RESUMO
Ex vivo ocular perfused models have been described in the past and were applied in different mammalian species as platforms to test drug delivery systems and surgical techniques. However, reproduction of those methods is challenging because extensive and precise description of the protocols used is lacking. In this technical paper we provide a detailed description of all the steps to be followed from the enucleation of porcine eyes to cannulation of the ophthalmic artery and perfusion. This model can contribute to the reduction of use of living animals in ophthalmology research, whereas as opposed to in vitro models, it preserves tissue complexity and integrity.
Assuntos
Olho/irrigação sanguínea , Artéria Oftálmica , Perfusão/métodos , Vasos Retinianos , Animais , Técnicas In Vitro/métodos , Modelos Animais , SuínosRESUMO
BACKGROUND: Since the early '80s, the pulsed dye laser has been the standard treatment tool for non-invasive port wine stain (PWS) removal. In the last three decades, a considerable amount of research has been conducted to improve clinical outcomes, given that a fraction of PWS patients proved recalcitrant to laser treatment. Whether this research actually led to increased therapeutic efficacy has not been systematically investigated. OBJECTIVE: To analyse therapeutic efficacy in PWS patients globally from 1986 to date. METHODS: PubMed was searched for all available PWS trials. Studies with a quartile percentage improvement scale were included, analysed and plotted chronologically. Treatment and patient characteristics were extracted. A mean clearance per study was calculated and plotted. A 5-study simple moving average was co-plotted to portray the trend in mean clearance over time. The data were separately analysed for multiple treatment sessions in previously untreated patients. RESULTS: Sixty-five studies were included (24.3% of eligible studies) comprising 6207 PWS patients. Of all patients, 21% achieved 75-100% clearance. Although a few studies reported remarkably good outcomes in a subset of carefully selected patients, there was no upward trend over time in mean clearance. CONCLUSION: The efficacy of PWS therapy has not improved in the past decades, despite numerous technical innovations and pharmacological interventions. With an unwavering patient demand for better outcomes, the need for development and implementation of novel therapeutic strategies to clear all PWS is as valid today as it was 30 years ago.
Assuntos
Lasers de Corante/uso terapêutico , Mancha Vinho do Porto/terapia , Humanos , Terapia a Laser/métodos , Terapia a Laser/tendências , Fotoquimioterapia , Resultado do TratamentoRESUMO
Endosomolytic peptides are often coupled to drug delivery systems to enhance endosomal escape, which is crucial for the delivery of macromolecular drugs that are vulnerable to degradation in the endolysosomal pathway. Melittin is a 26 amino acid peptide derived from bee venom that has a very high membranolytic activity. However, such lytic peptides also impose a significant safety risk when applied in vivo as they often have similar activity against red blood cells and other nontarget cell membranes. Our aim is to control the membrane-disrupting capacity of these peptides in time and space by physically constraining them to a nanocarrier surface in such a way that they only become activated when delivered inside acidic endosomes. To this end, a variety of chemical approaches for the coupling of lytic peptides to liposomes via functionalized PEG-lipids were explored, including maleimide-thiol chemistry, click-chemistry, and aldehyde-hydrazide chemistry. The latter enables reversible conjugation via a hydrazone bond, allowing for release of the peptide under endosomal conditions. By carefully choosing the conjugation site and by using a pH activated analog of the melittin peptide, lytic activity toward a model membrane is completely inhibited at physiological pH. At endosomal pH the activity is restored by hydrolysis of the acid-labile hydrazone bond, releasing the peptide in its most active, free form. Furthermore, using an analogue containing a nonhydrolyzable bond as a control, it was shown that the activity observed can be completely attributed to release of the peptide, validating dynamic covalent conjugation as a suitable strategy to maintain safety during circulation.
Assuntos
Preparações de Ação Retardada/metabolismo , Endossomos/metabolismo , Lipossomos/metabolismo , Meliteno/metabolismo , Sequência de Aminoácidos , Química Click , Preparações de Ação Retardada/química , Hidrazonas/química , Hidrazonas/metabolismo , Concentração de Íons de Hidrogênio , Lipossomos/química , Maleimidas/química , Maleimidas/metabolismo , Meliteno/química , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismoRESUMO
Cancer vaccines can be utilized in combination with checkpoint inhibitors to optimally stimulate the anti-tumor immune response. Uptake of vaccine antigen by antigen presenting cells (APCs) is a prerequisite for T cell priming, but often relies on non-specific mechanisms. Here, we have developed a novel vaccination strategy consisting of cancer antigen-containing liposomes conjugated with CD169- or DC-SIGN-specific nanobodies (single domain antibodies) to achieve specific uptake by APCs. Our studies demonstrate efficient nanobody liposome uptake by human and murine CD169+ and DC-SIGN+ APCs in vitro and in vivo when compared to control liposomes or liposomes with natural ligands for CD169 and DC-SIGN. Uptake of CD169 nanobody liposomes resulted in increased T cell activation by human APCs and stimulated naive T cell priming in mouse models. In conclusion, while nanobody liposomes have previously been utilized to direct drugs to tumors, here we show that nanobody liposomes can be applied as vaccination strategy that can be extended to other receptors on APCs in order to elicit a potent immune response against tumor antigens.
Assuntos
Células Apresentadoras de Antígenos , Vacinas Anticâncer , Lipossomos , Camundongos Endogâmicos C57BL , Anticorpos de Domínio Único , Linfócitos T , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/administração & dosagem , Humanos , Linfócitos T/imunologia , Camundongos , Células Apresentadoras de Antígenos/imunologia , Feminino , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/administração & dosagem , Ativação Linfocitária/efeitos dos fármacosRESUMO
Despite being the most widely used biomaterials in orthopedic surgery, metallic implants do not induce new bone growth because they are bioinert. Surface biofunctionalization of implants with immunomodulatory mediators is a recent approach to promote osteogenic factors that facilitate bone regeneration. Liposomes (Lip) can be used as a low-cost, efficient and simple immunomodulator to stimulate immune cells in favor of bone regeneration. Even though liposomal coating systems have been reported previously, their main disadvantage is their limited ability to preserve liposome integrity after drying. In order to address this issue, we developed a hybrid system in which liposomes could be embedded in a polymeric hydrogel namely gelatin methacryloyl (GelMA). Specifically, we have developed a novel versatile coating strategy using electrospray technology to coat implants with GelMA/Liposome without using adhesive intermediate layer. The two differently charged Lip (i.e., anionic and cationic) were blended with GelMA and coated via electrospray technology on the bone-implant surfaces. The results showed that the developed coating withstood mechanical stress during surgical replacement, and Lip inside GelMA coating stayed intact in different storage conditions for a minimum of 4 weeks. Surprisingly, bare Lip, either cationic or anionic, improved the osteogenesis of human Mesenchymal Stem Cells (MSCs) by inducing pro-inflammatory cytokines, even at a low dosage of Lip released from the GelMA coating. More importantly, we showed that the inflammatory response could be fine-tuned by selecting the Lip concentration, Lip/hydrogel ratio, and coating thickness to determine the timing of the release such that we can accommodate different clinical needs. These promising results pave the way to use these Lip coatings to load different types of therapeutic cargo for bone-implant applications.
Assuntos
Regeneração Óssea , Lipossomos , Humanos , Osteogênese , Gelatina , Hidrogéis/farmacologiaRESUMO
Angiogenesis, the sprouting of new blood vessels from the pre-existing vasculature, is a well established target in anti-cancer therapy. It is thought that the Rho GTPase Rac1 is required during vascular endothelial growth factor (VEGF)-mediated angiogenesis. In the present study, we have used a clinically relevant RNA interference approach to silence Rac1 expression. Human umbilical vein endothelial cells were transiently transfected with non-specific control siRNA (siNS) or Rac1 siRNA (siRac1) using electroporation or Lipofectamine 2000. Functional assays with transfected endothelial cells were performed to determine the effect of Rac1 knockdown on angiogenesis in vitro. Silencing of Rac1 inhibited VEGF-mediated tube formation, cell migration, invasion and proliferation. In addition, treatment with Rac1 siRNA inhibited angiogenesis in an in vivo Matrigel plug assay. Intratumoral injections of siRac1 almost completely inhibited the growth of grafted Neuro2a tumors and reduced tumor angiogenesis. Together, these data indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis. Knockdown of Rac1 may represent an attractive approach to inhibit tumor angiogenesis and growth.
Assuntos
Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Análise de Variância , Sequência de Bases , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno , Combinação de Medicamentos , Eletroporação , Humanos , Laminina , Dados de Sequência Molecular , Proteoglicanas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ensaio de Radioimunoprecipitação , Transfecção , Veias Umbilicais/citologia , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Doxorubicin is an anthracycline anticancer agent that is commonly used in the treatment of a variety of cancers, but its application is associated with severe side effects. Biodegradable and thermosensitive polymeric micelles based on poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-p(HPMAmLac(n))) have been studied as drug delivery systems for therapeutic and imaging agents and have shown promising in vitro and in vivo results. The purpose of this study was to investigate the covalent coupling of a doxorubicin-glucuronide prodrug (DOX-propGA3) to the core of mPEG-b-p(HPMAmLac(2)) micelles. This prodrug is specifically activated by human ß-glucuronidase, an enzyme that is overexpressed in necrotic tumor areas. To this end, an azide modified block copolymer (mPEG(5000)-b-p(HPMAmLac(2)-r-AzEMA)) was synthesized and characterized, and DOX-propGA3 was coupled to the polymer via click chemistry with a high (95%) coupling efficiency. Micelles formed by this DOX containing polymer were small (50 nm) and monodisperse and released 40% of the drug payload after 5 days incubation at 37 °C in the presence of ß-glucuronidase, but less than 5% in the absence of the enzyme. In vitro cytotoxicity experiments demonstrated that DOX micelles incubated with 14C cells showed the same cytotoxicity as free DOX only in the presence of ß-glucuronidase, indicating full conversion of the polymer-bound DOX into the parent drug. Overall, this novel system is very promising for enzymatically responsive anticancer therapy.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Metacrilatos/química , Micelas , Polietilenoglicóis/química , Pró-Fármacos/administração & dosagem , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Química Click , Doxorrubicina/química , Doxorrubicina/farmacologia , Humanos , Neoplasias/tratamento farmacológico , Pró-Fármacos/química , Pró-Fármacos/farmacologia , TemperaturaRESUMO
Cancer vaccines aim to efficiently prime cytotoxic CD8+ T cell responses which can be achieved by vaccine targeting to dendritic cells. CD169+ macrophages have been shown to transfer antigen to dendritic cells and could act as an alternative target for cancer vaccines. Here, we evaluated liposomes containing the CD169/Siglec-1 binding ligand, ganglioside GM3, and the non-binding ligand, ganglioside GM1, for their capacity to target antigens to CD169+ macrophages and to induce immune responses. CD169+ macrophages demonstrated specific uptake of GM3 liposomes in vitro and in vivo that was dependent on a functional CD169 receptor. Robust antigen-specific CD8+ and CD4+ T and B cell responses were observed upon intravenous administration of GM3 liposomes containing the model antigen ovalbumin in the presence of adjuvant. Immunization of B16-OVA tumor bearing mice with all liposomes resulted in delayed tumor growth and improved survival. The absence of CD169+ macrophages, functional CD169 molecules, and cross-presenting Batf3-dependent dendritic cells (cDC1s) significantly impaired CD8+ T cell responses, while B cell responses were less affected. In conclusion, we demonstrate that inclusion of GM3 in liposomes enhance immune responses and that splenic CD169+ macrophages and cDC1s are required for induction of CD8+ T cell immunity after liposomal vaccination.
Assuntos
Lipossomos , Linfócitos T , Animais , Linfócitos T CD8-Positivos , Células Dendríticas , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , OvalbuminaRESUMO
Drug targeting systems are nanometre-sized carrier materials designed for improving the biodistribution of systemically applied (chemo)therapeutics. Various different tumour-targeted nanomedicines have been evaluated over the years, and clear evidence is currently available for substantial improvement of the therapeutic index of anticancer agents. Here, we briefly summarise the most important targeting systems and strategies, and discuss recent advances and future directions in the development of tumour-targeted nanomedicines.
Assuntos
Antineoplásicos/uso terapêutico , Sistemas de Liberação de Medicamentos , Nanomedicina , Neoplasias/tratamento farmacológico , Humanos , Lipossomos , PolímerosRESUMO
Vascular disrupting agents (VDAs) are able to affect selectively tumour endothelial cell morphology resulting in vessel occlusion and widespread tumour cell necrosis. However, single-agent antitumour activity of VDAs is typically limited, as tumour regrowth occurs rapidly following drug treatment. To improve the therapeutic effectiveness of VDAs, we investigated liposomal targeting using ZD6126 as a model VDA. ZD6126 is a phosphate-prodrug of the tubulin-binding vascular disrupting agent ZD6126 phenol. ZD6126 was encapsulated into long circulating PEG-liposomes for passive targeting and PEG-liposomes conjugated with peptide ligands containing the RGD-motif for active targeting to alpha(v)-integrins on tumour endothelial cells. ZD6126 could be stably encapsulated, and liposomes displayed minimal leakage in vitro (<10% in 3 weeks). In vivo, upon intravenous injection, free ZD6126 was rapidly converted into ZD6126 phenol, which was cleared from the circulation within minutes. In contrast, ZD6126 encapsulated into either RGD-targeted or PEG liposomes showed prolonged blood circulation times (t(1/2)=10 h), and ZD6126 phenol exposure was also prolonged (t(1/2)=8 h). Both liposomal formulations displayed tumour accumulation plus hepatosplenic uptake by local macrophages. The altered pharmacokinetics and tissue distribution profiles of both liposomal ZD6126 formulations resulted both in single-dose and multiple-dose regimes, in improved therapeutic efficacy in established murine B16.F10 melanomas compared with free ZD6126. The passively and actively targeted liposomes showed equal antitumour efficacy, indicating that delivery of ZD6126 to the tumour tissue may suffice to disrupt tumour blood vessels without the need for specific targeting to the tumour endothelium.
Assuntos
Antineoplásicos/administração & dosagem , Melanoma Experimental/tratamento farmacológico , Compostos Organofosforados/administração & dosagem , Compostos Organofosforados/farmacocinética , Animais , Células Endoteliais/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Lipossomos , Macrófagos/efeitos dos fármacos , Melanoma Experimental/patologia , Camundongos , Neovascularização Patológica/tratamento farmacológico , Distribuição Tecidual , Veias UmbilicaisRESUMO
Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within tubular cells and reduced unwanted systemic effects. In our approach kinase inhibitors are linked to the renal carrier lysozyme using a platinum-based linker that binds drugs via a coordinative linkage. Many kinase inhibitors contain aromatic nitrogen atoms able to bind to this linker without the need of prior derivatization. The resulting drug-lysozyme conjugates are rapidly filtered in the glomerulus into the tubular lumen and subsequently reabsorbed via the endocytic pathway for clearance of low-molecular weight proteins. An important property of the formed conjugates is their in vivo stability and the sustained drug release profile within target cells. This review summarizes the state-of-the-art of drug targeting to the kidney. Furthermore, we will highlight recent results obtained with kinase inhibitor-lysozyme conjugates targeted to different kinases, i.e. the transforming growth factor (TGF)-beta-receptor kinase, p38 MAPkinase and Rho-associated kinase. Both in vitro and in vivo results demonstrated their efficient tubular uptake and beneficial therapeutic effects, superior to treatment with free kinase inhibitors. These proof-of-concept studies clearly indicate the feasibility of drug targeting for improving the renal specificity of kinase inhibitors.
Assuntos
Sistemas de Liberação de Medicamentos , Nefropatias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Fibrose/tratamento farmacológico , Fibrose/patologia , Marcação de Genes/métodos , Humanos , Rim/citologia , Rim/metabolismo , Nefropatias/fisiopatologia , Túbulos Renais/metabolismo , Muramidase/química , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinéticaRESUMO
In the field of nanomedicine, nanoparticles are developed to target antibiotics to sites of bacterial infection thus enabling adequate drug exposure and decrease development of resistant bacteria. In the present study, we investigated the encapsulation of two antibiotics with different polarity into different PEGylated polymeric nanoparticles based on aliphatic polyesters, to obtain a better understanding of critical factors determining encapsulation and release. The nanoparticles were prepared from diblock copolymers comprising of a poly(ethylene glycol) block attached to an aliphatic polyester block of varying polarity: poly(lactic-co-glycolic acid) (mPEG-PLGA), poly(lactic-co-hydroxymethyl glycolic acid) (mPEG-PLHMGA) and poly(lactic-co-benzyloxymethyl glycolic acid) (mPEG-PLBMGA). Hydrophobic bedaquiline and hydrophilic vancomycin were encapsulated via single and double-emulsion solvent evaporation techniques, respectively. Encapsulation, degradation and release studies at physiological simulating conditions were performed. Drug polarity and preparation techniques influenced encapsulation efficiency into polymer nanoparticles, giving almost complete encapsulation of bedaquiline and approx. 30% for vancomycin independent of the polymer type. The nonpolar bedaquiline showed a predominantly diffusion-controlled release independent of polymer composition. However, polar vancomycin was released by a combination of diffusion and polymer degradation, which was significantly affected by polymer composition, the most hydrophilic polymer displaying the fastest release.
Assuntos
Antibacterianos/química , Ácidos Graxos/química , Nanopartículas/química , Poliésteres/química , Polímeros/química , Antibacterianos/farmacocinética , Composição de Medicamentos , Liberação Controlada de Fármacos , Ácidos Graxos/farmacocinética , Nanopartículas/metabolismo , Poliésteres/farmacocinética , Polímeros/farmacocinéticaRESUMO
BACKGROUND: von Willebrand factor (VWF) serves a critical role as a carrier of factor (F)VIII in circulation. While it is generally believed that FVIII and VWF assemble in circulation after secretion from different cells, an alternative view is that cells should exist that co-express FVIII and VWF. OBJECTIVES: In this study, intracellular co-expression of FVIII and VWF was studied, with particular reference to complex assembly and high-affinity interaction. METHODS: Using yellow fluorescent protein-tagged FVIII (FVIII-YFP) and cyan fluorescent protein-tagged VWF (VWF-CFP), we studied intracellular trafficking in human embryonic kidney (HEK293) cells and human umbilical vein endothelial cells (HUVEC). The role of the high-affinity interaction between FVIII and VWF was assessed using a FVIII-YFP variant carrying a Tyr1680Phe substitution, which abolishes high-affinity binding to VWF. Cellular trafficking studies were complemented by binding studies employing purified proteins. RESULTS: Solid phase binding assays employing FVIII-YFP demonstrated that the presence of the fluorescent moiety did not compromise high-affinity binding (K(d) = 0.065 +/- 0.008 nm) whereas the binding of the Tyr1680Phe FVIII-YFP variant was significantly reduced. Co-expression studies in HEK293 cells revealed intracellular co-storage of both FVIII-YFP and Tyr1680Phe FVIII-YFP within VWF-containing storage organelles. In addition, expression of FVIII-YFP and Tyr1680Phe FVIII-YFP in HUVEC demonstrated co-trafficking with endogenous VWF to authentic Weibel-Palade bodies (WPBs). CONCLUSIONS: Our findings demonstrate that FVIII trafficking to WPBs is independent of Tyr1680 and high-affinity binding to VWF. We therefore conclude that the structural requirements that determine intracellular co-trafficking differ from those that determine complex assembly in circulation.
Assuntos
Fator VIII/metabolismo , Corpos de Weibel-Palade/metabolismo , Fator de von Willebrand/metabolismo , Substituição de Aminoácidos , Linhagem Celular , Corantes Fluorescentes , Humanos , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Transdução GenéticaRESUMO
The main constituent of bone is hydroxyapatite (HAP). Since HAP is only present in 'hard' tissues like bone and teeth, it represents a promising target for the selective drug delivery to bone. Due to the exceptional affinity of bisphosphonates (BP) for HAP, cholesteryl-trisoxyethylene-bisphosphonic acid (CHOL-TOE-BP), a new tailor-made BP derivative, was used as bone targeting moiety for liposomes. CHOL-TOE-BP-targeted liposomes were designed for the treatment of bone-related diseases to achieve prolonged local exposure to high concentrations of the bioactive compounds, thereby enhancing therapeutic efficacy and minimizing systemic side effects. The CHOL-TOE-BP-targeted liposomes were characterized regarding particle size and zeta potential. To study the bone targeting potential of these conjugates, an in vitro HAP binding assay was established. The obtained binding data indicate that CHOL-TOE-BP is useful as targeting device for liposomal drug delivery to bone.
Assuntos
Doenças Ósseas/tratamento farmacológico , Osso e Ossos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/farmacocinética , Sítios de Ligação , Ésteres do Colesterol/metabolismo , Difosfonatos/metabolismo , Durapatita/metabolismo , Lipossomos/uso terapêutico , Tamanho da PartículaRESUMO
PURPOSE: The combination of ultrasound and microbubbles can facilitate cellular uptake of (model) drugs via transient permeabilization of the cell membrane. By using fluorescent molecules, this process can be studied conveniently with confocal fluorescence microscopy. This study aimed to investigate the relation between cellular uptake and fluorescence intensity increase of intercalating model drugs. PROCEDURES: SYTOX Green, an intercalating fluorescent dye that displays >500-fold fluorescence enhancement upon binding to nucleic acids, was used as a model drug for ultrasound-induced cellular uptake. SYTOX Green uptake was monitored in high spatiotemporal resolution to qualitatively assess the relation between uptake and fluorescence intensity in individual cells. In addition, the kinetics of fluorescence enhancement were studied as a function of experimental parameters, in particular, laser duty cycle (DC), SYTOX Green concentration and cell line. RESULTS: Ultrasound-induced intracellular SYTOX Green uptake resulted in local fluorescence enhancement, spreading throughout the cell and ultimately accumulating in the nucleus during the 9-min acquisition. The temporal evolution of SYTOX Green fluorescence was substantially influenced by laser duty cycle: continuous laser (100 % DC) induced a 6.4-fold higher photobleaching compared to pulsed laser (3.3 % DC), thus overestimating the fluorescence kinetics. A positive correlation of fluorescence kinetics and SYTOX Green concentration was found, increasing from 0.6 × 10-3 to 2.2 × 10-3 s-1 for 1 and 20 µM, respectively. Finally, C6 cells displayed a 2.4-fold higher fluorescence rate constant than FaDu cells. CONCLUSIONS: These data show that the temporal behavior of intracellular SYTOX Green fluorescence enhancement depends substantially on nuclear accumulation and not just on cellular uptake. In addition, it is strongly influenced by the experimental conditions, such as the laser duty cycle, SYTOX Green concentration, and cell line.
Assuntos
Substâncias Intercalantes/metabolismo , Microbolhas , Microscopia de Fluorescência/métodos , Ultrassom , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Sobrevivência Celular , Humanos , Cinética , Compostos Orgânicos/metabolismo , Fotodegradação , Processamento de Sinais Assistido por ComputadorRESUMO
Liposomes have been investigated extensively as carriers for drugs in attempts to achieve selective deposition and/or reduced toxicity. Liposomes radiolabeled with gamma emitters such as (67)Ga, (111)In and (99m)Tc, can be used for imaging purposes. Liposomes as formulated in the past, are rapidly taken up by cells of the mononuclear phagocyte system (MPS), primarily those located in liver and spleen. The recent development of long-circulating liposomes (LCLs), yielded liposomes that oppose recognition by the MPS. The development of these LCLs with enhanced circulatory half-lives has broadened the potential of liposomes to scintigraphically visualize pathologic processes in vivo. Liposomes have been proposed for tumor imaging, infection imaging and blood pool imaging. Strategies have been developed that allow rapid, easy and efficient labeling of preformed liposomes with (111)In and (99m)Tc. There is now a vast body of preclinical evidence showing that LCLs can be used to image a wide variety of tumors as well as inflammatory lesions. The first studies in patients show that radiolabeled liposomes can image tumor and inflammatory lesions with good sensitivity and good specificity. Here, the present status of liposome-based radiopharmaceuticals for scintigraphic application is reviewed.
Assuntos
Infecções Bacterianas/diagnóstico por imagem , Lipossomos , Neoplasias/diagnóstico por imagem , Compostos Radiofarmacêuticos , Animais , Quelantes , Imagem do Acúmulo Cardíaco de Comporta , Meia-Vida , Humanos , Inflamação/diagnóstico por imagem , Marcação por Isótopo , Lipossomos/farmacocinética , Camundongos , Camundongos Nus , Modelos Animais , CoelhosRESUMO
The effect of changes in lipid composition on the antitumor activity of doxorubicin (DXR)-containing liposomes was studied in immunoglobulin solid immunocytoma-bearing Lou/M Wsl rats. Rats bearing a tumor with a diameter between 20 and 30 mm were treated i.v. with 2 mg/kg free DXR or different DXR-containing liposome types for 5 consecutive days followed by one injection more at day 11 after start of therapy. A similar pattern of tumor regression was observed for free DXR and DXR entrapped in "fluid" liposome types. However, DXR entrapped in "solid" liposome types expressed an antitumor activity which was significantly delayed; during the first 3 days after start of therapy solid DXR-containing liposomes were less effective in inducing antitumor activity than fluid DXR-containing liposomes. In order to gain more insight into the mode of action of DXR-containing liposomes, one of the solid liposome types [composed of distearoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and cholesterol (chol)] was compared with one of the fluid liposome types [composed of egg phosphatidylcholine, phosphatidylserine, and chol] with respect to distribution and integrity in vivo. Results obtained after i.v. administration of [3H]inulin-labeled vesicles to tumor-bearing animals suggested that a differential liposome uptake by the tumor was not relevant for the explanation of the delayed antitumor effect. To monitor the structural integrity of liposomes after i.v. injection, the liposomes were double radiolabeled with [3H]inulin as a marker of the aqueous phase and cholesteryl [14C]oleate as a marker of the lipid phase. The bilayer structure of both liposome types remained intact during their presence in the blood compartment. Intact liposomes were taken up primarily by liver and spleen with subsequent degradation of the liposome structure. The degradation rate appeared to be dependent on the lipid composition of the liposomal membranes; phosphatidylcholine/phosphatidylserine/chol liposomes were degraded much faster than distearoylphosphatidylcholine/dipalmitoylphosphatidylglycerol/chol liposomes. The difference in degradation rate was manifested more clearly in the spleen than in the liver. In vitro investigations on uptake and processing of liposomes by liver macrophages indicated that the difference in degradation rate between liver and spleen was caused by intrahepatic reutilization of [14C]oleate liberated from the liposome structures.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Doxorrubicina/administração & dosagem , Lipossomos/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Animais , Disponibilidade Biológica , Transporte Biológico , Colesterol/administração & dosagem , Ésteres do Colesterol/metabolismo , Lisossomos/metabolismo , Permeabilidade , Fosfolipídeos/administração & dosagem , Ratos , Relação Estrutura-AtividadeRESUMO
Specific binding of immunoliposomes to target tumor cells was investigated in a xenograft model (athymic nude mice) of i.p. growing human ovarian carcinoma (OVCAR-3). For the first time, quantitative evidence is presented that attachment of a tumor-specific antibody (OV-TL 3) dramatically enhances the association of liposomes with i.p. growing OVCAR-3 cells. The OV-TL 3-mediated binding of liposomes to the OVCAR-3 cells was rapid; 30 min after i.p. injection approximately 70% of the injected dose of OV-TL 3 immunoliposomes was associated with the OVCAR-3 cells while for unconjugated liposomes a value of only approximately 3% was obtained. At 2 h after injection, a maximal binding level of 84% was achieved in case of the OV-TL 3 immunoliposomes whereas the binding level of unconjugated liposomes was still about 3%. Twenty-four h after injection about 83% of the injected dose OV-TL 3 immunoliposomes still was associated with the OVCAR-3 cells, compared to about 10% of the injected dose of unconjugated liposomes. Accordingly, unconjugated liposomes disappeared from the peritoneal cavity much faster than the OV-TL 3 immunoliposomes. By comparison with immunoliposomes bearing irrelevant antibody, the specificity of the binding of the OV-TL 3 immunoliposomes to the OVCAR-3 cells was demonstrated. In addition, it was observed that the sustained high OV-TL 3 immunoliposome levels found in the peritoneal cavity are the result of both reduced particle clearance from the peritoneal cavity and the tenacious binding of the immunoliposomes to the tumor cells. Finally, data are presented showing that the degree of binding of OV-TL 3 immunoliposomes to OVCAR-3 cells in vitro and in vivo correlates positively with the antibody (Fab') density on the liposomes.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Neoplasias Ovarianas/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Radioisótopos de Carbono , Linhagem Celular , Colesterol/administração & dosagem , Colesterol/análogos & derivados , Colesterol/farmacocinética , Portadores de Fármacos , Feminino , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Cinética , Lipossomos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Ovarianas/imunologia , Sacarose/administração & dosagem , Sacarose/farmacocinética , Distribuição Tecidual , Transplante Heterólogo , TrítioRESUMO
Preferential localization of liposomes at sites of infection or inflammation has been demonstrated in a variety of experimental models. Most studies report enhanced localization at the target site of poly(ethyelene) glycol (PEG)-coated liposomes as compared to conventional non-coated liposomes. It is generally accepted that the prolonged circulation time of PEG-coated liposomes increases target site exposure, which results in increased target localization. A quantitative relationship between circulation kinetics and localization at the pathological site has not been defined as yet. Besides, an effect of the PEG coating itself has been suggested, as theoretically the PEG coating may facilitate liposome extravasation. In the present study, in a rat model of an acute unilateral Klebsiella pneumoniae pneumonia, circulation kinetics of PEG-coated liposomes were manipulated by incorporation of different amounts of phosphatidylserine (PS) and variation of lipid dose, additionally allowing evaluation of the saturability of the localization process. In addition, this paper addresses the effect of the PEG coating, by comparing the circulation kinetics and target localization of long-circulating 'PEG-free' and PEG-coated liposomes. It is shown that the degree of liposome localization at the target site is positively linearly related to the area under the blood concentration time curve (AUC) of the liposome formulations, irrespective of PEG coating. This finding is discussed in relation to the equation of Kedem and Katchalsky, which describes protein influx into sites of infection or inflammation.
Assuntos
Infecções por Klebsiella/metabolismo , Klebsiella pneumoniae , Lipossomos/farmacocinética , Pneumonia Bacteriana/metabolismo , Polietilenoglicóis , Animais , Feminino , Radioisótopos de Gálio , Infecções por Klebsiella/sangue , Lipossomos/análise , Pulmão/metabolismo , Pulmão/patologia , Tamanho do Órgão , Fosfatidilserinas , Pneumonia Bacteriana/sangue , RatosRESUMO
Intravenous injection of an endotoxin-free solution of poloxamine-908 to rats can enhance the phagocytic clearance capacity of tissue macrophages, particularly those of the liver and the spleen. Such stimulated cells were able to clear a significant portion of intravenously injected methoxypoly(ethyleneglycol)2000 liposomes (mean size of 87 nm), labelled with technetium-99m via the N-hydroxysuccinimidyl hydrazine nicotinate hydrochloride derivative of distearoyl phosphatidylethanolamine, within 4 h post administration. These liposomes, otherwise, exhibit long circulatory behaviour in control animals, with poor localization to the liver and spleen. We suggest that such technetium-99m-labelled engineered vesicles may be of aid for detection of the liver and spleen macrophages with enhanced phagocytic clearance capacity by gamma scintigraphy. Alterations in the phagocytic activity of liver and spleen macrophages is known to occur during cancer. Therefore, such diagnostic procedures may prove useful for patient selection or for monitoring the progress of treatment with long circulating nanoparticles carrying anti-cancer agents, thus minimizing damage to this important line of body's defence cells, and are discussed.