RESUMO
Several methods for the preparation of sterically stabilized immunoliposomes (SIL) have recently been described. This report examines an established method for coupling anti-CD34 My10 mAb to poly(ethylene glycol)-liposomes (PEG-liposomes) containing the anchor pyridyldithiopropionylamino-PEG-phosphatidylethanolamine (PDP-PEG-PE) via a cleavable disulfide bond. Efficient attachment of pyridyldithio-derivatized mAb took place (equivalent to coupling ca. 70% of total input protein) at 2 mol percent of the functionalized PEG-lipid. The My10-SIL bound specifically to CD34+ cells (human leukemic KG-1a and hematopoietic progenitor cells) and the extent of binding was a function of liposomal lipid concentration, the mAb density in the liposome surface and the CD34 cell expression. In mixtures with CD34- cells (CHO or Jurkat), CD34+KG-1a cells were determined by flow cytometry at percentages (1-4%) similar to those reported in clinical samples (such as cord blood, mobilized peripheral blood and bone marrow) using a direct immunostaining with My10-SIL. The disulfide bond was stable in cell culture medium (10% of fetal calf serum) during 8 h and cell-bound SIL can be released from cells by treatment with dithiothreitol as reducing agent under mild conditions (1 h of incubation with 50 mM DTT at 20 degrees C). SIL binding and subsequent dithiothreitol treatment did not influence the cell viability. Our approach should contribute to the development of targetable liposomal vehicles to CD34+ cells for use in ex vivo conditions as sorting of hematopoietic stem cells.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD34/imunologia , Dissulfetos/química , Células-Tronco Hematopoéticas/imunologia , Lipossomos/síntese química , Animais , Anticorpos Monoclonais/química , Reações Antígeno-Anticorpo , Células CHO , Separação Celular/métodos , Sobrevivência Celular , Cricetinae , Reagentes de Ligações Cruzadas , Meios de Cultura , Ditiotreitol , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Humanos , Lipossomos/imunologia , Microscopia Confocal , Polietilenoglicóis/química , Succinimidas , Células Tumorais CultivadasRESUMO
Liposomes containing negatively-charged phospholipid, N-palmitoylphosphatidylethanolamine (NPPE) were examined for stability in the presence of human serum, using the release of the entrapped 5,6-carboxyfluorescein as an aqueous marker. Either small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV) were used. Incorporation of NPPE into PC SUV decreases leakage in the presence of serum or phosphate-buffered saline, no strictly related to size increase observed and to the surface negative charge present. The stabilizing effect of NPPE and Chol were synergistic. Inhibition of destabilization induced by serum of PC/Chol liposomes was observed when NPPE concentrations were above 12 mol%. Change in the membrane fluidity or incorporation of a monosialoganglioside into liposomes do not significantly change the half-life of liposomes in the presence of a high NPPE concentration. Incorporation of NPPE into PC/Chol liposomes increases membrane rigidity which does not change after serum incubation. The presence of NPPE in liposomes decreases lipid transfer/exchange between liposomes and lipoproteins although the same amount of serum proteins were incorporated as in PC/Chol liposomes. As expected, these proteins are accessible to trypsin digestion. In accordance with these results, the liposome agglutination assay shows no steric barrier activity. As a whole, the results obtained in this paper suggest a complex mechanism for stabilization of NPPE containing liposomes in human serum.
Assuntos
Sangue , Lipídeos/análise , Lipossomos/química , Lisofosfolipídeos/farmacologia , Proteínas Sanguíneas/metabolismo , Colesterol/metabolismo , Colesterol/farmacologia , Estabilidade de Medicamentos , Sinergismo Farmacológico , Eletroquímica , Gangliosídeo G(M1)/farmacologia , Gangliosídeos/química , Meia-Vida , Humanos , Lipossomos/metabolismo , Lisofosfolipídeos/química , Lisofosfolipídeos/metabolismo , Fluidez de Membrana , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/farmacologia , Fótons , Análise Espectral , Tripsina/metabolismoRESUMO
Several methodologies for the preparation of polyethylene glycol-grafted immunoliposomes have been developed by attaching antibodies to the terminus of the polymer. Unilamellar liposomes were prepared containing a combination of a functionalized polyethylene glycol(3400) and an inert polyethylene glycol(2000) phosphatidylethanolamine derivate up to 5 mol%. The greater length of the functionalized polyethylene glycol derivate did not alter the liposomal sterical stability or the remote loading of doxorubicin. Anti-CD34 immunoliposomes were prepared by the reaction of maleimide-derivatized My10 antibody with generated thiol groups at the periphery of the liposomes and efficiencies of nearly 100% were obtained. The greater accessibility of the reactive group makes this strategy more efficient than others described. The immunoliposomes prepared bound specifically to CD34+ cells.
Assuntos
Anticorpos Monoclonais/química , Lipossomos/síntese química , Polietilenoglicóis/química , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Antígenos CD34/imunologia , Portadores de Fármacos , Estabilidade de MedicamentosRESUMO
The My-10 monoclonal antibody has facilitated the search of haematopoietic stem cells by recognizing selectively the human CD34 antigen. In the present work, My-10 immunoliposomes directed specifically against CD34+ cells were prepared, characterized and tested in vitro. Binding to target cells at 4 degreesC of immunoliposomes containing carboxyfluorescein as aqueous marker was evaluated by flow cytometry and fluorescence microscopy. These immunoliposomes demonstrated their capacity to bind specifically to CD34+ cells. Studies have shown that 9 antibodies/vesicle were sufficient to obtain a good binding efficiency. The product was stable over one month at 4 degreesC in terms of leakage of encapsulated carboxyfluorescein, particle size and antigen binding capacity.
Assuntos
Anticorpos Monoclonais , Antígenos CD34/análise , Células-Tronco Hematopoéticas/química , Lipossomos , Linhagem Celular , Citometria de Fluxo , Fluoresceínas , Corantes Fluorescentes , Humanos , Microscopia de FluorescênciaRESUMO
The expression of CD34 antigen in acute myelogenous leukemias is considered an unfavourable prognosis marker for response to anticancer drugs and duration of remission. This study investigated the applicability of long-circulating immunoliposomes loaded with doxorubicin targeted to CD34 antigen present on MDR(+) human myelogenous leukemia KG-1a cell line. Immunoliposomal doxorubicin showed a higher cytotoxicity against KG-1a cells than non-targeted liposomal doxorubicin, but it did not improve over that of free drug. Although no reversal of doxorubicin resistance was found to occur through its liposomal encapsulation, a therapeutic benefit can be obtained by the selective cytotoxicity observed. Endocytosis studies demonstrated that, after binding to CD34 antigen, the immunoliposomes are not internalized by the KG-1a cells and so the cytotoxic effect might be due to drug released into the space near the cell membrane. Thus, immunotargeting of liposomal doxorubicin to CD34(+) leukemic cells may only provide an ex vivo strategy for site-selective CD34(+) leukemia cell killing.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Antígenos CD34/imunologia , Doxorrubicina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacocinética , Anticorpos Monoclonais , Cápsulas , Divisão Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Endocitose/efeitos dos fármacos , Citometria de Fluxo , Humanos , Imunoquímica , Leucemia Mieloide Aguda/patologia , Lipossomos , Microscopia Confocal , Tamanho da PartículaRESUMO
Poly(ethylene glycol)-coated liposomes were prepared with two new synthesised pegylated cholesterol (Chol) derivatives linked via carbamate bond. Poly(ethylene glycol) (PEG) was directly linked to Chol (PEG-Chol) or through a space arm of diaminebutane (PEG-L-Chol). In buffer, the physicochemical properties of PC/Chol liposomes (2/1, molar ratio) containing up to 10 mol% of pegylated Chol derivatives did not change significantly and the PEG layer at liposome surface inhibited the agglutination of biotin-liposomes induced by streptavidin. On the other hand, in serum, PEG-L-Chol seemed to reduce the interactions of liposomes with serum proteins, much more than PEG-Chol. The low steric hindrance of PEG-Chol derivative may be due to the slow conformational transition rate of the polymer, since PEG may be deeper located in the membrane. The coupling efficiency of the ligand to the functionalised amino group at the polymer end was also affected, but, its antigen-binding activity was preserved. The basic physical-chemical characteristics studied in this work are relevant to assess the application of pegylated Chol liposomes as drug delivery systems.
Assuntos
Anticorpos Monoclonais/imunologia , Colesterol/análogos & derivados , Colesterol/química , Lipossomos/imunologia , Polietilenoglicóis/química , Anticorpos Monoclonais/química , Reações Antígeno-Anticorpo/efeitos dos fármacos , Antígenos CD34/imunologia , Proteínas Sanguíneas/metabolismo , Colesterol/farmacologia , Reagentes de Ligações Cruzadas/química , Sistemas de Liberação de Medicamentos , Humanos , Lipossomos/química , Lipossomos/metabolismo , Permeabilidade/efeitos dos fármacos , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/farmacologia , Fosfolipídeos/química , Polietilenoglicóis/farmacologia , Ligação Proteica/efeitos dos fármacosRESUMO
The ability of photosensitisers to give reactive oxygenated products is considered decisive for photodynamic applications, but the hydrophobic nature of many porphyrins makes necessary to obtain suitable pharmaceutical formulations. This paper reports the structural photosensitiser features that allow the preparation of stable liposomal formulations. Metallated and non-metallated TPPs and TPyPs and different lipid/porphyrin ratios were considered in order to procure liposomal preparations containing porphyrin concentrations adequate to necessary doses. The results show that the incorporation of porphyrins into liposomes can be related with their ability to form aggregates in a watery media. Thus, ZnTPP, which structural properties avoid the formation of aggregates, was efficiently incorporated into stable liposomes. Moreover, the efficient generation of singlet oxygen by ZnTPP liposomal suspensions has been shown. Because of this, the synthesis of hydrophobic porphyrin derived structures or other sensitisers, which do not aggregate in a watery media and with Q-bands shifted to higher lambda values than ZnTPP, will be efficiently incorporated into liposomes and useful for clinical applications.
Assuntos
Fármacos Fotossensibilizantes/química , Porfirinas/química , Varredura Diferencial de Calorimetria , Estabilidade de Medicamentos , Interações Hidrofóbicas e Hidrofílicas , Lipossomos , Manganês , Tamanho da Partícula , Fármacos Fotossensibilizantes/administração & dosagem , Porfirinas/administração & dosagem , Oxigênio Singlete/química , Espectrometria de Fluorescência , Relação Estrutura-Atividade , ZincoRESUMO
N-Acylphosphatidylethanolamines and N-acylphosphatidylserines have been isolated from mammalian cells and have been associated with some tissue degenerative changes, although the relationship between their synthesis and the uncontrolled sequence of events that ends in irreversible tissue damage is not completely established. Our results show that monovalent and divalent cations induce aggregation and fusion of liposomes constituted by N-palmitoylphosphatidylethanolamine (NPPE) and N-palmitoylphosphatidylserine (NPPS). The effectiveness of cations to induce the aggregation of NPPE and NPPS liposomes is Ca2+ > Mg2+ >> Na+. NPPS liposomes aggregate at lower concentrations of divalent cations than NPPE liposomes, but with sodium NPPE liposomes aggregate to a higher extent than NPPS liposomes. The reaction order for the aggregation processes depends on the lipid and the cation nature and range from 1.04 to 1.64. Dynamic light scattering shows an irreversible increase of the size of the aggregates in the presence of all cations tested. The irreversibility of the aggregation process and the intermixing of bilayer lipids, as studied by resonance energy transfer assay, suggest that fusion, rather than aggregation, occurs. The existence of a real fusion was demonstrated by the coalescence of the aqueous contents of both NPPS and NPPE liposomes in the presence of either monovalent or divalent cations. The different binding sensitivity of Ca2+ to NPPS and NPPE liposomes, determined by zeta potential measurements, agrees with the results obtained in the aggregation and fusion assays. Our results suggest that the synthesis in vivo of N-acylated phospholipids can introduce important changes in membrane-mediated processes.
Assuntos
Membrana Celular/metabolismo , Ácido Palmítico/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Cálcio/metabolismo , Membrana Celular/química , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Cinética , Luz , Lipídeos/química , Lipossomos/química , Magnésio/metabolismo , Fosfatidilserinas/metabolismo , Espalhamento de Radiação , Sódio/metabolismo , Cloreto de Sódio/farmacologia , Succinimidas/metabolismo , Temperatura , Fatores de TempoRESUMO
N-Acylphosphatidylserines have been isolated from intact and injured tissues, but the participation of such acidic phospholipids in membrane aggregation and fusion has not been demonstrated. We have synthesized N-stearoylphosphatidylserine (NSPS) and examined divalent-cation-induced aggregation of NSPS-liposomes, which leads to membrane destabilization and fusion. The purified lipid was characterized by its chromatographic and spectroscopic (infrared and 1H nuclear magnetic resonance) properties and by its chemical degradation pattern. Aggregation of unilamellar NSPS-liposomes was studied as a function of calcium and magnesium concentration. The ability of calcium and magnesium to induce vesicle aggregation is higher for phosphatidylserine (PS)-liposomes (threshold concentration 1.5 mM for calcium and 4.6 mM for magnesium) than for NSPS-liposomes (threshold concentration 2.8 mM for calcium and 6.6 mM for magnesium). The irreversibility of the aggregation reactions after adding EDTA suggests that vesicle fusion might occur in the presence of calcium and magnesium. Preliminary studies, based on mixing of both lipid and internal aqueous contents, show that fusion rather than aggregation of NSPS-liposomes occurs in the presence of calcium ions. The tendency of NSPS-liposomes to aggregate at higher cation concentrations than PS-liposomes suggests that N-acylation of phosphatidylserine protects the membrane against degenerative damage caused by aggregation and fusion.
Assuntos
Fusão de Membrana , Lipídeos de Membrana/química , Fosfatidilserinas/química , Fosfatidilserinas/síntese química , Cátions Bivalentes/farmacologia , Técnicas In Vitro , Lipossomos , Espectroscopia de Ressonância Magnética , Fusão de Membrana/efeitos dos fármacos , Microscopia Eletrônica , Tamanho da Partícula , Fosfatidilserinas/metabolismo , Fosfolipase D/metabolismo , Espectrofotometria Infravermelho , Streptomyces/enzimologiaRESUMO
Immunomagnetic systems have been used for positive selection of a cell fraction from a mixture using appropriate surface markers with satisfactory results, as haematopoietic CD34+ cells. This work reports on the development of poly(ethylene glycol)-grafted (PEG) immunoliposomes loaded with citrate-magnetite stabilized particles as the separation vehicles for immunomagnetic separations. The magnetic ferrofluid was encapsulated into PEG-liposomes by the DRV methodology. The magnetoliposomes had a liposomal size of approximately 450 nm and a Fe/lipid molar ratio of 1.52+/-0.26, and were retained in the magnetic field created by the MiniMACS system. Anti-CD34 immunomagnetoliposomes with 100 mAb/vesicle were prepared by coupling the My10 mAb and bound specifically for CD34+ KG-1a cells in culture and in mixtures with CD34-cells (CHO or Jurkat). The magnetic cell sorting was carried out in cell mixtures KG-1a/CHO or KG-1a/Jurkat with different initial% of CD34+ Kg-1a cells. For 10(6) positive cells and 100 microM of immunomagnetoliposomes, the capture efficiency was > 85% and independent of the starting percentage of CD34+ cells. The decrease of the final purity, when the starting percentage of CD34+ cells decreases and, dependent of the CD34- cell line used, point to the degree of non-specific cell binding of My10-immunomagnetoliposomes as being crucial, among of the methodological aspects as the number of starting CD34+ cells. The CD34+ cells isolated retained the viability with an estimated recovery of 45-50%.