are performed. 1H-NMR nuclear magnetic resonance (DOSY and STD) were used as the main tools. The displacements observed in the 1H-NMR presented the complexion between LAs and HP-ßCD. The diffusion coefficients of free BTB and RVC were 7.70 × 10-10 m2 s-1 and 4.07 × 10-10 m2 s-1, and in the complex with HP-ßCD were 1.90 × 10-10 m2 s-1 and 3.64 × 10-10 m2 s-1, respectively, which indicate a strong interaction between the BTB molecule and HP-ßCD (98.3% molar fraction and Ka = 72.279 L/mol). With STD-NMR, the encapsulation of the BTB/HP-ßCD and RVC/HP-ßCD in SL vesicles was proven. Beyond the saturation transfer to the LAs, there is the magnetization transfer to the hydrogens of HP-ßCD. BTB and RVC have already been studied in normal liposome systems; however, little is known of their behavior in SL.

Liposome surface modification by phospholipid chemical reactions.

Liposomal systems are well known for playing an important role as drug carriers, presenting several therapeutic applications in different sectors, such as in drug delivery, diagnosis, and in many other academic areas. A novel class of this nanoparticle is the actively target liposome, which is constructed with the surface modified with appropriated molecules (or ligands) to actively bind a target molecule of certain cells, system, or tissue. There are many ways to functionalize these nanostructures, from non-covalent adsorption to covalent bond formation. In this review, we focus on the strategies of modifying liposomes by glycerophospholipid covalent chemical reaction. The approach used in this text summarizes the main reactions and strategies used in phospholipid modification that can be carried out by chemists and researchers from other areas. The knowledge of these methodologies is of great importance for planning new studies using this material and also for manipulating its properties.

Physicochemical characterization and cytotoxicity of articaine-2-hydroxypropyl-ß-cyclodextrin inclusion complex.

Articaine (ATC) is one of the most widely used local anesthetics in dentistry. Despite its safety, local toxicity has been reported. This study aimed to develop an ATC-2- hydroxypropyl-ß-cyclodextrin inclusion complex (ATC HPßCD) and to assess its toxicity in vitro. The inclusion complex was performed by solubilization, followed by a fluorimetric and job plot assay to determine the complex stoichiometry. Scanning electron microscopy, DOSY- 1 H-NMR, differential scanning calorimetry (DSC), and sustained release kinetics were used to confirm the inclusion complex formation. In vitro cytotoxicity was analyzed by MTT assay and immunofluorescence in HGF cells. Fluorimetric and job plot assay determined the inclusion complex stoichiometry (ATC:HPßCD = 1:1) and complex formation time (400 min), as indicated by a strong host/guest interaction (Ka = 117.8 M - 1), complexed fraction (f = 41.4%), and different ATC and ATC HPßCD melting points (172 °C e 235 °C, respectively). The mean of cell viability was 31.87% and 63.17% for 20-mM ATC and 20-mM ATC HPßCD, respectively. Moreover, remarkable cell toxicity was observed with free ATC by immunofluorescence. These results indicate the ATC HPßCD complex could be used to improve the safety of ATC. Further research are needed to establish the anesthetic safety and effectiveness in vivo .

Anaesthetic benefits of a ternary drug delivery system (Ropivacaine-in-Cyclodextrin-in-Liposomes): in-vitro and in-vivo evaluation.

OBJECTIVES: To evaluate whether a ternary system composed of hydroxypropyl-ß-cyclodextrin (HP-ßCD) further encapsulated into egg phosphatidylcholine liposomes (LUV) could prolong the action and reduce the toxicity of ropivacaine (RVC). METHODS: Dynamic light scattering and NMR were used to characterize the inclusion complex (RVC : HP-ßCD), liposomal (RVC : LUV) and ternary (LUV : RVC : HP-ßCD) systems containing 0.25% RVC. Their encapsulation efficiency, release kinetics, in-vitro cytotoxicity and in-vivo anaesthetic effect (paw-withdraw tests in mice) were also evaluated. KEY FINDINGS: 1 : 1 RVC : HP-ßCD inclusion complex was encapsulated in liposomes (220.2 ± 20.3 nm size, polydispersity <0.25, zeta potentials = -31.7 ± 1.4 mV). NMR (diffusion-ordered spectroscopy (DOSY)) revealed stronger anaesthetic binding to LUV : RVC : HP-ßCD (Ka  = 342 m-1 ) than to RVC : HP-ßCD (Ka  = 128 m-1 ) or liposomal formulation (Ka  = 22 m-1 ). The formulations promoted in-vitro sustained drug release and partially reverted the cytotoxicity of RVC against 3T3 fibroblasts in the profile: LUV : RVC : HP-ßCD ≥ RVC : HP-ßCD > RVC : LUV. Accordingly, in-vivo sensory block of free RVC (180 min) was prolonged ca. 1.7 times with the ternary system and RVC : HP-ßCD (300 min) and 1.3 times with RVC : LUV (240 min). CONCLUSIONS: These results confirm the suitability of this double-carrier system in clinical practice, to decrease the toxicity and prolong the anaesthesia time evoked by RVC.

Combining nuclear magnetic resonance with molecular dynamics simulations to address sumatriptan interaction with model membranes.

The goal of this work is to obtain a complete map on the interactions between sumatriptan, an amphiphilic ionizable anti-migraine drug, with lipid bilayers. To this end, we combined two physico-chemical techniques - nuclear magnetic resonance and molecular dynamics simulations - to obtain a detailed picture at different pH values. Both approaches were used considering the strength and constraints of each one. NMR experiments were performed at pH 7.4 where at least 95% of the drug molecules are in their protonated state. From NMR, sumatriptan shows partition on the interfacial region of model membranes (near the head groups and intercalating between adjacent lipids), inducing changes in chemical environment and affecting lipid dynamics of liposomes, in a dose dependent manner. Due to the experimental instability of lipid bilayers at high pH, we took advantage of the molecular dynamics power to emulate different pH values, to simulate sumatriptan in bilayers including at fully uncharged state. Simulations show that the neutral species have preferential orientation within the bilayer interface while the distribution of protonated drugs is independent on the initial conditions. In summary, several properties depicted the interfacial partition of the anti-migraine drug at the water-lipid interface at different conditions. Both techniques were found complementary to shed light on the structural and dynamics of sumatriptan-lipid bilayer interactions.

In situ study of the magnetoelectrolysis phenomenon during copper electrodeposition using time domain NMR relaxometry.

Although the effect of magnetic field (B) on electrochemical reactions (magnetoelectrolysis phenomenon) has been long known, it has not been considered in electrochemical reactions analyzed in situ by magnetic resonance methods, such as nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), and magnetic resonance imaging (MRI), which are intrinsically performed in the presence of B. In this report, the effect of B on the copper electrodeposition reaction, measured by a low-field (0.23 T) NMR spectrometer, was demonstrated. As expected, an enhancement in the reaction rate in comparison to the ex situ electrodeposition reaction was observed. Such enhancement was not dependent on electrodes/magnetic field orientations. Parallel and perpendicular orientations showed similar electrodeposition rates, which is explained by the cyclotron flows generated by distortions in electric and magnetic field lines near the electrode and the electrode edge. Therefore, NMR spectroscopy is not a passive analytical method, as assumed in preceding in situ spectroelectrochemical studies. Although the magnetoelectrolysis phenomenon demonstrated in this report used a paramagnetic ion, it can also be observed for diamagnetic species, since the magnetoelectrolysis phenomenon is independent of the nature of the species. Consequently, similar convection effects may occur in other electrochemical nuclear magnetic resonance (EC-NMR) experiments, such as the electrochemical reaction of organic molecules, as well as in electrocatalysis/fuel cells, lithium-ion batteries, and experiments that use electrochemical electron paramagnetic resonance (EC-EPR) and electrochemical magnetic resonance imaging (EC-MRI).

Improvement of tetracaine antinociceptive effect by inclusion in cyclodextrins.

Local anesthetics (LA) are among the most important pharmacological compounds used to attenuate or eliminate pain. However, systemic toxicity is still a limitation for LA application, especially for ester-type drugs, such as tetracaine (TTC) that presents poor chemical stability (due to hydrolysis by plasma esterases). Several approaches have been used to improve LA pharmaceutical properties, including the employment of drug-delivery systems. Here we used beta-cyclodextrin (ß-CD) or hydroxypropyl-beta-cyclodextrin (HP-ß-CD) to develop two new TTC formulations (TTC:ß-CD and TTC:HP-ß-CD). The inclusion complexes formation, in a 1:1 stoichiometry, was confirmed by differential scanning calorimetry, X-ray diffraction, UV-VIS absorption and fluorescence. Nuclear magnetic resonance (DOSY experiments) revealed that TTC association with HP-ß-CD is stronger (Ka=1200 mol/L(-1)) than with ß-CD (Ka=845 mol/L(-1)). Moreover, nuclear Overhauser effect (NOE) experiments provided information on the topology of the complexes, where TTC aromatic ring is buried inside the CD hydrophobic cavity. In vitro tests with 3T3 fibroblast cells culture revealed that complexation decreased TTC cytotoxicity. In addition, the total analgesic effect of TTC, tested in rats through the infraorbital nerve test, was improved in 36% with TTC:ß-CD and TTC:HP-ß-CD. In conclusion, these formulations presented potential for future clinical use, by reducing the toxicity and increasing the antinociceptive effect of tetracaine.

Prilocaine-cyclodextrin-liposome: effect of pH variations on the encapsulation and topology of a ternary complex using 1H NMR.

A better comprehension of the prilocaine (PLC)-ß-cyclodextrin (ß-CD) complex liberation to membranes was provided by studying the architectural supramolecular arrangements of PLC, ß-CD and egg phosphatidylcholine (EPC) liposomes, a membrane model. The topologies and possible interactions of mixtures of PLC, ß-CD and EPC liposomes were investigated by nuclear magnetic resonances combining experimental (1)H-NMR (1D ROESY, STD and DOSY) at different pHs. The results indicate that in the mixture PLC/ß-CD/EPC at pH 10 the PLC molecules are almost totally embedded into the liposomes and little interaction was observed between PLC and ß-CD. However, at pH 5.5 not only was PLC imbedded in the EPC bilayer, but PLC was also interacting with ß-CD. These results were rationalized as a spontaneous PLC release from ß-CD to liposomes vesicles, whereas the PLC/EPC complex formation was higher at pH 10 than pH 5.5.

Topology of a ternary complex (proparacaine-beta-cyclodextrin-liposome) by STD NMR.

The topologies of proparacaine (PPC) in beta-cyclodextrin (beta-CD), PPC in egg phosphatidylcholine (EPC) liposomes and PPC in beta-CD in EPC were investigated using NMR experiments (1D ROESY and saturation transfer difference (STD)). This is the first description of the STD technique applied to PPC-EPC-beta-CD system, revealing that not only PPC was imbedded in EPC bilayer, but beta-CD was also interacting with liposome vesicles. These results are novel and were rationalized as the spontaneous formation of a ternary complex with some beta-CD molecules bound to external liposome vesicles surfaces.