Partial solubilization of the erythrocyte and synaptosome membranes induced by nonionic detergents

Orientador: Eneida de PaulaTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Lipids rafts são microdomínios, da ordem de 10 a 200 nm, enriquecidos em esfingolipídios, colesterol e proteínas específicas, responsáveis por diversos processos celulares e que possuem características similares à fase líquido-ordenada (Lo) encontrada em membranas modelo. Esses microdomínios têm sido estudados através do preparo de membranas resistentes à detergentes (DRMs). Neste trabalho nós investigamos a ação de detergentes não iônicos em duas membranas biológicas (de eritrócito humano e de sinaptossomas de córtex cerebral suíno) e a obtenção de DRMs com o fim de identificar os mecanismos determinantes da associação entre os componentes das membranas biológicas, que levam a separação lateral de fases. Medidas em monocamadas de Langmuir e em bicamadas (vesículas unilamelares gigantes, GUVs) com lipídios extraídos de membranas eritrocitárias possibilitaram avaliar a cinética de incorporação e o processo de solubilização induzido pelos detergentes TX-100 e Brij 98. Nessas membranas, o TX-100 foi capaz de separar domínios de fase Lo e, em concentrações acima da concentração micelar crítica (CMC), solubilizar a fração líquido-desordenada, enquanto o Brij 98 ¿ de maior afinidade pelos lipídios que o TX-100 - em concentrações menores que a CMC, já foi capaz de fragmentar a membrana em microvesículas. Numa segunda abordagem experimental, utilizamos sinaptossomas de córtex cerebral suíno. O tratamento dessas biomembranas com TX-100 ou Brij 98, levou a obtenção de DRMs tanto à 4 quanto à 37 ºC; as frações resistentes a TX-100 mostraram-se enriquecidas em colesterol e esfigomielinas, enquanto as DRMs de Brij 98 continham maior teor proteico e de fosfolipídios, e menor conteúdo de colesterol. O elevado teor de colesterol nas DRMs de TX-100 concorda com seu maior empacotamento lipídico, em relação às DRMs de Brij 98. Os resultados mostram que a composição de DRMs é totalmente dependente do detergente, indicando um processo de solubilização diferencial dos componentes da membrana eritrocitária e sinaptossomal induzido pelo TX-100 e pelo Brij-98. Desta forma, considerando a composição, o tamanho e a presença da fase Lo nos DRMs de TX-100, concluímos que este detergente não iônico é capaz de isolar mais eficientemente os microdomínios lipid rafts dessas duas biomembranas modeloAbstract: Lipids rafts are 10-200nm microdomains found in biological membranes, which are enriched in cholesterol, sphingolipids and specific proteins, being related to diverse cellular processes such as fusion, endocytosis,¿ Lipid rafts have properties also found in the liquid ordered (Lo) phase of model membranes. One approach to study such microdomains is offered by detergent resistant membranes (DRMs). In this thesis we have investigated the effect of non-ionic detergents in two biomembranes (human erythrocytes and pig brain cortex synaptosomes) and the preparation of their DRMs in order to identify the mechanisms that rule the association between membrane components, leading to phase separation. Studies with lipids extracted from erythrocyte membranes (Langmuir monolayers and giant unilamellar vesicles, GUV) allowed determination of the kinetics of membrane insertion and solubilization process induced by TX-100 and Brij 98. In such membranes, TX-100 was able to induce the formation of Lo phase resistant fractions and, above its critical micelle concentration (CMC), to solubilize such domains. As for Brij 98, which binds stronger to the membrane lipids, even below the CMC it disrupted the membranes in microvesicles (DRMs). We have also prepared synapsomal membranes from pig brain cortex. Treatment of such membranes with TX-100 or Brij 98 gave rise to DRMs, both at cold (4 ºC) and at 37 ºC; the TX-100 resistant fractions were enriched in cholesterol and sphingolipids, while the Brij 98 DRMs contained higher protein and phospholipid levels and low cholesterol content. The higher cholesterol level of the TX-100 DRMs agreed well with their improved lipid packing, detected by Electron Paramagnetic Resonance, in comparison to Brij 98 DRMs. In conclusion, the differential solubilization of erythrocyte and synaptosomal membrane components induced by TX-100 and Brij 98 show that is mainly the surfactant agent that determines DRMs composition. More specifically, considering the composition, size and presence of the Lo phase in TX-100 DRMs we conclude that this detergent is able to more efficiently isolate lipid rafts microdomains from the two model biomembranes studiedDoutoradoBioquimicaDoutora em Biologia Funcional e MolecularCAPE

Estudo de membranas eritrocitárias resistentes a detergentes da série éter de polioxietileno (Brij)

Orientadores: Eneida de Paula, Cleyton Crepaldi DominguesDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A visão atual sobre membranas biológicas abarca descrições cada vez mais complexas devido, principalmente, à descoberta de novos papéis atribuídos aos lipídios e sua heterogeneidade. Em particular a associação de esfingolipídios e colesterol, acrescida de proteínas específicas, constitui a base da formação de domínios membranares conhecidos como lipid rafts. Rafts são microdomínios funcionais de biomembranas e estão envolvidos em diversos processos biológicos como reconhecimento celular, endocitose, transdução de sinal, entre outros processos. Uma estratégia experimental para estudar esses domínios é a preparação de frações de membrana parcialmente resistentes ao tratamento com detergentes, a baixa temperatura (4°C). Neste trabalho demonstramos pela primeira vez o preparo e caracterização de frações resistentes a detergente (DRMs) extraídas de membranas de eritrócito humano, a partir do tratamento com os detergentes não iônicos polioxietileno 20-oleil éter (Brij 98) e polioxietileno 20-cetil éter (Brij 58), seguida de separação por ultracentrifugação em gradiente de sacarose. Essas DRMs foram obtidas a 4°C e 37°C, a partir de membranas intactas e com conteúdo reduzido de colesterol (após tratamento com metil-beta-ciclodextrina) e foram comparadas com DRMs de Triton X-100 (TX-100) em relação ao tamanho, conteúdo proteico e lipídico e grau de organização da bicamada. As frações de DRM de Brij mostraram-se enriquecidas em colesterol e fosfolipídios com ácidos graxos de cadeia saturada (em especial ácido lignocérico das esfingomielinas), características consistentes com lipid rafts. No entanto, DRMs de TX-100 apresentaram maior proporção de esfingomielinas/glicerofosfolipídios que as frações obtidas com Brij, além de menor proporção de fosfatidiletanolamina, um lipídio preferencial da monocamada interna da membrana de eritrócitos. Em relação à solubilização proteica, a membrana do eritrócito foi mais resistente ao tratamento com Brij 98 do que aos outros dois detergentes. Flotilina-2 e estomatina foram encontradas nas frações de DRMs de Brij 98 e Brij 58, porém a redução do conteúdo de colesterol da membrana eritrocitária resultou numa menor associação da flotilina-2 àquelas DRMs. Resultados de Ressonância Paramagnética Eletrônica, com uso de marcadores de spin do tipo doxil-estearato não acusaram variação significativa no grau de empacotamento dos lipídios nas bicamadas de DRMs de Brij 98 e Brij 58 em relação à membrana eritrocitária, diferentemente do observado em DRMs de TX-100 e do que seria esperado para domínios lipídicos na fase líquido-ordenada. Em conclusão, DRMs de eritrócitos humanos, com características compatíveis aos domínios funcionais de biomembranas (rafts), foram obtidas tanto a 4ºC quanto a 37ºC; essas frações resistentes aos Brij apresentaram tamanho, composição proteica e lipídica e grau de empacotamento da bicamada diferente das DRMs de TX-100, indicando um processo de extração diferencial dos componentes da membrana eritrocitária induzida por esses detergentesAbstract: Depiction of biological membranes has been turning more and more complex lately due to the new roles assigned to their lipid components and their heterogeneity. The association between sphingolipids and cholesterol is the basis of lipid rafts formation. Rafts are functional microdomains of biological membranes which have been associated to different biological processes such as cellular recognition, endocytosis and signal transduction, among others. An useful experimental approach to study lipid rafts is the preparation of membrane fractions partially resistant to detergents under low temperature (4ºC). In this work we report the isolation of detergent resistant membranes (DRMs) from human erythrocytes treated with the non-ionic detergents polioxyethylene 20-oleoyl ether (Brij 98) and polioxyethylene 20-cetyl ether (Brij 58) followed by sucrose gradient ultracentrifugation. Such DRMs were obtained at 4°C and 37°C, from cholesterol-depleted (treated with methyl-beta-cyclodextrin) and intact erythrocyte membranes and they compared to DRMs obtained with Triton X-100 (TX-100) regarding the size, protein and lipid content and membrane fluidity. Brij DRMs were found to be enriched in cholesterol and phospholipids containing saturated fatty acids (especially those from sphingomyelin), features commonly associated to lipid rafts. Nevertheless, TX-100 DRMs presented higher sphingomyelin/phospholipid ratios and lower phosphatidylethanolamine content (a glycerophospholipid mainly present in the inner leaflet) than the Brij's. As for protein solubilization, erythrocyte membranes were more resistant to Brij 98 than to the other two detergents. Flotillin-2 and stomatin were found in both Brij 98 and Brij 8 DRMs. However, flotillin-2 was partially solubilized when DRMs were prepared from cholesterol-depleted erythrocyte membrane. Electron paramagnetic resonance experiments, with doxyl stearic acid spin labels incorporated in the DRMs showed no significant changes in the bilayer compactness of Brij 98 and 58 DRMs in comparison to intact membrane, a unexpected result for lipid domains existing in a liquid-ordered phase, and in contrast to results previously observed with TX-100 DRMs. Altogether, these results show the isolation of DRMs from human erythrocytes treated with Brij 98 and Brij 58 at low (4°C) and physiological temperature (37°C). These detergent-resistant membrane fractions, obtained with Brij 98 and 58 presented some lipid rafts features, although with differences in protein and lipid contents, size and membrane fluidity in comparison to TX-100 DRMs. Besides, these results suggest that TX-100, Brij 98 and Brij 58 induced a different solubilization process on the erythrocyte membraneMestradoBioquimicaMestre em Biologia Funcional e Molecula

X-Ray Characterization of Pharmaceutical and Cosmetic Lipidic Nanoparticles for Cutaneous Application

Starting from the second half of the 1900s, the advent of nanotechnology in medicine has provoked a profound revolution in this area; at present, nanomedicine delivered a remarkably large set of research and clinically useful tools as diagnostic devices, contrast agents, analytical tools, physical therapy applications, and drug-delivery vehicles. Concerning nanoformulations for drug delivery, they are constituted by nanoparticles with dimensions lower than 1 mu m, usually characterized by improved pharmacokinetics, taking advantage of specific targeting. and reduced side effects. The contributors to the present chapter arc reviewing a range of papers related to the structural characterization of nanoformulations by X-ray diffraction techniques. The whole of the considered papers underlines the essential role that biophysical techniques have acquired as an essential prerequisite to understanding stability, bioavailability, and lipid, biopolymer, and drug organization in nanoformulations

Molecular features of nonionic detergents involved in the binding kinetics and solubilization efficiency, as studied in model (Langmuir films) and biological (Erythrocytes) membranes

The effect of the nonionic detergents Brij-98 and Brij-58 over human erythrocytes was studied through quantitative hemolysis and in Langmuir films. Hemolytic tests revealed that Brijs are stronger membrane solubilizers than Triton X-100 (TX-100), with effective detergent/lipid ratios of 0.18 and 0.37 for Brij-98 and Brij-58, respectively. Experiments with Langmuir films provided significant information on the kinetics and thermodynamics of detergent-membrane interaction. The adsorption (k(a)) and desorption (k(d)) rate constants of Brijs were lower than those of TX-100. In the case of k(a), that is probably due to their larger hydrophilic head (with twice (20) the oxyethylene units of TX-100). As for the thermodynamic binding constant, the linear and longer hydrophobic acyl chains of Brijs favor their stabilization in-between the lipids, through London van der Waals forces. Consequently, K-b,K-m values of Brij-98 (12,500 M-1) and Brij-58 (19,300 M-1) resulted higher than TX-100 (7500 M-1), in agreement with results from the hemolytic tests. Furthermore, Brij-58 binds with higher affinity than Brij-98 to bilayers and monolayers, despite its shorter (palmitic) hydrocarbon chain, showing that unsaturation restrains the detergent insertion into these environments. Our results provide significant information about the mechanism of interaction between Brijs and membranes, supporting their distinct solubilization effect. (C) 2018 Elsevier B.V. All rights reserved.Capes (Brazil)MincytSeCyT-Universidad Nacional de CordobaCONICETFoncyt (Argentina)CNPqCAPESUniv Estadual Campinas UNICAMP, Inst Biol, Dept Bioquim Biol & Tecidual, Campinas, SP, BrazilUniv Fed Sao Paulo, Dept Biofis, Sao Paulo, SP, BrazilGeorge Washington Univ, Dept Med, Washington, DC USAUniv Nacl Cordoba, Fac Ciencias Exactas Fis & Nat, Dept Quim, Catedra Quim Biol, Cordoba, ArgentinaUniv Nacl Cordoba, CONICET, Cordoba, ArgentinaUniv Fed Sao Paulo, Dept Biofis, Sao Paulo, SP, BrazilCNPq: 479993/2011-4CNPq: 308621/2013-1Web of Scienc

Bio-ecologia e produção do poliqueta Nereis diversicolor O.F. Müller - recurso zoológico de ambientes costeiros

A presente dissertação teve como objectivo investigar alguns aspectos da biologia e ecologia do poliqueta Nereis diversicolor na costa Sudoeste de Portugal, nomeadamente nas ribeiras de Odeceixe, Aljezur e Carrapateira, durante o período de Abril de 1993 até Maio de 1994, bem como a tentativa de produção deste recurso em pequena escala e em condições controladas. Esta espécie de poliqueta muito comum e frequentemente dominante em substratos móveis de ambientes estuarinos, apresentou algumas flutuações temporais das suas densidades. Estas oscilações estão associadas à própria biologia reprodutora da espécie, mas também às interacções inter- e intra-específicas e à dinâmica sedimentar provocada pela movimentação de massas de água de origem marinha e continental, que interagem e modificam consequentemente as condições ambientais destes sistemas. A produção obtida para N. diversicolor em cada local de amostragem, bem como as taxas de renovação da biomassa (P/B) na costa Sudoeste, situam-se numa posição intermédia entre as encontradas para outros sistemas da costa Atlântica. No entanto a ribeira da Carrapateira apresenta-se como um caso à parte, verificando-se uma produção baixa com um P/B elevado. A fase de maior crescimento, seja em animais selvagens ou de laboratório, foi obtida nos primeiros 12 meses de vida. A partir do segundo ano de idade há uma redução no crescimento somático e um investimento na reprodução, nomeadamente na produção de gâmetas. O estabelecimento do período reprodutivo baseou-se nos resultados da recolha de amostras de estados larvares, bem como da análise do conteúdo celomático dos adultos. Neste estudo registou-se a presença contínua de fêmeas maduras e estados juvenis ao longo do ano, com um evento de recrutamento intenso em Setembro, seguido de um segundo pico de menores proporções em Maio. Na Carrapateira, ao contrário de Odeceixe e Aljezur, foi observado um amadurecimento precoce de N. diversicolor e consequentemente indivíduos de menores dimensões. Em todas as estações a razão sexual estimada foi sempre favorável às fêmeas. O estudo da ecologia alimentar de N. diversicolor revelou hábitos maioritariamente filtradores, embora na estação da Carrapateira esta espécie evidenciou tendências carnívoras e canibais. Não foram encontradas diferenças significativas nos conteúdos digestivos de machos e fêmeas, mas sim uma variação na composição da dieta consoante o local de estudo, época do ano e tamanho dos indivíduos. Através do cultivo de juvenis N. diversicolor alimentados com variadas dietas e em diferentes condições controladas de salinidade, temperatura e luminosidade, pôde constatar-se que esta espécie parece não apresentar uma relação entre as taxas de crescimento e o tipo de dieta fornecida. A análise dos ácidos gordos realizadas aos indivíduos reflectiu o perfil das dietas testadas. Os indivíduos que foram alimentados com dietas ricas em DHA (ácido docosahexaenoico) podem metabolizar apenas 50%, por outro lado, se a quantidade for baixa ou ausente, podem biosintetizar de novo. N. diversicolor retém aproximadamente todo EPA (ácido eicosapentanoico) presente nas dietas, porém, se a quantidade de EPA for baixa, pode biosintetizar de novo. Este facto demonstra que N. diversicolor não necessita DHA em quantidade, contudo, dietas ricas em EPA e DHA permitem uma alta assimilação de HUFA (ácidos-gordos insaturados de cadeia longa) o que é essencial para um bom crescimento e uma alta sobrevivência. Os estudos de cultivo realizados, também demonstraram que N. diversicolor não apresenta diferenças significativas quanto à preferência dos sedimentos testados. Sob condições de salinidade de 15 %o e temperatura de 25°C esta espécie atinge as mais altas taxas de crescimento e uma sobrevivência de 100%, alcançando em poucos meses o "tamanho comercial". Em todas as combinações testadas, os indivíduos atingiram um elevado estado de maturação em somente três meses. Estas condições de cultivo, podem ser aplicadas em futuros estudos para a obtenção intensiva de larvas, no entanto será necessário um estudo pormenorizado acerca da viabilidade destes gâmetas e a certificação de que a maturação extremamente acelerada dos progenitores não tenha sido prejudicial à gametogénese

Development of egg Pc/cholesterol/α-tocopherol liposomes with ionic gradients to deliver ropivacaine

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULORopivacaine (RVC) is an aminoamide local anesthetic widely used in surgical procedures. Studies with RVC encapsulated in liposomes and complexed in cyclodextrins have shown good results, but in order to use RVC for lengthy procedures and during the postoperative period, a still more prolonged anesthetic effect is required. This study therefore aimed to provide extended RVC release and increased upload using modified liposomes. Three types of vesicles were studied: (i) large multilamellar vesicle (LMV), (ii) large multivesicular vesicle (LMVV) and (iii) large unilamellar vesicle (LUV), prepared with egg phosphatidylcholine/cholesterol/α-tocopherol (4:3:0.07 mol%) at pH 7.4. Ionic gradient liposomes (inside: pH 5.5, pH 5.5 + (NH4)2SO4 and pH 7.4 + (NH4)2SO4) were prepared and showed improved RVC loading, compared to conventional liposomes (inside: pH 7.4). An high-performance liquid chromatography analytical method was validated for RVC quantification. The liposomes were characterized in terms of their size, zeta potential, polydispersion, morphology, RVC encapsulation efficiency (EE(%)) and in vitro RVC release. LMVV liposomes provided better performance than LMV or LUV. The best formulations were prepared using pH 5.5 (LMVV 5.5in) or pH 7.4 with 250 mM (NH4)2SO4 in the inner aqueous core (LMVV 7.4in + ammonium sulfate), enabling encapsulation of as much as 2% RVC, with high uptake (EE(%) ∼70%) and sustained release (∼25 h). The encapsulation of RVC in ionic gradient liposomes significantly extended the duration of release of the anesthetic, showing that this strategy could be a viable means of promoting longer-term anesthesia during surgical procedures and during the postoperative period.Ropivacaine (RVC) is an aminoamide local anesthetic widely used in surgical procedures. Studies with RVC encapsulated in liposomes and complexed in cyclodextrins have shown good results, but in order to use RVC for lengthy procedures and during the postoperative period, a still more prolonged anesthetic effect is required. This study therefore aimed to provide extended RVC release and increased upload using modified liposomes. Three types of vesicles were studied: (i) large multilamellar vesicle (LMV), (ii) large multivesicular vesicle (LMVV) and (iii) large unilamellar vesicle (LUV), prepared with egg phosphatidylcholine/cholesterol/α-tocopherol (4:3:0.07 mol%) at pH 7.4. Ionic gradient liposomes (inside: pH 5.5, pH 5.5 + (NH4)2SO4 and pH 7.4 + (NH4)2SO4) were prepared and showed improved RVC loading, compared to conventional liposomes (inside: pH 7.4). An high-performance liquid chromatography analytical method was validated for RVC quantification. The liposomes were characterized in terms of their size, zeta potential, polydispersion, morphology, RVC encapsulation efficiency (EE(%)) and in vitro RVC release. LMVV liposomes provided better performance than LMV or LUV. The best formulations were prepared using pH 5.5 (LMVV 5.5in) or pH 7.4 with 250 mM (NH4)2SO4 in the inner aqueous core (LMVV 7.4in + ammonium sulfate), enabling encapsulation of as much as 2% RVC, with high uptake (EE(%) ∼70%) and sustained release (∼25 h). The encapsulation of RVC in ionic gradient liposomes significantly extended the duration of release of the anesthetic, showing that this strategy could be a viable means of promoting longer-term anesthesia during surgical procedures and during the postoperative period261110FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2011/21735-

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

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)(2)SO4 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. In vitro dialysis experiments showed that the combined liposomal system provided significantly longer (72 h) release of ropivacaine, compared to conventional liposomes (similar to 45 h), or plain ropivacaine (similar to 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 in vitro cytotoxicity and extended anesthesia time1210CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP464708/2014-114/14457-5; 2011/21735-

Annatto Oil Loaded Nanostructured Lipid Carriers: A Potential New Treatment for Cutaneous Leishmaniasis

Annatto (Bixa orellana L.) is extensively used as food pigment worldwide. Recently, several studies have found it to have healing and antioxidant properties, as well as effective action against leishmaniasis. Therefore, the purpose of this study was to incorporate the oil obtained from annatto seeds into a nanostructured lipid carrier (NLC) and evaluate its physicochemical properties and biological activity against Leishmania major. Nanoparticles were prepared by the fusion-emulsification and ultrasonication method, with the components Synperonic™ PE (PL) as the surfactant, cetyl palmitate (CP) or myristyl myristate (MM) as solid lipids, annatto oil (AO) (2% and 4%, w/w) as liquid lipid and active ingredient, and ultra-pure water. Physicochemical and biological characterizations were carried out to describe the NLCs, including particle size, polydispersity index (PDI), and zeta potential (ZP) by dynamic light scattering (DLS), encapsulation efficiency (EE%), thermal behavior, X-ray diffraction (XRD), transmission electron microscopy (TEM), Electron Paramagnetic Resonance (EPR), cytotoxicity on BALB/c 3T3 fibroblasts and immortalized human keratinocyte cells, and anti-leishmaniasis activity in vitro. Nanoparticles presented an average diameter of ~200 nm (confirmed by TEM results), a PDI of less than 0.30, ZP between −12.6 and −31.2 mV, and more than 50% of AO encapsulated in NLCs. Thermal analyses demonstrated that the systems were stable at high temperatures with a decrease in crystalline structure due to the presence of AOs (confirmed by XRD). In vitro, the anti-leishmania test displayed good activity in encapsulating AO against L. major. The results indicate that the oily fraction of Bixa orellana L. in NLC systems should be evaluated as a potential therapeutic agent against leishmaniasis

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