<p>Physico-chemical characterisation of structure and stability of liposomes intended for drug delivery is the central issue in this thesis. In addition, targeted liposomes to be used in boron neutron capture therapy (BNCT) were developed.</p><p>Lysolipids and fatty acids are products formed upon hydrolysis of PC-lipids. The aggregate structure formed upon mixing lysolipids, fatty acids and EPC were characterised by means of cryo-TEM. A relatively monodisperse population of unilamellar liposomes was detected in mixtures containing equimolar concentration of the three components. </p><p>The interactions between alternative steric stabilisers (PEO-PPO-PEO copolymers) and conventional PC-and pH-sensitive PE-liposomes were investigated. Whereas the PE-liposomes could be stabilised by the PEO-PPO-PEO copolymers, the PC-liposomes showed an enhanced permeability concomitant with the PEO-PPO-PEO adsorption.</p><p>Permeability effects induced by different PEG-stabilisers on EPC liposomes were shown to be dependent on the length of the PEG chain but also on the linkage used to connect the PEG polymer with the hydrophobic membrane anchor.</p><p>An efficient drug delivery requires, in most cases, an accumulation of the drug in the cell cytoplasm. The mechanism behind cytosolic drug delivery from pH-sensitive liposomes was investigated. The results suggest that a destabilisation of the endosome membrane, due to an incorporation of non-lamellar forming lipids, may allow the drug to be released. </p><p>Furthermore, sterically stabilised liposomes intended for targeted BNCT have been characterised and optimised concerning loading and retention of boronated drugs. </p>

<p>The focus of this thesis has been on the effects of segregation in mixtures of amphiphilic molecules. Two different systems were investigated: fluorocarbon-hydrocarbon surfactant mixtures and lipid-surfactant mixtures.</p><p>In fluorocarbon-hydrocarbon surfactant mixtures the repulsive interactions between the chains can lead to a demixing into different types of coexisting micelles, fluorocarbon rich and hydrocarbon rich. From NMR self-diffusion measurements such a demixing was found to occur in the mixture of the partially fluorinated surfactant HFDePC and C₁₆TAC. We furthermore suggested a demixing also within the micelles to explain ¹⁹F-NMR line width data and results from neutron scattering.</p><p>In lipid-surfactant mixtures, a segregation of the molecules may instead be caused by a difference in the preferred curvature of the lipid and the surfactant residing within the same aggregate. Using a surfactant selective electrode, binding isoterms of four different cationic surfactants (C₁₂TAC, C₁₄TAC, C₁₆TAC and HFDePC) to preformed lipid (GMO) vesicles were determined. Perforated vesicles were observed by cryo-TEM in the mixture with C₁₆TAC. To explain the results from the binding isoterms, the formation of pores in the bilayer was regarded as a cooperative process, similar to micelle formation. The surfactant accumulates at the edges of the pores, and increasing the surfactant concentration results in an increased number of pores with a constant surfactant/lipid ratio at the edges.</p><p>The lipid-surfactant mixtures were also studied at the solid/solution interface using AFM. An adsorbed mesh structure, a counterpart to the bulk perforated lamellar phase, was observed for the first time.</p>

Physico-chemical characterisation of structure and stability of liposomes intended for drug delivery is the central issue in this thesis. In addition, targeted liposomes to be used in boron neutron capture therapy (BNCT) were developed. Lysolipids and fatty acids are products formed upon hydrolysis of PC-lipids. The aggregate structure formed upon mixing lysolipids, fatty acids and EPC were characterised by means of cryo-TEM. A relatively monodisperse population of unilamellar liposomes was detected in mixtures containing equimolar concentration of the three components. The interactions between alternative steric stabilisers (PEO-PPO-PEO copolymers) and conventional PC-and pH-sensitive PE-liposomes were investigated. Whereas the PE-liposomes could be stabilised by the PEO-PPO-PEO copolymers, the PC-liposomes showed an enhanced permeability concomitant with the PEO-PPO-PEO adsorption. Permeability effects induced by different PEG-stabilisers on EPC liposomes were shown to be dependent on the length of the PEG chain but also on the linkage used to connect the PEG polymer with the hydrophobic membrane anchor. An efficient drug delivery requires, in most cases, an accumulation of the drug in the cell cytoplasm. The mechanism behind cytosolic drug delivery from pH-sensitive liposomes was investigated. The results suggest that a destabilisation of the endosome membrane, due to an incorporation of non-lamellar forming lipids, may allow the drug to be released. Furthermore, sterically stabilised liposomes intended for targeted BNCT have been characterised and optimised concerning loading and retention of boronated drugs.

The focus of this thesis has been on the effects of segregation in mixtures of amphiphilic molecules. Two different systems were investigated: fluorocarbon-hydrocarbon surfactant mixtures and lipid-surfactant mixtures. In fluorocarbon-hydrocarbon surfactant mixtures the repulsive interactions between the chains can lead to a demixing into different types of coexisting micelles, fluorocarbon rich and hydrocarbon rich. From NMR self-diffusion measurements such a demixing was found to occur in the mixture of the partially fluorinated surfactant HFDePC and C16TAC. We furthermore suggested a demixing also within the micelles to explain 19F-NMR line width data and results from neutron scattering. In lipid-surfactant mixtures, a segregation of the molecules may instead be caused by a difference in the preferred curvature of the lipid and the surfactant residing within the same aggregate. Using a surfactant selective electrode, binding isoterms of four different cationic surfactants (C12TAC, C14TAC, C16TAC and HFDePC) to preformed lipid (GMO) vesicles were determined. Perforated vesicles were observed by cryo-TEM in the mixture with C16TAC. To explain the results from the binding isoterms, the formation of pores in the bilayer was regarded as a cooperative process, similar to micelle formation. The surfactant accumulates at the edges of the pores, and increasing the surfactant concentration results in an increased number of pores with a constant surfactant/lipid ratio at the edges. The lipid-surfactant mixtures were also studied at the solid/solution interface using AFM. An adsorbed mesh structure, a counterpart to the bulk perforated lamellar phase, was observed for the first time.

PEG-lipid stabilized bilayer disks have been found in lipid mixtures containing polyethylene glycol (PEG)-lipids where the combination of a high bending rigidity and low PEG-lipid/lipid miscibility favours disk formation. The disks are planar and circular in shape and their long-term stability is excellent. Theoretical calculations and experimental observations suggest that the micelle forming PEG-lipid are situated at the rim of the aggregate, protecting the hydrophobic lipid chains in the bulk of the aggregate from contact with water. This thesis deals with fundamental aspects concerning the lipid distribution in the disks, as well as with development, optimization, and initial evaluation of the disks as model membranes in partition and interaction studies. Small angle neutron scattering was used to study the partial segregation of components within the bilayer disk. The experiments verified that the PEG-lipids segregate and accumulate at the bilayer disk rim. The proof of component segregation is important from a fundamental point of view and useful, as exemplified in the below-mentioned study of melittin-lipid interaction, when interpreting partition or binding data obtained from studies based on bilayer disks. Today liposomes are often used as model membranes in partition and interaction studies. Using liposomes to predict, e.g., drug partitioning can however have certain drawbacks. In this thesis the disks were proven to be attractive alternatives to liposomes as model membranes in partition studies. The formation of bilayer disks by a technique based on detergent depletion enabled incorporation of a transmembrane protein in the bilayer disks and opened up for the use of disks as model membranes in membrane protein studies. Further, bilayer disks were used in a comparative study focused on the effect of aggregate curvature on the binding of the peptide melittin. Various techniques were used to perform initial evaluations of the bilayer disks as model membranes. Of these, capillary electrophoresis and biosensor-based technology had not been used before in combination with bilayer disks.

There has been growing interest in developing colloidal metal and semiconductor nanocrystals as biomedical imaging contrast agents and therapeutics, since light excitation can cause the nanocrystals to fluoresce or heat up. Recent advances in synthetic chemistry produced fluorescent 2-4 nm diameter silicon and 1-2 nm diaemeter CuInSSe nanocrystals, as well as 16 nm diameter copper selenide (Cu₂₋[subscript x]Se) nanocrystals exhibiting strong absorbance of near infrared light suitable for biomedical applications. However, the syntheses yield nanocrystals that are stabilized by an adsorbed layer of hydrocarbons, making the nanocrystals hydrophobic and non-dispersible in aqueous solution. Encapsulating these nanocrystals in amphiphilic polymer micelles enables the nanocrystals to disperse in water. Subsequently, the Si nanocrystals were injected into tissue to demonstrate fluorescence imaging, the photothermal transduction efficiency of copper selenide nanocrystals was characterized in water, and the copper selenide nanocrystals were used enhance the photothermal destruction of cancer cells in vitro. The polymer-encapsulated copper selenide nanocrystals were found to have higher photothermal transduction efficiency than 140 nm diameter Au nanoshells, which have been widely investigated for photothermal therapy. Combining the optical properties of metal and semiconductor nanocrystals with the drug-carrying capability of lipid vesicles has received attention lately since it may create a nanomaterial capable of performing simultaneous drug delivery, optical contrast enhancement, and photo-induced therapy. Hydrophobic, dodecanethiol-coated Au nanocrystals were dispersed in water with phosphatidylcholine lipids and characterized using cryo transmission electron microscopy. 1.8 nm diameter Au nanocrystals completely load the bilayer of unsaturated lipid vesicles when the vesicles contain residual chloroform, and without chloroform the nanocrystals do not incorporate into the vesicle bilayer. 1.8 nm Au nanocrystals dispersed in water with saturated lipids to form lipid-coated nanocrystal agglomerates, which sometimes adhered to vesicles, and the shape of the agglomerates varied from linear nanocrystal chains, to flat sheets, to spherical clusters as the lipid fatty acid length was increased from 12 to 18 carbons. Including squalene formed lipid-stabilized emulsion droplets which were fully loaded with the Au nanocrystals. Results with 4.1 nm Au and 2-3 nm diameter Si nanocrystals were similar, but these nanocrystals could not completely load the bilayers of unsaturated lipids. / text

O presente trabalho teve por objetivo a avaliação físico-química e biológica de uma fração de saponinas enriquecida, obtida a partir de frutos verdes de Ilex paraguariensis A. St. Hil (mate). A espécie apresenta grande importância econômica para vários países sul-americanos, dentre eles o Brasil. Os frutos verdes da espécie acumulam significativa quantidade de saponinas, entretanto, são considerados subproduto da indústria do beneficiamento das folhas de mate. O extrato liofilizado dos frutos (EX40) foi obtido por turboextração na proporção planta-solvente de 1:10 utilizando como solvente solução hidroalcoólica a 40%. Para análise do extrato bruto foi desenvolvido e validado um método analítico por CLAE-UV, que permitiu: 1) quantificar o marcador químico ilexosídeo II em EX40; 2) estimar o conteúdo em saponinas totais do extrato, tendo sido obtidos valores de 8,2 g% e 47,6 g% respectivamente. Uma fração enriquecida em saponinas, denominada MSF, foi obtida mediante fracionamento em fase sólida a partir de EX40, utilizando resina hidrofóbica poliaromática e gradiente metanol:água de polaridade decrescente. Os maiores teores de saponinas foram obtidos para as frações contendo 70 e 90 % de metanol. A reprodutibilidade do processo foi avaliada por CLAE-UV, considerando o desvio padrão relativo (DPR) entre a área dos dois picos majoritários de MSF produzidas em seis lotes distintos. Valores de DPR de 8,17% para o pico I (ilexosídeo II) e 5,96 % para o pico II (majoritário) foram obtidos. A toxicidade da fração foi avaliada pelo potencial hemolítico in vitro e citotoxicidade sobre cultura de células de mamífero (MDBK ATCC CCL-22). Para atividade hemolítica foi determinado um valor de IC50 de 0,2 g/L (0,22 mM, expresso em ilexosídeo II). O valor de IC50 para citotoxicidade foi 3,8 g/L (4,04 mM, expresso em ilexosídeo II), caracterizando um produto de baixa toxicidade. Dentre as várias atividades biológicas descritas para saponinas, selecionouse avaliar MSF quanto ao seu potencial como imunoadjuvante, assim como a atividade tricomonicida. Para o primeiro caso, MSF foi testada em uma vacina contendo herpesvírus bovino tipo 5 como antígeno viral, nas doses de 100 e 500 μg. Entretanto, o incremento no título de IgG totais, avaliado por ELISA, não foi significativo. A atividade tricomonicida foi comparada aos tensoativos polissorbato 80 e tiloxapol e a uma fração enriquecida em saponinas de Quillaja saponaria. Após 24 h de incubação foi observado para MSF um efeito dose-dependente, atingindo letalidade total dos trofozoítos na concentração de 0,6 g/L. Esse efeito foi superior ao observado para polissorbato 80 e tiloxapol e similar ao de quillaia. O tratamento com metronidazol, administrado tanto após tratamento prévio dos trofozoítos com MSF quanto em associação com essa fração, não revelou qualquer alteração significativa no efeito de ambos os produtos, o que sugere ausência de interação entre MSF e MTZ e a possível existência de mecanismos de ação distintos. A caracterização físico-química enfatizou o estudo do tamanho e forma das micelas, seu comportamento reológico e capacidade de solubilizar compostos hidrofóbicos. A concentração micelar crítica foi determinada em 0,41 g/L utilizando um corante hidrofóbico como marcador. A análise por microscopia eletrônica de transmissão (MET) e CRIO-MET revelou a presença simultânea de micelas alongadas, com tamanho superior a 500 nm, e micelas esféricas de tamanho menor. Para a fração de micelas esféricas, a análise por espalhamento de nêutrons a baixo ângulo permitiu estabelecer diâmetro de 17,9 Å, perfazendo 23,1 e 32,6 % das micelas observadas em soluções de MSF a 0,5 e 1,0 % m/v, respectivamente. O comportamento reológico de MSF nas concentrações de 0,25 a 4,0 %, determinado por reometria dinâmica com controle de deformação, foi do tipo viscoelástico para todas as concentrações. Esse fato corrobora a existência de agregados não-esféricos, filiformes, semelhantes a micelas tipo wormlike. A solubilização micelar de fármacos mediante MSF foi avaliada utilizando como substâncias-modelos os fármacos flurbiprofeno – FLB (ácido fraco) e carbamazepina - CBZ (base fraca), ambos pouco solúveis, e saponinas de quillaia como produto de comparação. Nas concentrações de 0,13 a 1,5 % as saponinas de quillaia foram mais eficientes frente ao flurbiprofeno, enquanto MSF foi capaz de aumentar significativamente a solubilidade da carbamazepina. O incremento na solubilização gerado por MSF foi de 0,0145 e 0,0129 g/L para CBZ e FLB, respectivamente. / The present work aimed the physicochemical and biological evaluation of a saponin enriched fraction from the green fruits of Ilex paraguariensis A. St. Hil. (mate), a species of great economical importance in several Southern American countries, including Brazil. Despite possessing high amounts of saponins, mate green fruits have no commercial use to date and are considered a waste product from the mate leaves processing companies. Mate fruits lyophilized extract (EX40) was produced using turbo-extraction having a 40:60 mixture of water and ethanol as solvent and a 1:10 drug/solvent proportion. An HPLC-UV method was developed and validated aiming at: I) Quantify the EX40 chemical marker, ilexoside II; II) Determine the EX40 total saponin content. The values obtained were of 8.20 wt% and 47.60 wt%, respectively. An enriched saponin fraction (MSF) was obtained from EX40 through a solid phase extraction process within a polyaromatic resin and a decreasing solvent polarity gradient of methanol and water. The higher recovery of mate saponins was achieved with the 70 and 90 % methanol-containing fractions. The process reproducibility was assessed by HPLC-UV through the analysis of the relative standard deviation (RSD) of the two major MSF saponins peak areas obtained in six different batches. RSD values of 8.17 % for peak I (ilexoside II) and 5.96 % for peak II (major peak) were obtained. The MSF toxicity was evaluated according to its haemolytical activity and in vitro cytotoxicity against a mammalian cell culture lineage (MDBK ATCC CCL-22). Values of IC50 of 0.2 g/L (0.22 mM expressed as ilexoside II) and 3.8 g/L (4.04 mM as Ilexoside II) were found for the haemolysis and cytotoxicity respectively. Therefore we could classify MSF as a low toxicity product. Among the several biological activities ascribed to saponins, this work focused on the investigation of MSF immune enhancer potential and its anti-trichomonads vaginalis activity. For the first evaluation, MSF wad added to a bovine herpesvirus 5 vaccine, at 100 and 500 μg doses. However total IGg titres determined by ELISA were not statistically significant. The antitrichomonads activity of MSF was compared a saponin enriched fraction from the species Quillaja saponaria (Quillaja) and to tyloxapol and polysorbate 80. After 24h of incubation MSF presented a dose-dependent activity with total trophozoites lethality at a concentration of 0.6 g/L. The activity was higher than the synthetic surfactants and similar to Quillaja. The treatment with metronidazole (MTZ), peformed in association and after exposing the trophozoites with MSF did not show any synergistic effect of MZT and MSF which suggest an absence of interaction between the compounds and also that they probably have distinct mechanisms of action. The physicochemical evaluation of MSF focused the determination of MSF micelles size and shape, their rheological behaviour and ability to increase the solubility of hydrophobic compounds. MSF critical micellar concentration was determined as 0.41 g/L using a hydrophobic dye as a probe. The transmission electron microscopy (TEM) and CRYO-TEM analysis revealed the occurrence of filiform micelles higher than 500 nm and smaller sizes spherical micelles simultaneously. The spherical micelles radius of 17.9 Å were determined using small angle neutron scattering.and they corresponded to 23.1 and 32.6 % of the total micelles in MSF solutions of 0.5 and 1.0 % w/v, respectively. The rheological behavior of MSF ranging in concentration from 0.25 to 4.0% was determined on a dynamic strain control rheometer and a viscoelastic behavior was observed for all concentrations. These findings corroborate the occurrence of nonspherical micelles very long in size (wormlike). MSF ability to improve the solubilisation of poor aqueous soluble drugs was assessed using carbamazepine (CBZ) and flurbiprofen (FLB) as a model of amphiphilic basic and acidic coumpounds. Saponin enriched fractions were evaluated in concentrations ranging from 0.13 to 1.5 %. Quillaja was more efficient toward the weak acid while MSF was able to increase significantly the CBZ solubility. The increase on CBZ and FLB solubility after MSF addition was of 0.0145 and 0.0129 g/L, respectively.

O presente trabalho teve por objetivo a avaliação físico-química e biológica de uma fração de saponinas enriquecida, obtida a partir de frutos verdes de Ilex paraguariensis A. St. Hil (mate). A espécie apresenta grande importância econômica para vários países sul-americanos, dentre eles o Brasil. Os frutos verdes da espécie acumulam significativa quantidade de saponinas, entretanto, são considerados subproduto da indústria do beneficiamento das folhas de mate. O extrato liofilizado dos frutos (EX40) foi obtido por turboextração na proporção planta-solvente de 1:10 utilizando como solvente solução hidroalcoólica a 40%. Para análise do extrato bruto foi desenvolvido e validado um método analítico por CLAE-UV, que permitiu: 1) quantificar o marcador químico ilexosídeo II em EX40; 2) estimar o conteúdo em saponinas totais do extrato, tendo sido obtidos valores de 8,2 g% e 47,6 g% respectivamente. Uma fração enriquecida em saponinas, denominada MSF, foi obtida mediante fracionamento em fase sólida a partir de EX40, utilizando resina hidrofóbica poliaromática e gradiente metanol:água de polaridade decrescente. Os maiores teores de saponinas foram obtidos para as frações contendo 70 e 90 % de metanol. A reprodutibilidade do processo foi avaliada por CLAE-UV, considerando o desvio padrão relativo (DPR) entre a área dos dois picos majoritários de MSF produzidas em seis lotes distintos. Valores de DPR de 8,17% para o pico I (ilexosídeo II) e 5,96 % para o pico II (majoritário) foram obtidos. A toxicidade da fração foi avaliada pelo potencial hemolítico in vitro e citotoxicidade sobre cultura de células de mamífero (MDBK ATCC CCL-22). Para atividade hemolítica foi determinado um valor de IC50 de 0,2 g/L (0,22 mM, expresso em ilexosídeo II). O valor de IC50 para citotoxicidade foi 3,8 g/L (4,04 mM, expresso em ilexosídeo II), caracterizando um produto de baixa toxicidade. Dentre as várias atividades biológicas descritas para saponinas, selecionouse avaliar MSF quanto ao seu potencial como imunoadjuvante, assim como a atividade tricomonicida. Para o primeiro caso, MSF foi testada em uma vacina contendo herpesvírus bovino tipo 5 como antígeno viral, nas doses de 100 e 500 μg. Entretanto, o incremento no título de IgG totais, avaliado por ELISA, não foi significativo. A atividade tricomonicida foi comparada aos tensoativos polissorbato 80 e tiloxapol e a uma fração enriquecida em saponinas de Quillaja saponaria. Após 24 h de incubação foi observado para MSF um efeito dose-dependente, atingindo letalidade total dos trofozoítos na concentração de 0,6 g/L. Esse efeito foi superior ao observado para polissorbato 80 e tiloxapol e similar ao de quillaia. O tratamento com metronidazol, administrado tanto após tratamento prévio dos trofozoítos com MSF quanto em associação com essa fração, não revelou qualquer alteração significativa no efeito de ambos os produtos, o que sugere ausência de interação entre MSF e MTZ e a possível existência de mecanismos de ação distintos. A caracterização físico-química enfatizou o estudo do tamanho e forma das micelas, seu comportamento reológico e capacidade de solubilizar compostos hidrofóbicos. A concentração micelar crítica foi determinada em 0,41 g/L utilizando um corante hidrofóbico como marcador. A análise por microscopia eletrônica de transmissão (MET) e CRIO-MET revelou a presença simultânea de micelas alongadas, com tamanho superior a 500 nm, e micelas esféricas de tamanho menor. Para a fração de micelas esféricas, a análise por espalhamento de nêutrons a baixo ângulo permitiu estabelecer diâmetro de 17,9 Å, perfazendo 23,1 e 32,6 % das micelas observadas em soluções de MSF a 0,5 e 1,0 % m/v, respectivamente. O comportamento reológico de MSF nas concentrações de 0,25 a 4,0 %, determinado por reometria dinâmica com controle de deformação, foi do tipo viscoelástico para todas as concentrações. Esse fato corrobora a existência de agregados não-esféricos, filiformes, semelhantes a micelas tipo wormlike. A solubilização micelar de fármacos mediante MSF foi avaliada utilizando como substâncias-modelos os fármacos flurbiprofeno – FLB (ácido fraco) e carbamazepina - CBZ (base fraca), ambos pouco solúveis, e saponinas de quillaia como produto de comparação. Nas concentrações de 0,13 a 1,5 % as saponinas de quillaia foram mais eficientes frente ao flurbiprofeno, enquanto MSF foi capaz de aumentar significativamente a solubilidade da carbamazepina. O incremento na solubilização gerado por MSF foi de 0,0145 e 0,0129 g/L para CBZ e FLB, respectivamente. / The present work aimed the physicochemical and biological evaluation of a saponin enriched fraction from the green fruits of Ilex paraguariensis A. St. Hil. (mate), a species of great economical importance in several Southern American countries, including Brazil. Despite possessing high amounts of saponins, mate green fruits have no commercial use to date and are considered a waste product from the mate leaves processing companies. Mate fruits lyophilized extract (EX40) was produced using turbo-extraction having a 40:60 mixture of water and ethanol as solvent and a 1:10 drug/solvent proportion. An HPLC-UV method was developed and validated aiming at: I) Quantify the EX40 chemical marker, ilexoside II; II) Determine the EX40 total saponin content. The values obtained were of 8.20 wt% and 47.60 wt%, respectively. An enriched saponin fraction (MSF) was obtained from EX40 through a solid phase extraction process within a polyaromatic resin and a decreasing solvent polarity gradient of methanol and water. The higher recovery of mate saponins was achieved with the 70 and 90 % methanol-containing fractions. The process reproducibility was assessed by HPLC-UV through the analysis of the relative standard deviation (RSD) of the two major MSF saponins peak areas obtained in six different batches. RSD values of 8.17 % for peak I (ilexoside II) and 5.96 % for peak II (major peak) were obtained. The MSF toxicity was evaluated according to its haemolytical activity and in vitro cytotoxicity against a mammalian cell culture lineage (MDBK ATCC CCL-22). Values of IC50 of 0.2 g/L (0.22 mM expressed as ilexoside II) and 3.8 g/L (4.04 mM as Ilexoside II) were found for the haemolysis and cytotoxicity respectively. Therefore we could classify MSF as a low toxicity product. Among the several biological activities ascribed to saponins, this work focused on the investigation of MSF immune enhancer potential and its anti-trichomonads vaginalis activity. For the first evaluation, MSF wad added to a bovine herpesvirus 5 vaccine, at 100 and 500 μg doses. However total IGg titres determined by ELISA were not statistically significant. The antitrichomonads activity of MSF was compared a saponin enriched fraction from the species Quillaja saponaria (Quillaja) and to tyloxapol and polysorbate 80. After 24h of incubation MSF presented a dose-dependent activity with total trophozoites lethality at a concentration of 0.6 g/L. The activity was higher than the synthetic surfactants and similar to Quillaja. The treatment with metronidazole (MTZ), peformed in association and after exposing the trophozoites with MSF did not show any synergistic effect of MZT and MSF which suggest an absence of interaction between the compounds and also that they probably have distinct mechanisms of action. The physicochemical evaluation of MSF focused the determination of MSF micelles size and shape, their rheological behaviour and ability to increase the solubility of hydrophobic compounds. MSF critical micellar concentration was determined as 0.41 g/L using a hydrophobic dye as a probe. The transmission electron microscopy (TEM) and CRYO-TEM analysis revealed the occurrence of filiform micelles higher than 500 nm and smaller sizes spherical micelles simultaneously. The spherical micelles radius of 17.9 Å were determined using small angle neutron scattering.and they corresponded to 23.1 and 32.6 % of the total micelles in MSF solutions of 0.5 and 1.0 % w/v, respectively. The rheological behavior of MSF ranging in concentration from 0.25 to 4.0% was determined on a dynamic strain control rheometer and a viscoelastic behavior was observed for all concentrations. These findings corroborate the occurrence of nonspherical micelles very long in size (wormlike). MSF ability to improve the solubilisation of poor aqueous soluble drugs was assessed using carbamazepine (CBZ) and flurbiprofen (FLB) as a model of amphiphilic basic and acidic coumpounds. Saponin enriched fractions were evaluated in concentrations ranging from 0.13 to 1.5 %. Quillaja was more efficient toward the weak acid while MSF was able to increase significantly the CBZ solubility. The increase on CBZ and FLB solubility after MSF addition was of 0.0145 and 0.0129 g/L, respectively.

O presente trabalho teve por objetivo a avaliação físico-química e biológica de uma fração de saponinas enriquecida, obtida a partir de frutos verdes de Ilex paraguariensis A. St. Hil (mate). A espécie apresenta grande importância econômica para vários países sul-americanos, dentre eles o Brasil. Os frutos verdes da espécie acumulam significativa quantidade de saponinas, entretanto, são considerados subproduto da indústria do beneficiamento das folhas de mate. O extrato liofilizado dos frutos (EX40) foi obtido por turboextração na proporção planta-solvente de 1:10 utilizando como solvente solução hidroalcoólica a 40%. Para análise do extrato bruto foi desenvolvido e validado um método analítico por CLAE-UV, que permitiu: 1) quantificar o marcador químico ilexosídeo II em EX40; 2) estimar o conteúdo em saponinas totais do extrato, tendo sido obtidos valores de 8,2 g% e 47,6 g% respectivamente. Uma fração enriquecida em saponinas, denominada MSF, foi obtida mediante fracionamento em fase sólida a partir de EX40, utilizando resina hidrofóbica poliaromática e gradiente metanol:água de polaridade decrescente. Os maiores teores de saponinas foram obtidos para as frações contendo 70 e 90 % de metanol. A reprodutibilidade do processo foi avaliada por CLAE-UV, considerando o desvio padrão relativo (DPR) entre a área dos dois picos majoritários de MSF produzidas em seis lotes distintos. Valores de DPR de 8,17% para o pico I (ilexosídeo II) e 5,96 % para o pico II (majoritário) foram obtidos. A toxicidade da fração foi avaliada pelo potencial hemolítico in vitro e citotoxicidade sobre cultura de células de mamífero (MDBK ATCC CCL-22). Para atividade hemolítica foi determinado um valor de IC50 de 0,2 g/L (0,22 mM, expresso em ilexosídeo II). O valor de IC50 para citotoxicidade foi 3,8 g/L (4,04 mM, expresso em ilexosídeo II), caracterizando um produto de baixa toxicidade. Dentre as várias atividades biológicas descritas para saponinas, selecionouse avaliar MSF quanto ao seu potencial como imunoadjuvante, assim como a atividade tricomonicida. Para o primeiro caso, MSF foi testada em uma vacina contendo herpesvírus bovino tipo 5 como antígeno viral, nas doses de 100 e 500 μg. Entretanto, o incremento no título de IgG totais, avaliado por ELISA, não foi significativo. A atividade tricomonicida foi comparada aos tensoativos polissorbato 80 e tiloxapol e a uma fração enriquecida em saponinas de Quillaja saponaria. Após 24 h de incubação foi observado para MSF um efeito dose-dependente, atingindo letalidade total dos trofozoítos na concentração de 0,6 g/L. Esse efeito foi superior ao observado para polissorbato 80 e tiloxapol e similar ao de quillaia. O tratamento com metronidazol, administrado tanto após tratamento prévio dos trofozoítos com MSF quanto em associação com essa fração, não revelou qualquer alteração significativa no efeito de ambos os produtos, o que sugere ausência de interação entre MSF e MTZ e a possível existência de mecanismos de ação distintos. A caracterização físico-química enfatizou o estudo do tamanho e forma das micelas, seu comportamento reológico e capacidade de solubilizar compostos hidrofóbicos. A concentração micelar crítica foi determinada em 0,41 g/L utilizando um corante hidrofóbico como marcador. A análise por microscopia eletrônica de transmissão (MET) e CRIO-MET revelou a presença simultânea de micelas alongadas, com tamanho superior a 500 nm, e micelas esféricas de tamanho menor. Para a fração de micelas esféricas, a análise por espalhamento de nêutrons a baixo ângulo permitiu estabelecer diâmetro de 17,9 Å, perfazendo 23,1 e 32,6 % das micelas observadas em soluções de MSF a 0,5 e 1,0 % m/v, respectivamente. O comportamento reológico de MSF nas concentrações de 0,25 a 4,0 %, determinado por reometria dinâmica com controle de deformação, foi do tipo viscoelástico para todas as concentrações. Esse fato corrobora a existência de agregados não-esféricos, filiformes, semelhantes a micelas tipo wormlike. A solubilização micelar de fármacos mediante MSF foi avaliada utilizando como substâncias-modelos os fármacos flurbiprofeno – FLB (ácido fraco) e carbamazepina - CBZ (base fraca), ambos pouco solúveis, e saponinas de quillaia como produto de comparação. Nas concentrações de 0,13 a 1,5 % as saponinas de quillaia foram mais eficientes frente ao flurbiprofeno, enquanto MSF foi capaz de aumentar significativamente a solubilidade da carbamazepina. O incremento na solubilização gerado por MSF foi de 0,0145 e 0,0129 g/L para CBZ e FLB, respectivamente. / The present work aimed the physicochemical and biological evaluation of a saponin enriched fraction from the green fruits of Ilex paraguariensis A. St. Hil. (mate), a species of great economical importance in several Southern American countries, including Brazil. Despite possessing high amounts of saponins, mate green fruits have no commercial use to date and are considered a waste product from the mate leaves processing companies. Mate fruits lyophilized extract (EX40) was produced using turbo-extraction having a 40:60 mixture of water and ethanol as solvent and a 1:10 drug/solvent proportion. An HPLC-UV method was developed and validated aiming at: I) Quantify the EX40 chemical marker, ilexoside II; II) Determine the EX40 total saponin content. The values obtained were of 8.20 wt% and 47.60 wt%, respectively. An enriched saponin fraction (MSF) was obtained from EX40 through a solid phase extraction process within a polyaromatic resin and a decreasing solvent polarity gradient of methanol and water. The higher recovery of mate saponins was achieved with the 70 and 90 % methanol-containing fractions. The process reproducibility was assessed by HPLC-UV through the analysis of the relative standard deviation (RSD) of the two major MSF saponins peak areas obtained in six different batches. RSD values of 8.17 % for peak I (ilexoside II) and 5.96 % for peak II (major peak) were obtained. The MSF toxicity was evaluated according to its haemolytical activity and in vitro cytotoxicity against a mammalian cell culture lineage (MDBK ATCC CCL-22). Values of IC50 of 0.2 g/L (0.22 mM expressed as ilexoside II) and 3.8 g/L (4.04 mM as Ilexoside II) were found for the haemolysis and cytotoxicity respectively. Therefore we could classify MSF as a low toxicity product. Among the several biological activities ascribed to saponins, this work focused on the investigation of MSF immune enhancer potential and its anti-trichomonads vaginalis activity. For the first evaluation, MSF wad added to a bovine herpesvirus 5 vaccine, at 100 and 500 μg doses. However total IGg titres determined by ELISA were not statistically significant. The antitrichomonads activity of MSF was compared a saponin enriched fraction from the species Quillaja saponaria (Quillaja) and to tyloxapol and polysorbate 80. After 24h of incubation MSF presented a dose-dependent activity with total trophozoites lethality at a concentration of 0.6 g/L. The activity was higher than the synthetic surfactants and similar to Quillaja. The treatment with metronidazole (MTZ), peformed in association and after exposing the trophozoites with MSF did not show any synergistic effect of MZT and MSF which suggest an absence of interaction between the compounds and also that they probably have distinct mechanisms of action. The physicochemical evaluation of MSF focused the determination of MSF micelles size and shape, their rheological behaviour and ability to increase the solubility of hydrophobic compounds. MSF critical micellar concentration was determined as 0.41 g/L using a hydrophobic dye as a probe. The transmission electron microscopy (TEM) and CRYO-TEM analysis revealed the occurrence of filiform micelles higher than 500 nm and smaller sizes spherical micelles simultaneously. The spherical micelles radius of 17.9 Å were determined using small angle neutron scattering.and they corresponded to 23.1 and 32.6 % of the total micelles in MSF solutions of 0.5 and 1.0 % w/v, respectively. The rheological behavior of MSF ranging in concentration from 0.25 to 4.0% was determined on a dynamic strain control rheometer and a viscoelastic behavior was observed for all concentrations. These findings corroborate the occurrence of nonspherical micelles very long in size (wormlike). MSF ability to improve the solubilisation of poor aqueous soluble drugs was assessed using carbamazepine (CBZ) and flurbiprofen (FLB) as a model of amphiphilic basic and acidic coumpounds. Saponin enriched fractions were evaluated in concentrations ranging from 0.13 to 1.5 %. Quillaja was more efficient toward the weak acid while MSF was able to increase significantly the CBZ solubility. The increase on CBZ and FLB solubility after MSF addition was of 0.0145 and 0.0129 g/L, respectively.

The main objective of this study was the formulation of food-grade water-in-oil (W/O) nano-dispersions based mainly on medium or long-chain triglycerides. Two types of dispersions were formulated and structurally compared, namely emulsions and microemulsions. The systems were used as matrices for encapsulating targeted bioactive molecules with specific characteristics such as antioxidants or peptides. The structural characterization of the formulated systems was investigated using techniques such as Electron Paramagnetic Resonance (EPR) spectroscopy, Dynamic Light Scattering (DLS), Cryogenic Transmission Electron Microscopy (Cryo-TEM) and Small Angle Xray Scattering (SAXS). The existence of swollen inverse micelles was revealed for the case of microemulsions whereas larger droplets still at the nano-scale were observed for the case of emulsions. Structural differences in the presence of the bioactive molecules or induced by the alteration of components were also observed. In order to study the efficacy of the formulations, the proposed loaded systems were assessed either using EPR spectroscopy or Well Diffusion Assay (WDA) depending on the bioactive molecule. It was found that the encapsulated molecules retained their claimed characteristics when encapsulated to the proposed matrices. Finally, some of the formulated dispersions were investigated for their behavior under gastrointestinal (GI) conditions. A two-step digestion model using recombinant Dog Gastric Lipase (rDGL) and Porcine Pancreatic Lipase (PPL) was proposed to simulate lipid hydrolysis in humans. The studies revealed significant decrease of the rDGL specific activity in the presence of the microemulsion while in the presence of lower percent of surfactants (case of emulsion) no alterations were observed.