562 resultados para Mda
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Human gene therapy has faced many setbacks due to the immunogenicity and oncogenity of viruses. Safe and efficient alternative gene delivery vehicles are needed to implement gene therapy in clinical practice. Polymeric vectors are an attractive option due to their availability, simple chemistry, and low toxicity and immunogenicity. Our group has previously reported biodegradable polyethylenimines (PEI) that show high transfection efficiency and low toxicity by cross-linking 800 Da PEI with diacrylate cross-linkers using Michael addition. However, the synthesis was difficult to control, inconsistent, and resulted in polymers with a narrow range of molecular weights. In the present work, we utilized a heterogenous PVP(Fe(III)) catalyst to provide a more controllable PEI crosslinking reaction and wider range of biodegradable PEIs. The biodegradable PEIs reported here have molecular weights ranging from 1.2 kDa to 48 kDa, are nontoxic in MDA-MB-231 cells, and show low toxicity in HeLa cells. At their respective optimal polymer:DNA ratios, these biodegradable PEIs demonstrated about 2-5-fold higher transfection efficiency and 2-7-fold higher cellular uptake, compared unmodified 25 kDa PEI. The biodegradable PEIs show similar DNA condensation properties as unmodified PEI but more readily unpackage DNA, based on ethidium bromide exclusion and heparan sulfate competitive displacement assays, which could contribute to their improved transfection efficiency. Overall, the synthesis reported here provides a more robust, controlled reaction to produce cross-linked biodegradable PEIs that show enhanced gene delivery, low toxicity, and high cellular uptake and can potentially be used for future in vivo studies.
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Extreme abiotic factors, such drought combined with heat waves and/or high UVB radiation are predicted to become more frequent in the future. The impact on plant production of these challenges on multipurpose Moringa oleifera L. remains unclear. A susceptibility of this species may lead to increase poverty in endangered regions. M. oleifera is a woody species native from sub-Himalaya regions under high climate stress pressure. The interest on this species is emerging due to its several medicinal properties and its nutritional value. Agropharmaceutical industry is interest in this species too. To understand the impact of increased climate factors, young (2 months old) plants of this species were exposed to water deficit (WD) and UVB (alone or combined). WD and WD+UVB imposition consists of unwater for 4 days. After 1 day withholding water, UVB and WD+UVB were irradiated with 26.3 kJ m-2 distributed per 3 days. Immediately after treatment exposition (1 day) and after 10 days, plant water status, growth, carbon metabolism and oxidative stress were measured. Overall no significant differences were observed in WD, regarding the parameters analysed, except on gas exchanges, MDA and phenols. The plants exposed to UVB showed, in general, more severe effects, as higher pigment content, MDA and membrane permeability, while no changes were observed in the total antioxidant activity. Plants exposed to UVB+WD, despite changes observed, the impact was lower than the one observed in UVB exposed plants, meaning that a protective/adaptive mechanism was developed in the plants under combined stressors. On the other hand, in all treatments the net CO2 assimilation rate decreased. Results suggest that M. oleifera has some tolerance to WD and UVB, and that develops mechanism of adaptation to these two types of stress that often arise in combination under a climate change scenario.
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Os nanomateriais de carbono como o fulereno (C60) apresenta comportamentos bioquímicos distintos, podendo atuar como antioxidante ou pró-oxidante em diferentes sistemas biológicos. Outra evidência ao C60 refere-se a sua característica lipofilica, na qual oferece ação mais direta a diferentes tipos de membranas celulares. Do mesmo modo ácidos graxos poliinsaturados (AGPs) como o ômega-3 (DHA) e o ômega-6 (LA) são importantes para funções celulares da membrana, sendo considerados antioxidantes clássicos. Dessa forma este estudo avaliou em suspensões celulares de cérebro da carpa (Cyprinus carpio, Cyprinidae), o efeito de C60 após um pré-tratamento com DHA ou LA. Para tal avaliação os ensaios consistiram em um pré-tratamento com AGPs (48h) e após exposição a C60 (2h). Como resultados observamos que a viabilidade celular e a capacidade antioxidante total não apresentaram diferença (p> 0.05) entre todos os grupos. Em relação a valores de espécies ativas de oxigênio e dano lipídico foi observado redução nos seus valores nos grupos expostos ao C60 pré – tratados com AGPs (p<0.05). Em termos de cisteína, ocorre uma redução da sua concentração em todos os grupos expostos ao C60. Porém para glutationa a exposição ao C60 provoca um aumento de sua concentração nos grupo controle (sem AGPs) e no grupo pré – tratado com DHA. Dessa forma consideramos que o pré – tratamento com AGPs é benéfico às células, uma vez que um aumento nos níveis de glutationa e uma diminuição na concentração de espécies ativas de oxigênio e peroxidação lipídica foram observados nos grupos expostos ao C60. Sendo assim um bom estado nutritivo em termos da concentração de AGPs foi considerado benéfico na exposição ao fulereno.
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Cellular models are important tools in various research areas related to colorectal biology and associated diseases. Herein, we review the most widely used cell lines and the different techniques to grow them, either as cell monolayer, polarized two-dimensional epithelia on membrane filters, or as three-dimensional spheres in scaffoldfree or matrix-supported culture conditions. Moreover, recent developments, such as gut-on-chip devices or the ex vivo growth of biopsy-derived organoids, are also discussed. We provide an overview on the potential applications but also on the limitations for each of these techniques, while evaluating their contribution to provide more reliable cellular models for research, diagnostic testing, or pharmacological validation related to colon physiology and pathophysiology.
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A chymotrypsin inhibitor was purified from Erythrina velutina seeds by ammonium sulphate fractionation, affinities chromatographies on Trypsin-Sepharose, Quimotrypsin-Sepharose and reversed phase C-18 FPLC/AKTA system. The inhibitor, named EvCI, shown molecular mass of 17 kDa, as determined by SDSPAGE. 2D-PAGE showed four isoinhibitors with pI values of 4,42, 4,63, 4,83 and 5,06, with molecular mass of 17 kDa each. The aminoacid sequence of EvCI was determined by MALDI-TOF-MS and showed a high similarity with other Kunitz-type inhibitor of Erythrina variegata. EvCI competitively inhibited chymotrypsin, with Ki of 4 x10-8 M, but did not inhibited trypsin, pancreatic elastase, bromelain and papain. The inhibitory activity of EvCI was stable over wide pH and temperature ranges. In the presence of DTT 100 mM for 120 min, EvCI lost 50 % of activity. Cytotoxicity was studied in HeLa, MDA, HepG2, K562 and PC3 cells after 72-h incubation period. EvCl inhibited HeLa cells growth with an IC50 value of 50 μg/ml. Subsequent studies in HeLa cells analysis of cell death by annexin V/PI double-staining and cell cycle, using flow cytometry. The results provide evidence for a cytostatic activity of EvCl and support further studies on potential application of this inhibitors as an antiproliferative agent in combined therapy against cervical cancer
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A 140,0 kDa lectin was purified and characterized from the mushroom Clavaria cristata. The purification procedures from the crude extract of the mushroom comprised gel filtration chromatography on Sephacryl s200 and ion exchange on Resource Q column. The purified lectin agglutinated all types of human erythrocytes with preference for trypsinized type O erythrocytes. The haemagglutinating activity is dependent of Ca 2+ ions and was strongly inhibited by the glycoprotein bovine submaxillary mucin (BSM) up to the concentration of 0, 125 mg/mL. The C. cristata lectin (CcL) was stable in the pH range of 2,5-11,5 and termostable up to 80 °C. CcL molecular mass determined by gel filtration on a Superose 6 10 300 column was approximately 140,3 kDa. SDS polyacrilamide gel electrophoresis revealed a single band with a molecular mass of approximately 14,5 kDa, when the lectin was heated at 100 ⁰C in the presence or absence of β-mercaptoethanol. CcL induced activation of murine peritoneal macrophages in vitro resulting in the release of nitric oxide (NO), reaching the maximum production at 24 h. In experimental paw oedema model in mice, CcL showed proinflammatory activity being able to induce oedema formation. Cell viability of HepG2, MDA 435 e 3T3 cell lines was examined after 72 h of incubation with CcL in different concentrations (0,5-50 μg/mL). CcL inhibited HepG2 cells growth with an IC50 value of 50 μg/mL. In the present work, the observed immunomodulatory and antiproliferative effects indicate CcL as a possible immunomodulator compound, interfering in the macrophages immune response, taking possible anti-parasitic, anti-tumoral effects or diagnostic and/or therapeutic
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La muqueuse intestinale est exposée à des agents oxydants provenant de l’ingestion d’aliments modifiés, de cellules immuno-inflammatoires et de la flore intestinale. Une diète élevée en fruits et légumes peut diminuer le stress oxydant (SOx) ainsi que l’inflammation via plusieurs mécanismes. Ces effets bénéfiques peuvent être attribuables à leur contenu élevé en polyphénols. La première étude de mon doctorat consistait à tester l’hypothèse que les polyphénols extraits de pelures de pomme (DAPP) pouvaient diminuer le stress oxydant et l'inflammation impliqués dans les maladies inflammatoires de l'intestin (MII). Nous avons caractérisé les polyphénols des DAPP par spectrométrie de masse (LC-MS) et examiné leur potentiel antioxydant et anti-inflammatoire au niveau des cellules intestinales. L’identification des structures chimiques des polyphénols a été effectuée par LC-MS. Le SOx a été induit par l’ajout du complexe fer/ascorbate (Fe/Asc, 200 µM/2 mM) et l’inflammation par la lipopolysaccharide (LPS, 200µg/mL) à des cellules intestinales Caco-2/15 pré-incubées avec les DAPP (250 µg/mL). L’effet du SOx est déterminé par le dosage du malondialdéhyde (MDA), de la composition des acides gras polyinsaturés et de l’activité des enzymes antioxydantes endogènes (SOD et GPx). L’impact des DAPP sur l’inflammation a été testé par l’analyse de l’expression des marqueurs inflammatoires: cyclooxygénase-2 (COX-2), le facteur de nécrose tumorale alpha (TNF-a et l’interleukine-6 (IL-6) et les facteurs de transcription NF-KB, Nrf-2 et PGC1α par immunobuvardage. Nos données ont montré que les flavonols et les flavan-3-ols constituent les composés polyphénoliques majoritaires des DAPP. L’ajout de Fer2+/Asc a provoqué une augmentation de la peroxidation lipidique comparativement aux cellules contrôles, un appauvrissement des acides gras polyinsaturés n-3 et n-6, et une modulation des enzymes antioxydantes, se traduisant par une augmentation de l’activité de la SOD et une diminution de la GPx. En contrepartie, les DAPP ont exhibé leur potentiel à corriger la plupart des perturbations, y compris l’expression protéique anormalement élevée du COX-2 et la production de la prostaglandine E2 (PGE2), ainsi que l’inflammation telle que réflétée par les facteurs NF-κB, TNF-α et IL-6. Par ailleurs, les mécanismes sous-jacents à ces changements bénéfiques des DAPP ont fait intervenir les facteurs de transcription antioxydants (Nrf-2, PGC1α). Vraisemblablement, cette première étude a permis de démontrer la capacité des DAPP à amoindrir le SOx et à réduire l’inflammation, deux processus étroitement impliqués dans les MII. Dans la deuxième étape de mon doctorat, nous avons voulu comparer les résultats de DAPP à ceux des polyphénols dérivant de la canneberge qui est considérée par la communauté scientifique comme le fruit ayant le plus fort potentiel antioxydant. À cette fin, nous avons caractérisé l’effet des composés polyphénoliques de la canneberge (CPC) sur le SOx, la défense antioxydante et l’inflammation au niveau intestinal tout en définissant leur métabolisme intraluminal. Les différents CPC ont été séparés selon leur poids moléculaire par chromatographie et leurs structures chimiques ont été identifiées par LC-MS. Suite à une pré-incubation des cellules Caco-2/15 avec les extraits CPC (250 µg/mL), le Fe/Asc et la LPS ont été administrés comme inducteurs du SOx et de l’inflammation, respectivement. La caractérisation globale des CPC a révélé que les acides phénoliques composaient majoritairement l’extrait de canneberge de petit poids moléculaire (LC) alors que les flavonoïdes et les procyanidines dimériques/trimériques représentaient l’extrait de poids moléculaire moyen (MC) tout en laissant les procyanidines oligo et polymériques à l’extrait de haut poids moléculaire (HC). Les CPC ont permis de restaurer la plupart des perturbations engendrées dans les Caco-2/15 par le Fe/Asc et le LPS. Les CPC exhibaient le potentiel d’abaisser les niveaux de MDA, de corriger la composition des acides gras polyinsaturés n-3 et n-6, d’augmenter l’activité des enzymes antioxydantes (SOD, GPx et CAT) et d’élever l’expression de Nrf2 et PGC1α. En outre, les CPC pouvaient aussi réduire les niveaux élevés des protéines inflammatoires COX-2, TNF-α et IL-6 ainsi que la production des PGE2 par un mécanisme impliquant le NF-κB. Au niveau mitochondrial, les procyanidines oligomériques ont réussi à corriger les dysfonctions reliées à la production d’énergie (ATP), l’apoptose (Bcl-2, Cyt C et AIF) et le statut des facteurs de transcription mitochondriaux (mtTFA, mtTFB1, mtTFB2). Dans le but de bien comprendre les mécanismes d’action des CPC, nous avons défini par LC-MS les composés polyphénoliques qui ont été transportés ou absorbés par l’entérocyte. Nos analyses soulignent le transport (i) des acides cinnamiques et benzoïques (LC); (ii) la quercétine glycosylée et conjuguée et les procyanidines dimériques de type A (MC); et (iii) l’épicatéchine et les procyanidines oligomériques (HC). Les processus de métabolisation (méthylation, glucuronidation et sulfatation) au niveau de l’entérocyte ont probablement permis le transport de ces CPC surtout sous leur forme conjuguée. Les procyanidines oligomériques ayant un degré de polymérisation supérieur à 2 (HC) ont semblé adhérer aux cellules Caco-2/15. L’épicatéchine suivi par les procyanidines dimériques de type A ont été trouvés majoritaires au niveau des mitochondries. Même si nous ignorons encore l’action biologique de chaque composé polyphénolique, nous pouvons suggérer que leurs effets combinatoires exercent des fonctions antioxydantes, anti-inflammatoires et mitochondriales dans le modèle intestinal Caco-2/15. Dans une troisième étape, nous avons procédé à l’évaluation des aspects préventifs et thérapeutique des DAPP tout en sondant les mécanismes sous-jacents dans une étude préclinique. À cette fin, nous avons exploité le modèle de souris avec colite expérimentale provoquée par le Dextran Sulfate de Sodium (DSS). L’induction de l’inflammation intestinale chez la souris C57BL6 a été effectuée par l’administration orale de DSS à 2.5% pendant 10 jours. Des doses physiologiques et supra-physiologiques de DAPP (200 et 400 mg/kg/j, respectivement) ont été administrées par gavage pendant 10 jours pré- et post-DSS. L’inflammation par le DSS a provoqué une perte de poids, un raccourcissement du côlon, le décollement dystrophique de l’épithélium, l’exulcération et les infiltrations de cellules mono et polynucléaires au niveau du côlon. De plus, le DSS a induit une augmentation de la peroxidation lipidique, une régulation à la baisse des enzymes antioxydantes, une expression protéique à la hausse de la myéloperoxidase (MPO), du COX-2 et de la production des PGE2. Par ailleurs, les DAPP ont permis de corriger ou du moins d’alléger la plupart de ces anomalies en situation préventive ou thérapeutique, en plus d’abaisser l’expression protéique de NF-κB et des cytokines inflammatoires (TNF-a et l’IL-6) tout en stimulant les facteurs de transcription antioxydants (Nrf-2, PGC1α). Conséquemment, les polyphénols des DAPP ont exhibé leur puissant pouvoir antioxydant et anti-inflammatoire au niveau intestinal dans un modèle in vivo. Leurs actions sont associées à la régulation des voies de signalisation cellulaire et des changements dans la composition du microbiote. Ces trois projets de recherche permettent d’envisager l’évaluation des effets préventifs et thérapeutiques des DAPP cliniquement chez les patients avec des désordres inflammatoires de l’intestin.
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Background: Type 2 diabetes mellitus is associated with abnormal markers of inflammatory cytokines and oxidative stress markers. Although, these abnormalities could be modulated with weight reduction; there is limitation in clinical studies that have addressed the beneficial effects of weight reduction in modulating biomarkers of inflammatory cytokines and oxidative stress for obesity associated with type 2 diabetes mellitus. Objective: This study was designed to detect the effects of weight loss on the inflammatory cytokines, oxidative stress markers in obese type 2 diabetic patients. Material and Methods: Eighty obese patients with type 2 diabetes mellitus, their age ranged from 35-57 years and their body mass index ranged from 31-35 kg/m2 were equally assigned into 2 groups: the weight reduction group received aerobic exercises, diet regimen, where as the control group received medical treatment only for 12 weeks. Results: The mean values of body mass index (BMI), tumor necrosis factor–alpha (TNF-α), interleukin-6 (IL-6), C-reactive protein (sCRP), conjugated dienes (CD) and malondialdehyde (MDA) were significantly decreased, while the mean values of glutathione peroxidase (GPx), superoxide dismutase (SOD) and glutathione (GSH) were significantly increased in patients of group (A), while changes were not significant in group (B). Also, there were significant differences between mean levels of the investigated parameters in group (A) and group (B) at the end of the study. Conclusion: Weight loss ameliorates inflammatory cytokines and oxidative stress markers in obese type 2 diabetic patients.
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The dieletric relaxation properties of thermosetting material nanocomposites based on spherosilicate nanoplatforms were studied from room temperature to 170 degrees C, varying the frequency from 10 to 1000 KHz. Permittivity (epsilon'), dielectric loss (epsilon ''), and activation energy (E-a) were calculated. The results of dielectric relaxation were confirmed by those of the final properties. The dielectric loss amplitude decreases with increasing ODPG content until about 70-73 wt % and slightly increases at higher ODPG content. This means that the increasing of the ODPG content in the composite samples decreases the number of pendants groups and/or increases crosslink densitv, causing decreased motion of organic tethers, and subsequently decreasing of the dipolar mobility. The results of apparent activation energy, fracture toughness and tensile modulus mechanical properties show the same profile with respect to ODPG content, in the sense that they exhibit maxima around 70 wt % ODPG. For the ODPG/MDA composites, this formulation of 70 wt % ODPG containing excess of amine is not composition where the highest crosslinked density is reached. This implies that the best mechanical properties and E-a are provided by some degree of chain flexibility. (c) 2007 Wiley Periodicals, Inc.
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The brain is exposed throughout life to oxidative stress, and certain diseases of the brain and nervous system are thought to involve free radical processes and oxidative damage. This study is aimed at evaluating the effect of kolaviron on kolanut-induced oxidative stress in developing rat brain. Twenty-five adult pregnant Wistar rats weighing between 160 and 180g were used for the experiment. They were randomly divided into five groups of five animals each. The animals were fed with standard diets of mice cubes and water provided ad libitum. The control rats received water and cornoil, while the experimental animals received 200 mg/kg body weight of kolanut (kn), 200 mg/kg of kolaviron (kv), and 200 mg/kg body weight of vitamin E which served as a standard antioxidant with cornoil as vehicle orally in pre- and post-natal life. After birth, gross morphometry and behavioural changes of the pups of days 1, 7, 14, 21 and 28 postpartum were evaluated. Blood samples were collected from pups of day 21 for hematological, liver and renal function analyses, while the brains of pups of day 21 postpartum were preserved in phosphate buffer at a temperature of 4oC and pH 7.4 for biochemical analysis. There were significant alterations in the gross morphometry and behavioural parameters studied in the treated animals compared with the control at p< 0.05. There were elevated levels of RBC, WBC and platelets in the treated group compared with the control at p< 0.05. However, no significant change was observed in the PCV, Hb, liver and renal function parameters studied at p>0.05. A non-significant increase in levels of malondialdehyde, MDA, a bye-product of lipid peroxidation in the kolanut group was observed. However, administration of kolaviron and vitamin E non-significantly (p>0.05) reversed these changes. In conclusion, maternal consumption of kolanut induced mild oxidative stress and the administration of kolaviron and vitamin E decreased the rate at which kolanut induced oxidative stress in developing rat brain.
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253 p.
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Dissertação de Mestrado, Engenharia Informática, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014
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Dissertação de Mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014
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Purpose: To investigate the effect of ampicillin on rat intestinal microflora and liver in the presence of high carbohydrate and protein diets. Methods: Male Wistar albino rats were divided into four groups. The first group served as the control, the second group was treated with ampicillin (50 mg/kg for 3 weeks) and fed with a standard diet, while the third and fourth groups were treated with the same dose of ampicillin and fed with acarbohydrateand protein-rich diets, respectively, to observe the effect of diet on gut flora and liver. Fecal specimens were collected and used for qualitative determination of gut microbiota composition. Serum hepatospecific markers (AST, ALT and ALP) were estimated. The antioxidant status of liver tissues was estimated for GSH, MDA, GST, LDH and vitamin C l, in addition to sodium and potassium. Results: Administration of orogastric dose of ampicillin for 3 weeks induced inhibition of E.coli, yeasts, total anaerobes, and anaerobic lactobacilli with new growth of P. vulgaris and K. pneumonia. The levels of serum AST, ALT and ALP showed significant (p ˂ 0.05) increase to 163, 112.38 and 115.35 %, respectively in ampicillin-treated animals, compared to control. Also significant (p ˂ 0.05) increase in lipid peroxidation (120 %) and LDH (111 %) coupled with significant (p ˂ 0.05) decrease in glutathione (74.57 %), vitamin C (63.49 %) and glutathione S-transferase (41.51 %) were observed in ampicillintreated groups. No significant variation (p ˂ 0.05) in sodium and potassium levels were found between control and the treated group after 3 weeks of treatment. Conclusion: These results confirm that extended ampicillin therapy disrupts gut flora, which results in liver injury; hence, overuse of antibiotics should be avoid.
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Purpose: To develop liposome formulations containing monoclonal antibody anti-HER2 (MabHer2), and Paclitaxel (PTX). Methods: Seven different liposomal systems containing PTX, or MabHer2 or a combination of PTX and MabHer2 were made using lipid film hydration technique and sonication. The effects of liposome preparation conditions and extraction methods on antibody structure were investigated by polyacrylamide gel electrophoresis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The characteristics of the liposomes were determined by a zetasizer, while drug-loading efficiency was evaluated by high-performance liquid chromatography. The cytotoxic effect of the liposome formulations was evaluated on MDA-MB-453 (HER2+) and MCF-7 (HER2-) breast cancer cell lines by MTT assay. Results: The antibody was not significantly affected by the stress conditions and the method of extraction. The particle size of liposomes was < 200 nm while the amount of incorporated PTX was 97.6 % for liposome without cationic agent and 98.2 % for those with cationic agent. Recovery of MabHer2 was 94.38 % after extraction. Combined PTX/MabHer2 liposome was more toxic on HER2 overexpressing positive MDA-MB-453 cell line than PTX-loaded liposomes and MabHer2. MabHer2 and combined PTX/MabHer2 liposomes showed no toxic effects on HER2 overexpressing negative MCF-7 cells relative to cationic PTX-loaded liposomes. Conclusions: This results obtained show that PTX can be encapsulated successfully into liposoma systems and that owing to Her2 specific antibody, these systems can be delivered directly to the target cell.