Nanoscale Liposomes Research Articles

High-throughput continuous production of liposomes using hydrodynamic flow-focusing microfluidic devices

The microfluidic hydrodynamic flow-focusing is a simple technique for nanoscale liposome formation that provides several advantages compared to the traditional manufacturing techniques. This work aimed to perform a systematic study of the liposome formation using planar microfluidic devices with different channel aspect-ratios, as an alternative to enhance the throughput of liposome synthesis. In general, liposomes with a low polydispersity and a precise control of the size were successfully produced from alteration of the flow rate ratio and channel aspect-ratio. The higher aspect-ratio enabled the most rapid generation of liposomes with similar diameter and significant lower polydispersity index than the obtained by other batch technique. Besides, β-carotene was successfully incorporated into liposomes with efficiency of approximately 60% and the incorporation ability was not specific to a choice of microfluidic device aspect-ratio. The results suggest that the use of microfluidic devices could be employed for liposome production with a possible advantage to minimize the degree of parallelization of processes. These results demonstrate the potential technical feasibility of microfluidic processes for future industrial applications.

Competition between Bending and Internal Pressure Governs the Mechanics of Fluid Nanovesicles.

Nanovesicles (∼100 nm) are ubiquitous in cell biology and an important vector for drug delivery. Mechanical properties of vesicles are known to influence cellular uptake, but the mechanism by which deformation dynamics affect internalization is poorly understood. This is partly due to the fact that experimental studies of the mechanics of such vesicles remain challenging, particularly at the nanometer scale where appropriate theoretical models have also been lacking. Here, we probe the mechanical properties of nanoscale liposomes using atomic force microscopy (AFM) indentation. The mechanical response of the nanovesicles shows initial linear behavior and subsequent flattening corresponding to inward tether formation. We derive a quantitative model, including the competing effects of internal pressure and membrane bending, that corresponds well to these experimental observations. Our results are consistent with a bending modulus of the lipid bilayer of ∼14kbT. Surprisingly, we find that vesicle stiffness is pressure dominated for adherent vesicles under physiological conditions. Our experimental method and quantitative theory represents a robust approach to study the mechanics of nanoscale vesicles, which are abundant in biology, as well as being of interest for the rational design of liposomal vectors for drug delivery.

Integrated microfluidic devices for the synthesis of nanoscale liposomes and lipoplexes

In this work, pDNA/cationic liposome (CL) lipoplexes for gene delivery were prepared in one-step using multiple hydrodynamic flow-focusing regions. The microfluidic platform was designed with two distinct regions for the synthesis of liposomes and the subsequent assembly with pDNA, forming lipoplexes. The obtained lipoplexes exhibited appropriate physicochemical characteristics for gene therapy applications under varying conditions of flow rate-ratio (FRR), total volumetric flow rate (QT) and pDNA content (molar charge ratio, R±). The CLs were able to condense and retain the pDNA in the vesicular structures with sizes ranging from 140nm to 250nm. In vitro transfection assays showed that the lipoplexes prepared in one step by the two-stage configuration achieved similar efficiencies as lipoplexes prepared by conventional bulk processes, in which each step comprises a series of manual operations. The integrated microfluidic platform generates lipoplexes with liposome formation combined in-line with lipoplex assembly, significantly reducing the number of steps usually required to form gene carrier systems.

A nano-delivery system for bioactive ingredients using supercritical carbon dioxide and its release behaviors

For the purpose of ensuring the bioavailability of bioactive ingredients, a nano-delivery system with low toxicity was developed using supercritical carbon dioxide (SC-CO2). Compared to thin-film hydration (TFH), obtaining nano-scale liposomes is easier using SC-CO2. The characteristic of these liposomes was also demonstrated by the analysis of particle size and morphology. An in vitro release study showed that liposomes produced using SC-CO2 were resistant to low pH in simulated gastric conditions. In a simulated intestinal environment, enteric solubility of these liposomes was enhanced, which are important properties for controlled releasing bioactive ingredient. Furthermore, SC-CO2-produced liposomes had a higher storage stability than those produced using TFH. Analysis of the organic solvent residue in the liposomes by gas chromatography-mass spectrometry (GC-MS) indicated that SC-CO2-produced liposomes had lower toxicity than those produced by TFH. A chemical free nano-delivery system using SC-CO2 has been revealed for storage and controlled release of bioactive ingredients.

Quantitative Profiling of Nanoscale Liposome Deformation by a Localized Surface Plasmon Resonance Sensor.

Characterizing the shape of sub-100 nm, biological soft-matter particulates (e.g., liposomes and exosomes) adsorbed at a solid-liquid interface remains a challenging task. Here, we introduce a localized surface plasmon resonance (LSPR) sensing approach to quantitatively profile the deformation of nanoscale, fluid-phase 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes contacting a titanium dioxide substrate. Experimental and theoretical results validate that, due to its high sensitivity to the spatial proximity of phospholipid molecules near the sensor surface, the LSPR sensor can discriminate fine differences in the extent of ionic strength-modulated liposome deformation at both low and high surface coverages. By contrast, quartz crystal microbalance-dissipation (QCM-D) measurements performed with equivalent samples were qualitatively sensitive to liposome deformation only at saturation coverage. Control experiments with stiffer, gel-phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes verified that the LSPR measurement discrimination arises from the extent of liposome deformation, while the QCM-D measurements yield a more complex response that is also sensitive to the motion of adsorbed liposomes and coupled solvent along with lateral interactions between liposomes. Collectively, our findings demonstrate the unique measurement capabilities of LSPR sensors in the area of biological surface science, including competitive advantages for probing the shape properties of adsorbed, nanoscale biological particulates.

CPX-351 exhibits potent and direct ex vivo cytotoxicity against AML blasts with enhanced efficacy for cells harboring the FLT3-ITD mutation

PurposeIdentify AML patients most likely to respond to CPX-351, a nano-scale liposome formulation containing cytarabine and daunorubicin co-encapsulated at a 5:1 molar ratio. MethodsWe examined the ex vivo cytotoxic activity of CPX-351 against leukemic cells isolated from 53 AML patients and an additional 127 samples including acute lymphoblastic leukemia, myelodysplastic syndrome/myeloproliferative neoplasms, or chronic lymphocytic leukemia/lymphoma. We assessed activity with respect to common molecular lesions and used flow cytometry to assess CPX-351 cellular uptake. ResultsAML specimen sensitivity to CPX-351 was similar across conventional risk groups. FLT3-ITD cases were five-fold more sensitive to CPX-351. CPX-351 was active across other indications with nearly all cases exhibiting IC50 values markedly lower than reported 72-h plasma drug concentration in patients receiving CPX-351. The range and distribution of CPX-351 IC50 values were comparable for AML, CLL, and ALL, whereas MDS/MPN cases were less sensitive. CPX-351 uptake analysis revealed a correlation between uptake of CPX-351 and cytotoxic potency. ConclusionsOur findings are consistent with clinical data, in which CPX-351 activity is retained in high-risk AML patients. Ex vivo analysis of cytotoxic potency may provide a means to identify specific AML subsets, such as FLT3-ITD, that benefit most from CPX-351 and warrant additional clinical evaluation.

Hydrodynamic Propulsion of Liposomes Electrostatically Attracted to a Lipid Membrane Reveals Size-Dependent Conformational Changes.

The efficiency of lipid nanoparticle uptake across cellular membranes is strongly dependent on the very first interaction step. Detailed understanding of this step is in part hampered by the large heterogeneity in the physicochemical properties of lipid nanoparticles, such as liposomes, making conventional ensemble-averaging methods too blunt to address details of this complex process. Here, we contribute a means to explore whether individual liposomes become deformed upon binding to fluid cell-membrane mimics. This was accomplished by using hydrodynamic forces to control the propulsion of nanoscale liposomes electrostatically attracted to a supported lipid bilayer. In this way, the size of individual liposomes could be determined by simultaneously measuring both their individual drift velocity and diffusivity, revealing that for a radius of ∼45 nm, a close agreement with dynamic light scattering data was observed, while larger liposomes (radius ∼75 nm) displayed a significant deformation unless composed of a gel-phase lipid. The relevance of being able to extract this type of information is discussed in the context of membrane fusion and cellular uptake.

Manufacturing Man-Made Magnetosomes: High-Throughput In Situ Synthesis of Biomimetic Magnetite Loaded Nanovesicles

A new synthetic method for the production of artificial magnetosomes, i.e., lipid-coated vesicles containing magnetic nanoparticles, is demonstrated. Magnetosomes have considerable potential in biomedical and other nanotechnological applications but current production methods rely upon magnetotactic bacteria which limits the range of sizes and shapes that can be generated as well as the obtainable yield. Here, electrohydrodynamic atomization is utilized to form nanoscale liposomes of tunable size followed by electroporation to transport iron into the nanoliposome core resulting in magnetite crystallization. Using a combination of electron and fluorescence microscopy, dynamic light scattering, Raman spectroscopy, and magnetic susceptibility measurements, it is shown that single crystals of single-phase magnetite can be precipitated within each liposome, forming a near-monodisperse population of magnetic nanoparticles. For the specific conditions used in this study the mean particle size is 58 nm (±8 nm) but the system offers a high degree of flexibility in terms of both the size and composition of the final product.

Ultrasound Induced Fluorescence of Nanoscale Liposome Contrast Agents.

A new imaging contrast agent is reported that provides an increased fluorescent signal upon application of ultrasound (US). Liposomes containing lipids labelled with pyrene were optically excited and the excimer fluorescence emission intensity was detected in the absence and presence of an ultrasound field using an acousto-fluorescence setup. The acousto-fluorescence dynamics of liposomes containing lipids with pyrene labelled on the fatty acid tail group (PyPC) and the head group (PyPE) were compared. An increase in excimer emission intensity following exposure to US was observed for both cases studied. The increased intensity and time constants were found to be different for the PyPC and PyPE systems, and dependent on the applied US pressure and exposure time. The greatest change in fluorescence intensity (130%) and smallest rise time constant (0.33 s) are achieved through the use of PyPC labelled liposomes. The mechanism underlying the observed increase of the excimer emission intensity in PyPC labelled liposomes is proposed to arise from the “wagging” of acyl chains which involves fast response and requires lower US pressure. This is accompanied by increased lipid lateral diffusivity at higher ultrasound pressures, a mechanism that is also active in the PyPE labelled liposomes.

TRAIL-coated leukocytes that prevent the bloodborne metastasis of prostate cancer

Prostate cancer, once it has progressed from its local to metastatic form, is a disease with poor prognosis and limited treatment options. Here we demonstrate an approach using nanoscale liposomes conjugated with E-selectin adhesion protein and Apo2L/TRAIL (TNF-related apoptosis-inducing ligand) apoptosis ligand that attach to the surface of leukocytes and rapidly clear viable cancer cells from circulating blood in the living mouse. For the first time, it is shown that such an approach can be used to prevent the spontaneous formation and growth of metastatic tumors in an orthotopic xenograft model of prostate cancer, by greatly reducing the number of circulating tumor cells. We conclude that the use of circulating leukocytes as a carrier for the anti-cancer protein TRAIL could be an effective tool to directly target circulating tumor cells for the prevention of prostate cancer metastasis, and potentially other cancers that spread through the bloodstream.

High-Throughput Continuous Flow Production of Nanoscale Liposomes by Microfluidic Vertical Flow Focusing.

Liposomes represent a leading class of nanoparticles for drug delivery. While a variety of techniques for liposome synthesis have been reported that take advantage of microfluidic flow elements to achieve precise control over the size and polydispersity of nanoscale liposomes, with important implications for nanomedicine applications, these methods suffer from extremely limited throughput, making them impractical for large-scale nanoparticle synthesis. High aspect ratio microfluidic vertical flow focusing is investigated here as a new approach to overcoming the throughput limits of established microfluidic nanoparticle synthesis techniques. Here the vertical flow focusing technique is utilized to generate populations of small, unilamellar, and nearly monodisperse liposomal nanoparticles with exceptionally high production rates and remarkable sample homogeneity. By leveraging this platform, liposomes with modal diameters ranging from 80 to 200 nm are prepared at production rates as high as 1.6 mg min(-1) in a simple flow-through process.

Ultrasound-mediated drug release from nanoscale liposomes using nanoscale cavitation nuclei

We have previously presented a liposomal formulation of mean size 140 nm, manufactured using DSPE, cholesterol, DSPC and DSPE-PEG at ratios of 65:25:3:7, which exclusively releases encapsulated doxorubicin in the presence of inertial cavitation nucleated by microbubbles (SonoVue®, Bracco) at peak rarefactional pressures in excess of 1.2 MPa at 0.5 MHz (Graham et al., J. Controlled Release, 2014). However, the benefits of cavitation-sensitive liposomes small enough to pass through the leaky tumor vasculature can only be fully realized if they can be triggered by cavitation nuclei which are also small enough to extravasate into the tumor mass. In the present work, we demonstrate that liposomal release comparable to that mediated by SonoVue® microbubbles can be achieved using gas-stabilizing polymeric nanocups of mean diameter 400 nm, at peak rarefactional pressure amplitudes in excess of 1.5 MPa at 0.5 MHz or 4 MPa at 1.6 MHz. Mechanistically, we hypothesize that release occurs once a threshold peak shear rate is exceeded in the fluid surrounding the collapsing microbubble, thus exceeding the critical shear stress on the liposomal surface. This is confirmed experimentally by demonstrating a correlation between release and the maximum power of broadband acoustic emissions received by a passive cavitation detector.

Mimicking subsecond neurotransmitter dynamics with femtosecond laser stimulated nanosystems.

Existing nanoscale chemical delivery systems target diseased cells over long, sustained periods of time, typically through one-time, destructive triggering. Future directions lie in the development of fast and robust techniques capable of reproducing the pulsatile chemical activity of living organisms, thereby allowing us to mimic biofunctionality. Here, we demonstrate that by applying programmed femtosecond laser pulses to robust, nanoscale liposome structures containing dopamine, we achieve sub-second, controlled release of dopamine – a key neurotransmitter of the central nervous system – thereby replicating its release profile in the brain. The fast delivery system provides a powerful new interface with neural circuits, and to the larger range of biological functions that operate on this short timescale.

Microfluidic-Enabled Liposomes Elucidate Size-Dependent Transdermal Transport

Microfluidic synthesis of small and nearly-monodisperse liposomes is used to investigate the size-dependent passive transdermal transport of nanoscale lipid vesicles. While large liposomes with diameters above 105 nm are found to be excluded from deeper skin layers past the stratum corneum, the primary barrier to nanoparticle transport, liposomes with mean diameters between 31–41 nm exhibit significantly enhanced penetration. Furthermore, multicolor fluorescence imaging reveals that the smaller liposomes pass rapidly through the stratum corneum without vesicle rupture. These findings reveal that nanoscale liposomes with well-controlled size and minimal size variance are excellent vehicles for transdermal delivery of functional nanoparticle drugs.

A facile route to the synthesis of monodisperse nanoscale liposomes using 3D microfluidic hydrodynamic focusing in a concentric capillary array.

A novel microscale device has been developed to enable the one-step continuous flow assembly of monodisperse nanoscale liposomes using three-dimensional microfluidic hydrodynamic focusing (3D-MHF) in a concentric capillary array. The 3D-MHF flow technique displays patent advantages over conventional methods for nanoscale liposome manufacture (i.e., bulk-scale alcohol injection and/or sonication) through the on-demand synthesis of consistently uniform liposomes without the need for post-processing strategies. Liposomes produced by the 3D-MHF device are of tunable size, have a factor of two improvement in polydispersity, and a production rate that is four orders of magnitude higher than previous MHF methods, which can be attributed to entirely radially symmetric diffusion of alcohol-solvated lipid into an aqueous flow stream. Moreover, the 3D-MHF platform is simple to construct from low-cost, commercially-available components, which obviates the need for advanced microfabrication strategies necessitated by previous MHF nanoparticle synthesis platforms.

Automated formation of multicomponent‐encapuslating vesosomes using continuous flow microcentrifugation

Vesosomes - hierarchical assemblies consisting of membrane-bound vesicles of various scales - are potentially powerful models of cellular compartmentalization. Current methods of vesosome fabrication are labor intensive, and offer little control over the size and uniformity of the final product. In this article, we report the development of an automated vesosome formation platform using a microfluidic device and a continuous flow microcentrifuge. In the microfluidic device, water-in-oil droplets containing nanoscale vesicles in the water phase were formed using T-junction geometry, in which a lipid monolayer is formed at the oil/water interface. These water-in-oil droplets were then immediately transferred to the continuous flow microcentrifuge. When a water-in-oil droplet passed through a second lipid monolayer formed in the continuous flow microcentrifuge, a bilayer-encapsulated vesosome was created, which contained all of the contents of the aqueous phase encapsulated within the vesosome. Encapsulation of nanoscale liposomes within the outer vesosome membrane was confirmed by fluorescence microscopy. Laser diffraction analysis showed that the vesosomes we fabricated were uniform (coefficient of variation of 0.029). The yield of the continuous flow microcentrifuge is high, with over 60% of impinging water droplets being converted to vesosomes. Our system provides a fully automatable route for the generation of vesosomes encapsulating arbitrary contents. The method employed in this work is simple and can be readily applied to a variety of systems, providing a facile platform for fabricating multicomponent carriers and model cells.

Self-Destructing “Mothership” Capsules for Timed Release of Encapsulated Contents

We describe a new class of hierarchical containers that are formed via single-step assembly and, at a later time, self-destruct because of their packaged contents. These containers are spherical capsules formed by electrostatic complexation of the anionic biopolymer, gellan gum, with the cationic biopolymer, chitosan. The capsules are termed "motherships" and are engineered to carry a cargo of much smaller containers (e.g., nanoscale liposomes ("babyships")), within their lumen. Additionally, we package an enzyme, chitosanase, in the capsule that is capable of degrading polymeric chitosan into short oligomers. Thereby, we create motherships that self-destruct, liberating their cargo of babyships into the external solution. The time scale for self-destruction can be engineered based on the internal concentration of enzyme. The motherships are stable when stored in a freeze-dried form and can be readily dispersed into water or buffer solutions at a later time, whereupon their "internal clock" for self-destruction is initiated. The above concept could be useful for the triggered release of a variety of payloads including drugs, biological therapeutics, cosmetics, and flavor ingredients.

Comparative Physicochemical Characterization and Bioavailability of Nano-Liposomes Formed by Mixing Lipids at Different Ratios

Liposomes are small lipid vesicles that mimic biological membranes and have been spotlighted in the clinical field due to their ability to enclose a biologically active substance of any structure and to release it into the host's body. This study compares the physicochemical properties and biological activity of nano-liposomes with different compositions to determine the most effective formulation for further in vivo application. Nano-scale liposomes composed of different ratios of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), dihexadecyl phosphate (DCP), and cholesterol (Chol): DMPE, DMPE/DCP, DMPE/Chol, and DMPE/DCP/Chol were produced. The thermal phase transition was assessed via differential scanning calorimetry (DSC); the particle size, via dynamic light scattering (DLS); the colloidal stability, via the zeta potential; the direct morphological characterization, via transmission electron microscopy (TEM); and the protein encapsulation efficiency. The bioavailability was also investigated with respect to the immunological responses via porcine interferon gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay in peripheral blood mononuclear cells (PBMC) of immunocompetent pigs. All the liposomes can be expected to be stable in an in vivo physiological temperature, and the liposomes that were prepared from DMPE/DCP showed the best efficiency in the in vitro model that mimicked the release of a bioactive substance in vivo. In the result of DLS and the zeta potential for the investigation of the colloidal stability in the system, DMPE/DCP/Chol appeared better than the other formulations. The porcine IFN-gamma ELISPOT assay results postulated that DMPE/DCP most potently induced the IFN-gamma secretion by PBMC, followed by DMPE/DCP/Chol and DMPE alone, in that order.

Abstract 5057: Gene expression profile of chemoresponse of microdissected papillary serous tumors of the ovary identifies POLH and REV3L as potential therapeutic targets

Abstract Treatment of advanced stage ovarian carcinoma often involves cytoreductive surgery followed by adjuvant chemotherapy with paclitaxel and either cisplatin or carboplatin. Most tumors have either inherent or develop acquired resistance to chemotherapy, resulting in patient demise from their cancer. The mechanism of drug nonresponse (refractory/resistance) remains unknown and currently there is no clinically useful molecular predictor of response. Here, we determined the gene expression profile of 52 microdissected advanced stage papillary serous ovarian cancer and identified gene expression signatures predictive for nonresponse to chemotherapy. To generate a predictive gene signature, class prediction algorithms were applied to genes differentially expressed between resistant and sensitive tumors (p<0.001) using leave-one-out cross-validation. We developed a predictive 96-gene signature for tumor refractory and 29-gene signature for tumor resistance to chemotherapy. Real-time quantitative RT-PCR validated the results of the cDNA microarray analysis. To further validate our array results and demonstrate therapeutic value of these targets, we pursued several novel genes to determine whether over expression will render ovarian cancer cell lines resistance to chemotherapeutic drugs. Overexpression of EDIL3, GNA13 and POLH genes imparted cisplatin resistance to the ovarian cancer cell lines. As DNA Polymerases contribute to cell survival and the emergence of resistant cells by allowing cells to replicate beyond cisplatin adducts to avoid replication arrest and cell death, we decided to evaluate the potential therapeutic value of the genes POLH (DNA polymerase eta) and REV3L (DNA polymerase zeta) using short interfering RNA (siRNA) approach. Reduced gene expression via gene specific siRNA molecules renders ovarian cancer cell lines (A2780-CP20 and UCI 107) sensitive to cisplatin in vitro. Further, we validated these results in an orthotopic mouse model of ovarian cancer using liposomal (DOPC) siRNA approach as these nanoscale liposomes can penetrate deeply into the tumor. A combination of siREV3L/siPOLH-DOPC with cisplatin resulted in a significant reduction in mean tumor weight compared to mice treated with nonspecific control siRNA (77% reduction; Mann Whitney test, P=0.0140), compared to mice treated by nonspecific control siRNA with cisplatin (72% reduction; Mann Whitney test, P=0.0108), and compared to those mice treated with combination siREV3L/siPOLH-DOPC (68% reduction; Mann Whitney test, P=0.0070). In conclusion, here we report gene profile signatures of the chemoresponse of ovarian cancer. POLH and REV3L identified as potential therapeutic targets which can serve as targets for specific molecular therapeutic molecules that can increase the sensitivity of ovarian cancer to standard chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5057. doi:1538-7445.AM2012-5057

E-selectin liposomal and nanotube-targeted delivery of doxorubicin to circulating tumor cells

The presence of circulating tumor cells (CTCs) is believed to lead to the formation of secondary tumors via an adhesion cascade involving interaction between adhesion receptors of endothelial cells and ligands on CTCs. Many CTCs express sialylated carbohydrate ligands on their surfaces that adhere to selectin protein found on inflamed endothelial cells. We have investigated the feasibility of using immobilized selectin proteins as a targeting mechanism for CTCs under flow. Herein, targeted liposomal doxorubicin (L-DXR) was functionalized with recombinant human E-selectin (ES) and polyethylene glycol (PEG) to target and kill cancer cells under shear flow, both when immobilized along a microtube device or sheared in a cone-and-plate viscometer in a dilute suspension. Healthy circulating cells such as red blood cells were not targeted by this mechanism and were left to freely circulate, and minimal leukocyte death was observed. Halloysite nanotube (HNT)-coated microtube devices immobilized with nanoscale liposomes significantly enhanced the targeting, capture, and killing of cancer cells. This work demonstrates that E-selectin functionalized L-DXR, sheared in suspension or immobilized onto microtube devices, provides a novel approach to selectively target and deliver chemotherapeutics to CTCs in the bloodstream.