RESUMEN
Encapsulating chemotherapeutic drugs like doxorubicin (DOX) inside lipid nanoparticles (LNPs) can overcome their acute, systematic toxicity. However, a precise drug release at the tumor microenvironment for improving the maximum tolerated dose and reducing side effects has yet to be well-established by implementing a safe stimuli-responsive strategy. This study proposes an integrated nanoscale perforation to trigger DOX release from hybrid plasmonic multilamellar LNPs composed of 5 nm gold (Au) NPs clustered at the internal lamellae interfaces. To promote site-specific DOX release, a single pulse irradiation strategy is developed by taking advantage of the resonant interaction between nanosecond pulsed laser radiation (527 nm) and the plasmon mode of the hybrid nanocarriers. This approach enlarges the amount of DOX in the target cells up to 11-fold compared to conventional DOX-loaded LNPs, leading to significant cancer cell death. The simulation of the pulsed laser interactions of the hybrid nanocarriers suggests a release mechanism mediated by either explosive vaporization of thin water layers adjacent to AuNP clusters or thermo-mechanical decomposition of overheated lipid layers. This simulation indicates an intact DOX integrity following irradiation since the temperature distribution is highly localized around AuNP clusters and highlights a controlled light-triggered drug delivery system.
Asunto(s)
Antineoplásicos , Nanopartículas del Metal , Nanopartículas , Oro , Portadores de Fármacos , Antineoplásicos/farmacología , Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos , Rayos LáserRESUMEN
Liposomes, which consist of bilayer lipids surrounding interior aqueous compartment(s), were first characterized nearly 60 years ago. Remarkably, many fundamental properties of liposomes and their micellar-like "solid core" counterparts (a lipid monolayer surrounding a hydrophobic core) and transitions between these structures remain poorly understood. In this work, we examine the effects of basic variables on the morphology adopted by lipid-based systems produced by rapid mixing of lipids in ethanol with aqueous media. We show that, for lipids such as distearolyphosphatidylcholine (DSPC)-cholesterol mixtures that form bilayer vesicles on hydration, osmotic stress can induce regions of high positive membrane curvature, leading to fusion between unilamellar vesicles to produce bilamellar vesicles. Addition of lyso PC, an "inverted cone"-shaped lipid that supports regions of high positive curvature, can inhibit the formation of these bilamellar vesicles by stabilizing a hemifused intermediate structure. Conversely, the presence of "cone"-shaped lipids such as dioleoylphosphatidylethanolamine (DOPE) that results in negative membrane curvature promotes fusion events subsequent to vesicle formation (during the ethanol dialysis stage), leading to bilamellar and multilamellar systems even in the absence of osmotic stress. Alternatively, the presence of increasing amounts of triolein, a lipid that is insoluble in lipid bilayers, results in increasing internal solid core structures until micellar-like systems with a hydrophobic core of triolein are achieved. These results are interpreted in terms of the intrinsic membrane curvature that bilayer vesicles can stably maintain as well as the ability of bilayer lipids to first form a monolayer around a solid core of hydrophobic material such as triolein and then, as the proportion of bilayer lipids is increased, progressively form bilayer structures that can eventually form a complete bilayer encapsulating both a hydrophobic core and an aqueous compartment. These hybrid intermediate structures may have utility as novel drug delivery systems.
Asunto(s)
Liposomas , Trioleína , Liposomas/química , Membrana Dobles de Lípidos/química , MicelasRESUMEN
Hybrid lipid nanoparticles containing gold nanoparticles (LNP-GNPs) and drugs have potential for imaging applications as well as triggered release of LNP contents in response to pulsed laser or X-ray radiation mediated by the GNPs. However, methods to synthesize LNP-GNP systems that efficiently entrap GNPs (the potential triggered release and imaging agent) and then load and retain the drug cargo in a manner that may have clinical applications have proven elusive. Here, we develop a straightforward "bottom-up" approach to manufacture drug-loaded LNP-GNP systems. We show that negatively charged GNPs of 5 nm diameter can be stably loaded into LNPs containing 10 mol % ionizable cationic lipid using an ethanol dilution, rapid mixing approach and that these systems also exhibit aqueous compartments. Further, we show that such systems can also entrap ammonium sulfate, enabling pH-dependent loading of the weak base anti-cancer drug doxorubicin into the aqueous compartments. Cryo-transmission electron microscopy (Cryo-TEM) imaging clearly demonstrates the presence of GNPs in the interior of the resulting hybrid nanostructures as well as the formation of electron-dense drug precipitates in the aqueous core of the LNP-GNPs. The approach described here is a robust and straightforward method to generate hybrid LNP-GNP-drug and other LNP-metal nanoparticle-drug systems with potential applications for a variety of triggered release protocols.
Asunto(s)
Nanopartículas del Metal , Nanopartículas , Doxorrubicina/química , Oro/química , Liposomas/química , Nanopartículas del Metal/química , Nanopartículas/químicaRESUMEN
Lipid nanoparticles (LNPs) are playing a leading role in enabling clinical applications of gene therapies based on DNA or RNA polymers. One factor impeding clinical acceptance of LNP therapeutics is that LNP formulations of nucleic acid polymers can be immunostimulatory, necessitating co-administration of potent corticosteroid immunosuppressive agents. Here, we describe the development of hydrophobic prodrugs of a potent corticosteroid, dexamethasone, that can be readily incorporated into LNP systems. We show that the presence of the dexamethasone prodrug LD003 effectively suppresses production of cytokines such as KC-GRO, TNFα, IL-1ß and IL-6 following intravenous administration of LNP loaded with immune stimulatory oligodeoxynucleotides containing cytosine-guanine dinucleotide motifs. Remarkably, LD003 dose levels corresponding to 0.5â¯mg/kg dexamethasone achieve a greater immunosuppressive effect than doses of 20â¯mg/kg of free dexamethasone. Similar immunosuppressive effects are observed for subcutaneously administered LNP-siRNA. Further, the incorporation of low levels of LD003 in LNP containing unmodified mRNA or plasmid DNA significantly reduced pro-inflammatory cytokine levels following intravenous administration. Our results suggest that incorporation of hydrophobic prodrugs such as LD003 into LNP systems could provide a convenient method for avoiding the immunostimulatory consequences of systemic administration of genetic drug formulations.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Dexametasona/administración & dosificación , Portadores de Fármacos/química , Lípidos/química , Oligodesoxirribonucleótidos/administración & dosificación , Profármacos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Animales , Dexametasona/farmacología , Femenino , Células HeLa , Humanos , Ratones Endogámicos C57BL , Nanopartículas/química , Ácidos Nucleicos/administración & dosificación , Ácidos Nucleicos/farmacología , Oligodesoxirribonucleótidos/farmacología , Profármacos/farmacologíaRESUMEN
Previous studies from this group have shown that limit size lipid-based systems--defined as the smallest achievable aggregates compatible with the packing properties of their molecular constituents--can be efficiently produced using rapid microfluidic mixing technique. In this work, it is shown that similar procedures can be employed for the production of homogeneously sized unilamellar vesicular systems of 30-40 nm size range. These vesicles can be remotely loaded with the protonable drug doxorubicin and exhibit adequate drug retention properties in vitro and in vivo. In particular, it is demonstrated that whereas sub-40 nm lipid nanoparticle (LNP) systems consisting entirely of long-chain saturated phosphatidylcholines cannot be produced, the presence of such lipids may have a beneficial effect on the retention properties of limit size systems consisting of mixed lipid components. Specifically, a 33-nm diameter doxorubicin-loaded LNP system composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1,2-dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and PEGylated lipid (DSPE-PEG2000) demonstrated adequate, stable drug retention in the circulation, with a half-life for drug release of â¼ 12 h. These results indicate that microfluidic mixing is the technique of choice for the production of bilayer LNP systems with sizes less than 50 nm that could lead to development of a novel class of ultra-small drug delivery vehicles.
Asunto(s)
Sistemas de Liberación de Medicamentos , Liposomas/química , Nanopartículas/química , Animales , Portadores de Fármacos/química , Lípidos/sangre , Lípidos/química , Liposomas/sangre , Ratones , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Lipid nanoparticles (LNP) containing ionizable cationic lipids are the leading systems for enabling therapeutic applications of siRNA; however, the structure of these systems has not been defined. Here we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an ionizable cationic lipid), phospholipid, cholesterol and a polyethylene glycol (PEG) lipid formed using a rapid microfluidic mixing process. Techniques employed include cryo-transmission electron microscopy, (31)P NMR, membrane fusion assays, density measurements, and molecular modeling. The experimental results indicate that these LNP siRNA systems have an interior lipid core containing siRNA duplexes complexed to cationic lipid and that the interior core also contains phospholipid and cholesterol. Consistent with experimental observations, molecular modeling calculations indicate that the interior of LNP siRNA systems exhibits a periodic structure of aqueous compartments, where some compartments contain siRNA. It is concluded that LNP siRNA systems formulated by rapid mixing of an ethanol solution of lipid with an aqueous medium containing siRNA exhibit a nanostructured core. The results give insight into the mechanism whereby LNP siRNA systems are formed, providing an understanding of the high encapsulation efficiencies that can be achieved and information on methods of constructing more sophisticated LNP systems.
RESUMEN
Limit size systems are defined as the smallest achievable aggregates compatible with the packing of the molecular constituents in a defined and energetically stable structure. Here we report the use of rapid microfluidic mixing for the controlled synthesis of two types of limit size lipid nanoparticle (LNP) systems, having either polar or nonpolar cores. Specifically, limit size LNP consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC), cholesterol and the triglyceride triolein were synthesized by mixing a stream of ethanol containing dissolved lipid with an aqueous stream, employing a staggered herringbone micromixer. Millisecond mixing of aqueous and ethanol streams at high flow rate ratios (FRR) was used to rapidly increase the polarity of the medium, driving bottom-up synthesis of limit size LNP systems by spontaneous assembly. For POPC/triolein systems the limit size structures consisted of a hydrophobic core of triolein surrounded by a monolayer of POPC where the diameter could be rationally engineered over the range 20-80 nm by varying the POPC/triolein ratio. In the case of POPC and POPC/cholesterol (55/45; mol/mol) the limit size systems achieved were bilayer vesicles of approximately 20 and 40 nm diameter, respectively. We further show that doxorubicin, a representative weak base drug, can be efficiently loaded and retained in limit size POPC LNP, establishing potential utility as drug delivery systems. To our knowledge this is the first report of stable triglyceride emulsions in the 20-50 nm size range, and the first time vesicular systems in the 20-50 nm size range have been generated by a scalable manufacturing method. These results establish microfluidic mixing as a powerful and general approach to access novel LNP systems, with both polar or nonpolar core structures, in the sub-100 nm size range.
Asunto(s)
Lípidos/química , Microfluídica/métodos , Nanopartículas/química , Tamaño de la Partícula , Triglicéridos , AguaRESUMEN
Hydrophobic uncharged drugs such as docetaxel are difficult to encapsulate and retain in liposomal nanoparticles (LNP). In this work we show that a weak base derivative of docetaxel can be actively loaded into LNP using pH gradient loading techniques to achieve stable drug encapsulation and controlled release properties. Docetaxel was derivatized at the hydroxyl group in the C-2' position to form an N-methyl-piperazinyl butanoic acid ester. The free hydroxyl group in this position is essential for anticancer activity and the prodrug has, therefore, to be converted into the parent drug (docetaxel) to restore activity. Cytotoxicity testing against a panel of cancer cell lines (breast, prostate and ovarian cancer) demonstrated that the prodrug is readily converted into active drug; the derivative was found to be as active as the parent drug in vitro. The docetaxel derivative can be efficiently loaded at high drug-to-lipid ratios (up to 0.4 mg/mg) into LNP using pH loading techniques. Pharmacokinetic, tolerability and efficacy studies in mice demonstrate that the LNP-encapsulated prodrug has the long drug circulation half-life required for efficient tumor accumulation (50-100 times higher drug plasma levels compared with free derivative and Taxotere, the commercial docetaxel formulation), is active in a xenograft model of breast cancer (MDA-MB-435/LCC6), and is well tolerated at i.v. doses of 3 times higher than the maximum tolerated dose (MTD) of the parent drug. This is the first demonstration that a therapeutically active, remote-loaded, controlled-release LNP formulation of a taxane can be achieved. The approach reported here has broad applicability to other approved drugs as well as new chemical entities.
Asunto(s)
Antineoplásicos/administración & dosificación , Portadores de Fármacos/química , Lípidos/química , Nanopartículas/química , Taxoides/administración & dosificación , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Microscopía por Crioelectrón , Docetaxel , Composición de Medicamentos , Estabilidad de Medicamentos , Femenino , Humanos , Concentración de Iones de Hidrógeno , Ratones , Estructura Molecular , Solubilidad , Taxoides/química , Taxoides/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The development of procedures to enhance drug retention in liposomes is important in order to achieve therapeutically optimized rates of drug release from liposomal carriers. In this study, the ability of lipophilic weak base drugs to complex with arylsulfonates resulting in formation of intravesicular precipitates is investigated as a means to enhance drug retention. It is shown that the arylsulfonates benzenesulfonate and hydroxybenzenesulfonate (HBS) induce precipitation of ciprofloxacin and vinorelbine, two representative weak base drugs that are difficult to retain in liposomal systems. The most complete precipitation was observed at pH values corresponding to charge neutralization of the drug-arylsulfonate complex. HBS is shown to be a much more effective precipitating agent than benzenesulfonate. It is also shown that vinorelbine and ciprofloxacin can be loaded into large unilamellar vesicles (LUV) containing the calcium salt of HBS using an ionophore-based loading method. Following drug loading, the formation of intravesicular drug-arylsulfonate precipitates of vinorelbine and ciprofloxacin was observed by cryo-electron microscopy. In vitro release experiments showed substantial improvements in drug retention for both vinorelbine and ciprofloxacin when HBS was present as compared to standard loading procedures employing MgSO4 as the entrapped solute. In vivo release experiments for vinorelbine in NuNu mice indicated a half-time for release for HBS-containing LUV of approximately 30 h, compared to 6.4 h for LUV loaded employing MgSO4. It is suggested that encapsulation procedures employing HBS in the internal medium can improve the retention of drugs that are difficult to retain in liposomes, possibly leading to enhanced therapeutic properties.
Asunto(s)
Arilsulfonatos/química , Química Farmacéutica , Liposomas , Animales , Antibacterianos/administración & dosificación , Antibacterianos/química , Antibacterianos/farmacocinética , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacocinética , Ciprofloxacina/administración & dosificación , Ciprofloxacina/química , Ciprofloxacina/farmacocinética , Microscopía por Crioelectrón , Excipientes , Femenino , Células HT29 , Humanos , Ratones , Microscopía Electrónica de Transmisión , Trasplante de Neoplasias , Soluciones , Vinblastina/administración & dosificación , Vinblastina/análogos & derivados , Vinblastina/química , Vinblastina/farmacocinética , VinorelbinaRESUMEN
A comparative study of the loading and retention properties of three structurally very closely related vinca alkaloids (vincristine, vinorelbine and vinblastine) in liposomal formulations has been performed. All three vinca alkaloids showed high levels of encapsulation when accumulated into egg sphingomyelin/cholesterol vesicles in response to a transmembrane pH gradient generated by the use of the ionophore A23187 and encapsulated MgSO4. However, despite the close similarities of their structures the different vinca drugs exhibited very different release behavior, with vinblastine and vinorelbine being released faster than vincristine both in vitro and in vivo. The differences in loading and retention can be related to the lipophilicity of the drugs tested, where the more hydrophobic drugs are released more rapidly. It was also found that increasing the drug-to-lipid ratio significantly enhanced the retention of vinca alkaloids when the ionophore-based method was used for drug loading. In contrast, drug retention was not dependent on the initial drug-to-lipid ratio for vinca drugs loaded into liposomes containing an acidic citrate buffer. The differences in retention can be explained on the basis of differences in the physical state of the drug inside the liposomes. The drug-to-lipid ratio dependence of retention observed for liposomes loaded with the ionophore technique may provide a way to improve the retention characteristics of liposomal formulations of vinca drugs.
Asunto(s)
Vinblastina/análogos & derivados , Vinblastina/química , Vinblastina/farmacocinética , Vincristina/química , Vincristina/farmacocinética , Animales , Calcimicina/química , Femenino , Inyecciones Intravenosas , Ionóforos/química , Liposomas , Sulfato de Magnesio/química , Ratones , Ratones Endogámicos ICR , Solubilidad , Vinblastina/administración & dosificación , Vincristina/administración & dosificación , VinorelbinaRESUMEN
In many applications, an ability of liposomes to retain drug and then rapidly release it at some later time would be of benefit. In this work, we investigate the ability of cationic large unilamellar vesicles (LUV) to promote rapid release of doxorubicin from anionic LUV. It is shown that the addition of cationic liposomes containing cholesterol, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC) to doxorubicin-containing LUV composed of cholesterol, DOPE, DSPC and the anionic lipid dioleoyphosphatidylglycerol (DOPG) can result in release of more than 90% of the drug in times of 30 s or less. Further, it is shown that these release characteristics are exquisitely dependent on the presence of DOPE and cholesterol. In the absence of DOPE, much slower release rates are observed, with maximum release levels of 50% after a 2-h incubation at 20 degrees C. Remarkably, threshold levels of more than 10 mol% cholesterol are required before any appreciable release is observed. [31P]NMR spectroscopy and freeze-fracture electron microscopy studies reveal that systems giving rise to rapid release of doxorubicin exhibit limited formation of inverted hexagonal (H(II)) phase, suggesting that these lipids facilitate drug release by formation of local regions of non-bilayer structure. It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications.