RESUMO
Synthetic access to multiple surface decorations are a bottleneck in the development of liposomes for receptor mediated targeting. This opens a complex multiparameter space, exploration of which is severely limited in terms of sample numbers and turnaround times. Here, we unlock this technological barrier by a combination of a milligram-scale liposome formulation using dual centrifugation and orthogonal click chemistry on the liposomal surface. Application of these techniques to conceptually new amphiphilic compounds, which feature norbornene and alkyne groups at the apex of sterically stabilizing, hyperbranched polyglycerol moieties, revealed a particular influence of the membrane anchor of functional amphiphiles. Folic acid residues clicked to cholesterol-based amphiphiles were inefficient in folate-mediated cell targeting, while dialkyl-anchored amphiphiles remained stable in the liposomal membrane and imparted efficient targeting properties. These findings are of specific importance considering the popularity of cholesterol as a lipophilic anchor.
Assuntos
Colesterol/química , Ácido Fólico/química , Glicerol/química , Lipídeos/química , Lipossomos/química , Polímeros/química , Colesterol/sangue , Química Click , HumanosRESUMO
Herein we report on a liposomal system for siRNA delivery consisting of cholesterol (Chol), distearoylphosphatidylcholine (DSPC), and surfactant TF (1-hydroxy-50-amino-3,4,7,10,13,16,19,22-octaoxa-37,41,45-triaza-pentacontane), a novel spermine derivative (HO-EG8-C12-spermine) which has shown improved siRNA delivery to cells in vitro and in vivo. Predominantly single-walled liposomes with reproducible sizes and moderately broad size distributions were generated with an automated extrusion device. The liposomes remained stable when prepared in the presence of siRNA at N/P ratios of 17-34. However, when mixed with human serum in equal volumes, larger aggregates in the size range of several hundred nanometers were observed by dynamic light scattering. These larger aggregates could potentially limit prolonged in vivo applications. Aggregate formation could be reduced by the addition of a cholesterol-hyperbranched polyglycerol surfactant (hbPG) that sterically shields the liposomal surface against serum induced aggregation. In vitro experiments with murine macrophages utilizing macrophage-specific anti-CD68 siRNA loaded liposomes showed potent and sequence specific reduction of CD68 transcript levels without cytotoxicity. Experiments in mice using intravenous application of CW800 NHS ester labeled liposomes, near-infrared in vivo imaging, and fluorescent assisted cell sorting of inflammatory cells demonstrated an almost quantitative accumulation of these liposomes, with and without hbPG, in the liver and a specific knockdown of CD68 mRNA of up to 70% in liver resident macrophages. It was found that aggregate formation of TF liposomes in serum does not significantly affect in vivo siRNA delivery to these central inflammatory cells of the liver.
Assuntos
Lipossomos/química , Fígado/citologia , Macrófagos/metabolismo , RNA Interferente Pequeno/administração & dosagem , Espermina/química , Tensoativos/química , Animais , Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Células Cultivadas , Colesterol/química , Portadores de Fármacos/química , Citometria de Fluxo , Camundongos , Modelos Teóricos , Tamanho da Partícula , Fosfatidilcolinas/química , RNA Interferente Pequeno/química , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In this study, linear poly(ethylene glycol) (PEG) and novel linear-hyperbranched, amphiphilic polyglycerol (hbPG) polymers with cholesterol (Ch) as a lipid anchor moiety were radiolabeled with fluorine-18 via copper-catalyzed click chemistry. In vivo investigations via positron emission tomography (PET) and ex vivo biodistribution in mice were conducted. A systematic comparison to the liposomal formulations with and without the polymers with respect to their initial pharmacokinetic properties during the first hour was carried out, revealing remarkable differences. Additionally, cholesterol was directly labeled with fluorine-18 and examined likewise. Both polymers, Ch-PEG27-CH2-triazole-TEG-(18)F and Ch-PEG30-hbPG24-CH2-triazole-TEG-(18)F (TEG: triethylene glycol), showed rapid renal excretion, whereas the (18)F-cholesten displayed retention in lung, liver, and spleen. Liposomes containing Ch-PEG27-CH2-triazole-TEG-(18)F revealed a hydrodynamic radius of 46 nm, liposomal Ch-PEG30-hbPG24-CH2-triazole-TEG-(18)F showed a radius of 84 nm and conventional liposomes with (18)F-cholesten 204 nm, respectively. The results revealed fast uptake of the conventional liposomes by liver, spleen, and lung. Most importantly, the novel hbPG-polymer stabilized liposomes showed similar behavior to the PEG-shielded vesicles. Thus, an advantage of multifunctionality is achieved with retained pharmacokinetic properties. The approach expands the scope of polymer tracking in vivo and liposome tracing in mice via PET.
Assuntos
Éteres/química , Lipossomos/química , Polímeros/química , Compostos Radiofarmacêuticos/química , Animais , Colesterol/química , Éteres/farmacocinética , Radioisótopos de Flúor , Marcação por Isótopo , Masculino , Camundongos Endogâmicos C57BL , Micelas , Polímeros/farmacocinética , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
Cholesterol (Ch) linked to a linear-hyperbranched block copolymer composed of poly(ethylene glycol) (PEG) and poly(glycerol) (hbPG) was investigated for its membrane anchoring properties. Two polyether-based linear-hyperbranched block copolymers with and without a covalently attached rhodamine fluorescence label (Rho) were employed (Ch-PEG30-b-hbPG23 and Ch-PEG30-b-hbPG17-Rho). Compression isotherms of co-spread 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with the respective polymers were measured on the Langmuir trough and the morphology development of the liquid-condensed (LC) domains was studied by epi-fluorescence microscopy. LC domains were strongly deformed due to the localization of the polymers at the domain interface, indicating a line activity for both block copolymers. Simultaneously, it was observed that the presence of the fluorescence label significantly influences the domain morphology, the rhodamine labelled polymer showing higher line activity. Adsorption isotherms of the polymers to the water surface or to monolayers of DPPC and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), respectively, were collected. Again the rhodamine labelled polymer showed higher surface activity and a higher affinity for insertion into lipid monolayers, which was negligibly affected when the sub-phase was changed to aqueous sodium chloride solution or phosphate buffer. Calorimetric investigations in bulk confirmed the results found using tensiometry. Confocal laser scanning microscopy (CLSM) of giant unilamellar vesicles (GUVs) also confirmed the polymers' fast adsorption to and insertion into phospholipid membranes.
Assuntos
Colesterol/química , Fosfolipídeos/química , Polímeros/química , Lipossomas Unilamelares/química , Glicerilfosforilcolina/análogos & derivados , Ácido Láctico/química , Bicamadas Lipídicas/química , Microscopia de Fluorescência , Fosfatidilcolinas , Poliésteres , Polietilenoglicóis/química , Rodaminas/químicaRESUMO
To overcome the limited functionality of "stealth" lipids based on linear poly(ethylene glycol) (PEG) chains, hyperbranched polyether-based lipids that bear multiple hydroxyl groups for further chemical modification may be a suitable replacement. This study focuses on the development and characterization of "stealth" liposomes modified with a novel hyperbranched polyglycerol lipid (cholesterol-PEG30-hbPG23). An emphasis was placed on the stability of these liposomes in comparison to those containing a linear PEG derivative (cholesterol-PEG44) directly in human blood serum, characterized via dynamic light scattering (DLS). Polymer lipid contents were varied between 0 and 30 mol %, resulting in liposomes with sizes between 150 and 80 nm in radius, depending on the composition. DLS analysis showed no aggregation inducing interactions between serum components and liposomes containing 10-30 mol % of the hyperbranched lipid. In contrast, liposomes functionalized with comparable amounts of linear PEG exhibited aggregate formation in the size range of 170-330 nm under similar conditions. In addition to DLS, cryo-transmission electron microscopy (TEM) was employed for all liposome samples to prove the formation of unilamellar vesicles. These results demonstrate the outstanding potential of the introduction of hyperbranched polyglycerol into liposomes to stabilize the assemblies against aggregation while providing additional functionalization sites.
Assuntos
Análise Química do Sangue/métodos , Glicerol/química , Lipídeos/química , Lipossomos/sangue , Polímeros/química , Colesterol/química , Humanos , Lipossomos/química , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Estrutura MolecularRESUMO
Polyglycerols (sometimes also called "polyglycidols") represent a class of highly biocompatible and multihydroxy-functional polymers that may be considered as a multifunctional analogue of poly(ethylene glycol) (PEG). Various architectures based on a polyglycerol scaffold are feasible depending on the monomer employed. While polymerization of glycidol leads to hyperbranched polyglycerols, the precisely defined linear analogue is obtained by using suitably protected glycidol as a monomer, followed by removal of the protective group in a postpolymerization step. This review summarizes the properties and synthetic approaches toward linear polyglycerols (linPG), which are at present mainly based on the application of ethoxyethyl glycidyl ether (EEGE) as an acetal-protected glycidol derivative. Particular emphasis is placed on the manifold functionalization strategies including, e.g., the synthesis of end-functional linPGs or multiheterofunctional modifications at the polyether backbone. Potential applications like bioconjugation and utilization as a component in degradable biomaterials or for diagnostics, in which polyglycerol acts as a promising PEG substitute are discussed. In the last section, the important role of linear polyglycerol as a macroinitiator or as a highly hydrophilic segment in block co- or terpolymers is highlighted.
Assuntos
Materiais Biocompatíveis/química , Glicerol/química , Preparações Farmacêuticas/química , Polietilenoglicóis/química , Polímeros/química , Animais , Éteres/química , HumanosRESUMO
Aiming at controlled modification of liposomal surface structures, we describe a postpreparational approach for surface derivatization of a new type of multifunctional, sterically stabilized liposomes. Application of dual centrifugation (DC) resulted in high encapsulation efficiencies above 50% at very small batch sizes with a total volume of 150 µL, which were conductive to fast and efficient optimization of variegated surface modification reactions. Cholesterol-polymer amphiphiles, including complex hyperbranched polyether structures bearing 1-4 terminal alkynes, were used in DC formulations to provide steric stabilization. The alkyne moieties were explored as anchors for the conjugation of small molecules to the liposomal surface via click chemistry, binding 350-450 fluorophores per liposome as examples for surface active molecules. Using Förster resonance energy transfer (FRET) spectroscopy, the conjugation reaction as well as the uptake of FRET-labeled liposomes by RBE4 cells was monitored, and the distribution of the fluorescent lipids among cellular structures and membranes could be studied. Thus, the combination of clickable hyperbranched amphiphiles and dual centrifugation provides access to well-defined liposomal formulations with a variety of surface moieties.
Assuntos
Doxorrubicina/análogos & derivados , Polímeros/farmacologia , Alcinos/química , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Linhagem Celular , Química Click , Doxorrubicina/química , Doxorrubicina/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Transferência Ressonante de Energia de Fluorescência , Lipossomos , Microscopia Confocal , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Polímeros/química , RatosRESUMO
All polymeric chemosensitizers proposed thus far have a linear poly(ethylene glycol) (PEG) hydrophilic block. To testify whether precisely this chemical structure and architecture of the hydrophilic block is a prerequisite for chemosensitization, we tested a series of novel block copolymers containing a hyperbranched polyglycerol segment as a hydrophilic block (PPO-NG copolymers) on multi-drug-resistant (MDR) tumor cells in culture. PPO-NG copolymers inhibited MDR of three cell lines, indicating that the linear PEG can be substituted for a hyperbranched polyglycerol block without loss of the polymers' chemosensitizing activity. The extent of MDR reversal increased with the polymers affinity toward the cells and the expression level of P-glycoprotein. In contrast with Pluronic L61, which increases viability of tumor cells in the absence of drugs, PPO-NG chemosensitizers are completely devoid of this property undesired in cancer therapy, making them promising candidates for application as novel MDR reversal agents.
Assuntos
Antineoplásicos/farmacologia , Glicerol/farmacologia , Polietilenoglicóis/farmacologia , Polímeros/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Células K562 , Células MCF-7 , MicelasRESUMO
Poly(ethylene glycol) (PEG) with acid-sensitive moieties gained attention particularly for various biomedical applications, such as the covalent attachment of PEG (PEGylation) to protein therapeutics, the synthesis of stealth liposomes, and polymeric carriers for low-molecular-weight drugs. Cleavable PEGs are favored over their inert analogues because of superior pharmacodynamic and/or pharmacokinetic properties of their formulations. However, synthetic routes to acetal-containing PEGs published up to date either require enormous efforts or result in ill-defined materials with a lack of control over the molecular weight. Herein, we describe a novel methodology to implement a single acetaldehyde acetal in well-defined (hetero)functional poly(ethylene glycol)s with total control over its position. To underline its general applicability, a diverse set of initiators for the anionic polymerization of ethylene oxide (cholesterol, dibenzylamino ethanol, and poly(ethylene glycol) monomethyl ether (mPEG)) was modified and used to synthesize the analogous labile PEGs. The polyether bearing the cleavable lipid had a degree of polymerization of 46, was amphiphilic and exhibited a critical micelle concentration of 4.20 mg·L(-1). From dibenzylamino ethanol, three heterofunctional PEGs with different molecular weights and labile amino termini were generated. The transformation of the amino functionality into the corresponding squaric acid ester amide demonstrated the accessibility of the cleavable functional group and activated the PEG for protein PEGylation, which was exemplarily shown by the attachment to bovine serum albumin (BSA). Furthermore, turning mPEG into a macroinitiator with a cleavable hydroxyl group granted access to a well-defined poly(ethylene glycol) derivative bearing a single cleavable moiety within its backbone. All the acetal-containing PEGs and PEG/protein conjugates were proven to degrade upon acidic treatment.
Assuntos
Química Farmacêutica , Polietilenoglicóis/química , Polietilenoglicóis/síntese química , Proteínas/química , Acetaldeído/química , Álcoois/química , Colesterol/química , Compostos de Epóxi/química , Polímeros/síntese química , Soroalbumina Bovina/químicaRESUMO
Biocompatible, highly water-soluble, nonionic, amphiphilic block copolymers having different hydrophobic blocks and architectures, but similar molecular size and chemical nature of the hydrophilic blocks, were investigated to check for their ability to form hybrid giant unilamellar vesicles with proteins, and for their interactions with giant unilamellar phospholipid vesicles (GUV). PGM14-b-PPO34-b-PGM14 (PGM-PPO-PGM) consists of a poly(propylene oxide) middle block and outer poly(glycerol monomethacrylate) blocks. Ch-PEG32-b-IPG18 (Ch-PEG-IPG) and Ch-PEG30-b-hbPG17 (Ch-PEG-hbPG) have a linear poly(ethylene glycol) block, linked to a cholesterol end group and to a linear (IPG) or hyperbranched (hbPG) polyglycerol block. Fluorescently-labelled polymers were synthesised to image and analyse the self-assembling and interaction processes using confocal laser scanning microscopy (CLSM). By implementing a novel strategy for polymersomes formation the copolymers were found to spontaneously form giant unilamellar vesicles with proteins in aqueous solution. Furthermore, the investigation of the interaction of the block copolymers with different phospholipid GUVs provided detailed information about the structure-behaviour relationship. Additionally, it was found that these neutral copolymers are able to cross artificial and natural phospholipid membranes.