Article

Analysis of lipid nanoparticles by Cryo-EM for characterizing siRNA delivery vehicles

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Abstract

Lipid nanoparticles are self-assembling, dynamic structures commonly used as carriers of siRNA, DNA, and small molecular therapeutics. Quantitative analysis of particle characteristics such as morphological features can be very informative as biophysical properties are known to influence biological activity, biodistribution, and toxicity. However, accurate characterization of particle attributes and population distributions is difficult. Cryo-Electron Microscopy (Cryo-EM) is a leading characterization method and can reveal diversity in particle size, shape and lamellarity, however, this approach is traditionally used for qualitative review or low throughput image analysis due to inherent EM micrograph contrast characteristics and artifacts in the images which limit extraction of quantitative feature values. In this paper we describe the development of a semiautomatic image analysis framework to facilitate reliable image enhancement, object segmentation, and quantification of nanoparticle attributes in Cryo-EM micrographs. We apply this approach to characterize two formulations of siRNA-loaded lipid nanoparticles composed of cationic lipid, cholesterol, and poly(ethylene glycol)-lipid, where the formulations differ only by input component ratios. We found Cryo-EM image analysis provided reliable size and morphology information as well as the detection of smaller particle populations that were not detected by standard dynamic light scattering (DLS) analysis.

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... Our results, however, are in line with the results of Crawford et al., who also reported differences in liposome sizes measured by Cryo-EM and DLS. Cryo-EM was found to detect even the smallest liposomes having a diameter of 20 nm (a low-PEG/high-cholesterol liposome formulation), which the DLS method was not able to detect [29]. The differences in the measurement systems (Cryo-EM, DLS) could be explained by the bias of DLS in detecting small nano-scale lipid particles in a poly-dispersed suspension [30]. ...
... The PDI value for the BBR-loaded liposomes generated by the ethanol-injection method (FJ17) was 0.113, suggesting that the size distribution of liposomes is not monomo dal. The mean size of liposomes measured by the Cryo-EM and DLS techniques are shown to be similar only if the size distribution is monomodal and monodisperse [29]. Furthermore, there are many factors affecting the size and PDI results of liposomes obtained with DLS, such as temperature [31], concentration of particles [32], pH and viscosity of the buffer [33]. ...
... Cryo-EM has been reported to be a golden standard for liposome imaging, enabling the simultaneous size measurement and imaging of the morphology and substructure of liposomes [34,35]. The application of Cryo-EM, however, is still limited in the analysis of pharmaceutical liposomes due to the relatively complex sample preparation, cost and perhaps also challenges in the number of liposomes to be trapped in EM grids [29]. ...
Article
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Berberine (BBR) is a poorly water-soluble quaternary isoquinoline alkaloid of plant origin with potential uses in the drug therapy of hypercholesterolemia. To tackle the limitations associated with the oral therapeutic use of BBR (such as a first-pass metabolism and poor absorption), BBR-loaded liposomes were fabricated by ethanol-injection and thin-film hydration methods. The size and size distribution, polydispersity index (PDI), solid-state properties, entrapment efficiency (EE) and in vitro drug release of liposomes were investigated. The BBR-loaded liposomes prepared by ethanol-injection and thin-film hydration methods presented an average liposome size ranging from 50 nm to 244 nm and from 111 nm to 449 nm, respectively. The PDI values for the liposomes were less than 0.3, suggesting a narrow size distribution. The EE of liposomes ranged from 56% to 92%. Poorly water-soluble BBR was found to accumulate in the bi-layered phospholipid membrane of the liposomes prepared by the thin-film hydration method. The BBR-loaded liposomes generated by both nanofabrication methods presented extended drug release behavior in vitro. In conclusion, both ethanol-injection and thin-film hydration nanofabrication methods are feasible for generating BBR-loaded oral liposomes with a uniform size, high EE and modified drug release behavior in vitro.
... Cryo-EM is a method that requires vitrification of samples and therefore maintains in-solution structure [311]. With the recent advancements with detector sensitivity during the resolution revolution, structure may be determined to the molecular and atomic scale [312]. ...
... CryoEM has been used to characterize liposomes PEGylated with PEG-cholesterol structures. This process entails imaging of many lipid nanoparticles and image analysis to determine nanoparticle structure with advantage over DLS [311]. ...
Article
Poly(ethylene glycol) or PEG has a long history of use in medicine. Many conventional formulations utilize PEG as either an active ingredient or an excipient. PEG found its use in biotechnology therapeutics as a tool to slow down drug clearance and shield protein therapeutics from undesirable immunogenicity. Nanotechnology field applies PEG to create stealth drug carriers with prolonged circulation time and decreased recognition and clearance by the mononuclear phagocytic cells (MPS). Most nanomedicines approved for clinical use and experimental nanotherapeutics contain PEG. Among the most recent successful examples are two mRNA-based COVID-19 vaccines that are delivered by PEGylated lipid nanoparticles. The breadth of PEG use in a wide variety of over the counter (OTC) medications as well as in drug products and vaccines stimulated research which uncovered that PEG is not as immunologically inert as it was initially expected. Herein, we review the current understanding of PEG’s immunological properties and discuss them in the context of synthesis, biodistribution, safety, efficacy, and characterization of PEGylated nanomedicines. We also review the current knowledge about immunological compatibility of other polymers that are being actively investigated as PEG alternatives.
... This knowledge is remarkable in that it is feasible to start engineering nanobased carriers for smart delivery of small interfering RNA (siRNA) molecules to regulate or reduce the number of specific transcripts or gene products. Furthermore, Crawford et al. 85 promoted the understanding of the structure and assembly of lipid nanoparticles. This is also instrumental for drug delivery and therapeutics. ...
... This is also instrumental for drug delivery and therapeutics. 85 Besides ribosome-based cargoes, the combination of liposomes with gold nanoparticles is in increased use to develop robust drug delivery systems. Zǐvanovićet al. 86 used cryo-EM to reveal the 3D interactions among different lipid structures and nanoparticles (Figure 5a−f). ...
Article
Engineered nanomaterials undergo a dynamic interaction with matrices such as soil, water, and biological tissues in their lifetime. The investigation and assessment of these interactions is the key to understand the implications that nanotechnology has on different aspects, including biotoxicity, therapeutic efficacy, and overall environmental sustainability. Electron microscopy (EM) is a high-resolution imaging technique that has been rapidly developed to achieve in situ, in vitro, and real-time imaging of nanomaterials in biological matrices. This perspective is the first to review the recent progress on assessing nanobio interaction at different biological levels using EM. We provide a clear picture of different EM configurations with their overall properties and a systematic summary of three main sample preparation methods, such as nanomaterials in dehydrated, frozen, and hydrated matrices. This perspective also provides a thorough discussion on the findings of the past five years on nanobio assessments using EM, such as the identification of nanocorona formation inside seeds during nanoagricultural studies; near-native structural analysis of lipid nanoparticles in nanomedicine studies; and the real-time observation of tumor cell internalization of gold nanoparticle in cancer research. Solving how engineered nanomaterials behave in a biological matrix has made a qualitative leap in nanoresearch. By summarizing recent studies, this perspective discusses the benefits and overall pitfalls of EM in aspects such as the resolution, sampling, and cost.
... Crawford et al.57 report PLGA NPs, complexed with polyethylenimine and a PLGA scaffold, to enhance transfection efficiency.57 Specifically, treatment of an articular defect in a rabbit model with the scaffold, seeded with BMP-4 transfected adipose derived stem cell, significantly improves in vivo chondrogenesis.58 ...
... Crawford et al.57 report PLGA NPs, complexed with polyethylenimine and a PLGA scaffold, to enhance transfection efficiency.57 Specifically, treatment of an articular defect in a rabbit model with the scaffold, seeded with BMP-4 transfected adipose derived stem cell, significantly improves in vivo chondrogenesis.58 ...
Article
Osteoarthritis (OA) is a multifactorial disease of the entire joint which afflicts 140 million individuals worldwide regardless of economic or social status. Current clinical treatments for OA primarily center on reducing pain and increasing mobility, and there are limited therapeutic interventions to restore degraded cartilage or slow disease pathogenesis. This second installment of a two‐part review on nanotechnology and osteoarthritis focuses on novel treatment strategies. Specifically, Part 2 first discusses current surgical and non‐surgical treatments for OA and then summarizes recent advancements in nanotechnology‐based treatments, while Part 1 (insert DOI) described advances in imaging and diagnostics. We review nano delivery systems for small molecule drugs, nucleic acids, and proteins followed by nano‐based scaffolds for neocartilage formation and osteochondral regeneration, and lastly nanoparticle lubricants. We conclude by identifying opportunities for nanomedicine advances, and prospects for OA treatments. This article is protected by copyright. All rights reserved.
... Early attempts to visualize biological molecules within nuclei revealed that immunogold labels were unable to pass the nuclear envelope. Conjugating monoclonal antibodies to gold particles of various sizes, it was found that nuclear transport could be achieved using 0. 8 [7], indicating that prior efforts were limited by the size of the gold labels. This discovery should greatly enable further applications of TEM to the analysis of nucleic acid delivery, including to nuclei for gene therapy/transfer applications. ...
... Consequently, single-particle analyses, crystallography and tomography are more common with cryo-EM. In examining siRNA delivery vehicles, cryo-EM was used in conjunction with novel computational analyses to obtain quantitative morphological data on individual lipid nanoparticles [8]. A novel approach, Zernike phase contrast cryo-EM was applied to generate improved structural information on the interactions of Dicer with substrate RNAs, showing that the structure of Dicer changes based on the incorporation of the substrate and interactions of the substrate with other RNA-binding proteins [9]. ...
... LNP sizes measured from these micrographs indicate diameters of 44.5 ± 6.8 nm and 22.4 ± 3.9 nm for the 1 and 5 mol% formulations, respectively, in agreement with the light scattering measurements (Supplementary Table S1). We note that the LNP siRNA systems exhibit an electron dense interior core similar to that obtained elsewhere for LNP siRNA systems generated employing the T-tube method.25 Such morphology is distinctly different than observed for bilayer LNP systems and has been ascribed to a nanostructured lipid interior.23 ...
... The production of PFVs is also not required. Advantages of the microfluidics approach as compared to the T-tube mixer again includes the ability to produce smaller systems below 50-nm diameter (the smallest reported using the T-tube approach are of 50-nm diameter25) as well as the fact that high flow rates (>1 ml/sec) are required to achieve the velocities required for rapid mixing to occur using the T-tube mixer. The micromixer allows LNP siRNA formulation under well defined, reproducible conditions at much lower flow rates resulting in reduced losses due to dead volumes and straightforward preparation of small scale batches for LNP optimization and in vitro testing. ...
Article
Full-text available
Lipid nanoparticles (LNP) are the leading systems for in vivo delivery of small interfering RNA (siRNA) for therapeutic applications. Formulation of LNP siRNA systems requires rapid mixing of solutions containing cationic lipid with solutions containing siRNA. Current formulation procedures employ macroscopic mixing processes to produce systems 70-nm diameter or larger that have variable siRNA encapsulation efficiency, homogeneity, and reproducibility. Here, we show that microfluidic mixing techniques, which permit millisecond mixing at the nanoliter scale, can reproducibly generate limit size LNP siRNA systems 20 nm and larger with essentially complete encapsulation of siRNA over a wide range of conditions with polydispersity indexes as low as 0.02. Optimized LNP siRNA systems produced by microfluidic mixing achieved 50% target gene silencing in hepatocytes at a dose level of 10 µg/kg siRNA in mice. We anticipate that microfluidic mixing, a precisely controlled and readily scalable technique, will become the preferred method for formulation of LNP siRNA delivery systems.
... 8 Similar to other nanodrug delivery systems, efficient cellular delivery of lipid-based nanoparticles requires the particles to be within 30 to 200 nm. [9][10][11][12] This is because particles smaller than 30 nm can leak into liver sinusoidal capillaries 9,13 and particles larger than 200 nm are susceptible to mononuclear phagocytic uptake. 9,14 In response to the stringent particle size requirements, a number of nanoparticle production technologies have been developed for generating size-specific nanodrugs. ...
Preprint
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Lipid nanoparticles (LNPs) are drug carriers for protecting nucleic acids for cellular delivery. The first mRNA vaccines authorized by the United States Food and Drug Administration are the mRNA-1273 (Moderna) and BNT162b (BioNTech/Pfizer) vaccines against coronavirus disease 2019 (COVID-19). We designed a 3D printed Omnidirectional Sheath-flow Enabled Microfluidics (OSEM) Device for producing mRNA-loaded LNPs that closely resemble the Moderna vaccine: we used the same lipid formulations to encapsulate mRNA encoding SARS-CoV-2 spike protein. The OSEM device is made of durable methacrylate-based materials that can support flow rates in the mL/min range and was fabricated by stereolithography (SLA), incorporating readily adaptable interfaces using commercial fluidic connectors. Two key features of the OSEM device are: 1) a 4-way hydrodynamic flow focusing region and 2) a staggered herringbone mixer (SHM). Superior to conventional planar fluid junctions, the 4-way sheath flow channel generates an evenly focused, circular center flow that facilitates the formation of LNPs with low polydispersity. Downstream, fluid mixing in the SHM is intensified by incorporating a zig-zag fluidic pathway to deliver high mRNA encapsulation efficiency. We characterized the mRNA-loaded LNPs produced in the OSEM device and showed that the enhanced 3D microfluidic structures enable a 5-fold higher throughput production rate (60 mL/min) of LNPs than commercial multi-thousand-dollar micromixers. The device produced LNPs of diameter less than 90 nm, with low polydispersity (2-8%) and high mRNA encapsulation efficiency (> 90%). At a significantly lower cost (US $1.5) compared to commercial instruments, the OSEM device provides an unprecedented all-in-one solution to LNP production from lab to market.
... Furthermore, microphotographs were also useful for confirming the size of the liposomal vesicles prepared, although TEM typically measures mean sizes smaller than those determined by DLS. This trend is a consequence of the scattering of a small number of aggregated liposomes, which are also present at the high dilutions of the dispersion [42,43]. ...
Article
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Background: Oleuropein is already known for its numerous pharmacological properties, but its activity in the ocular field has not yet been investigated. The study aims to verify a possible use of oleuropein (OLE)-based eye drops both in terms of efficacy in dry eye syndrome and stability in aqueous solution. Methods: OLE was co-precipitated with HP-β-cyclodextrin, and the obtained complex was encapsulated into liposomes prepared by hydration of a lipid film composed of Lipoid S100 and cholesterol with different pH buffer solutions. The hydrated vesicles were shrunk by ultrasonication or extrusion. The preparations were characterized from the physicochemical point of view by subjecting them to differential scanning calorimetry, ATR-FTIR, dynamic light scattering analysis, and microscopy. Subsequently, OLE protective activity against hyperosmotic and oxidative stress on rabbit corneal epithelial cells (RCE) was evaluated. Results: The liposomal vesicles obtained after extrusion showed a tendency towards greater encapsulation efficiency (up to 80.77%) compared to that obtained by sonication, and the liposomes hydrated in pH 5.5 solution tended to incapsulate more than the neutral ones. Ultrasonication produced two-dimensional populations of liposomes, the largest of which reached 2149 nm. On the contrary, the extruded liposomes showed homogeneous diameters of about 250 nm. Complexation with cyclodextrin and subsequent encapsulation in liposomes greatly increased the OLE stability in aqueous solution, especially at 4 °C and for the extruded formulations. OLE aqueous solution (OLE7.4-sol, reference) and neutral extruded liposomes (F7.4-e) were well tolerated on RCE cells. Moreover, OLE was able to control the effects of hyperosmolarity on ocular surface cells and to prevent oxidative stress-induced loss of cell viability.
... Therefore, in this study, we propose a novel pH responsive MLVs loaded with vancomycin hydrochloride (VCM) drug delivery system for antibiotic delivery. We envisage the proposed the self-assembled MLVs having stacked layers with the lipidic layers formed with a hydrophobic layer of electrostatic complexed of the anionic oleic acid (OA) and stearyl amine (SA) cationic lipids stabilized and hydrated by tween 80, while the hydrophilic cargo is localized in the spaces between the onionlike lipid layers [12]. Moreover, the deportation and protonation of the complexed OA and SA will destabilize the system resulting in faster drug release at the lower pH, which is synonymous with bacterial sites of infection [13]. ...
Article
Fight against antimicrobial resistance calls for innovative strategies that can target infection sites and enhance activity of antibiotics. Herein is a pH responsive multilamellar vesicles (MLVs) for targeting bacterial infection sites. The vancomycin (VCM) loaded MLVs had 62.25 ± 8.7 nm, 0.15 ± 0.01 and -5.55 ± 2.76 mV size, PDI and zeta potential, respectively at pH 7.4. The MLVs had a negative ZP at pH 7.4 that switched to a positive charge and faster release of the drug at acidic pH. The encapsulation efficiency was found to be 46.34 ± 3.88 %. In silico studies of the lipids, interaction suggested an energetically stable system. Studies to determine the minimum inhibitory concentration studies (MIC) showed the MLVs had 2-times and 8-times MIC against Staphylococcus aureus (SA) and Methicillin resistance SA respectively at physiological pH. While at pH 6.0 there was 8 times reduction in MICs for the formulation against SA and MRSA in comparison to the bare drug. Fluorescence-activated Cell Sorting (FACS) studies demonstrated that even with 8-times lower MIC, MLVs had a similar elimination ability of MRSA cells when compared to the bare drug. Fluorescence microscopy showed MLVs had the ability to clear biofilms while the bare drug could not. Mice skin infection models studies showed that the colony finding units (CFUs) of MRSA recovered from groups treated with MLVs was 4,050 and 525-fold lower than the untreated and bare VCM treated groups, respectively. This study demonstrated pH-responsive multilamellar vesicles as effective system for targeting and enhancing antibacterial agents.
... 28,29 An example of this approach is hydrodynamic flow focusing, a microfluidic laminar flow method where particles are formed at the interface of laminar flowstreams; however, this process is limited in throughput (<10 mL/h) and mainly used for preparing liposomes, which have less structural complexity than LNPs. 30−33 Another type of rapid mixing process is Tjunction mixing, where turbulent mixing in a macroscopic channel (>1 mm) 5,20,29,34 can achieve small LNP sizes (<100 nm) 35 but cannot scale down to the small volumes (μL) needed for high-throughput library screening of nanomaterials. 21,24,29 In this paper, we focus on the staggered herringbone micromixer (SHM) design, a widely applied, low-throughput (<100 mL/h) microfluidic strategy for the production of precisely defined LNPs. ...
... within hydrophobic lipid layers or aqueous cores of liposomes, and provide structural information indirectly. In addition, lipid layers can be directly imaged and counted by several microscopy techniques, including differential interference contrast microscopy [131], freeze-fracture TEM [132], and cryo-TEM [29,133]. However, results from these image-based analyses are subjected to sample preparation processes, choices of representative images, and image quality. ...
Article
Lipid nanoparticles, especially liposomes and lipid/nucleic acid complexed nanoparticles have shown great success in the pharmaceutical industry. Their success is attributed to stable drug loading, extended pharmacokinetics, reduced off-target side effects, and enhanced delivery efficiency to disease targets with formidable blood-brain or plasma membrane barriers. Therefore, they offer promising formulation options for drugs limited by low therapeutic indexes in traditional dosage forms and current “undruggable” targets. Recent development of siRNA, antisense oligonucleotide, or the CRISPR complex-loaded lipid nanoparticles and liposomal vaccines also shed light on their potential in enabling versatile formulation platforms for new pharmaceutical modalities. Analytical characterization of these nanoparticles is critical to drug design, formulation development, understanding in vivo performance, as well as quality control. The multi-lipid excipients, unique core-bilayer structure, and nanoscale size all underscore their complicated critical quality attributes, including lipid species, drug encapsulation efficiency, nanoparticle characteristics, product stability, and drug release. To address these challenges and facilitate future applications of lipid nanoparticles in drug development, we summarize available analytical approaches for physicochemical characterizations of lipid nanoparticle-based pharmaceutical modalities. Furthermore, we compare advantages and challenges of different techniques, and highlight the promise of new strategies for automated high-throughput screening and future development.
... It is critical to accurately characterize liposomes and drug-liposome interactions as biophysical properties of liposomes are known to influence biological activity, biodistribution, and toxicity. Among the available techniques (Dynamic Light Scattering (DLS), Size Exclusion Chromatography (SEC), Atomic Force Microscopy (AFM), and cryo-EM), cryo-EM is the most precise and direct method to determine liposome lamellarity, size, shape and ultrastructure, which may reveal clues to mechanism of action toward the clinical endpoints of efficacy and toxicity [32][33][34][35][36][37][38][39][40][41]. ...
Article
Liposomes are widely used as delivery systems in pharmaceutical, cosmetics and food industries, as well as a system for structural and functional study of membrane proteins. To accurately characterize liposomes, cryo-Electron Microscopy (cryo-EM) has been employed as it is the most precise and direct method to determine liposome lamellarity, size, shape and ultrastructure. However, its use is limited by the number of liposomes that can be trapped in the thin layer of ice that spans holes in the perforated carbon film on EM grids. We report a long-incubation method for increasing the density of liposomes in holes. By increasing the incubation time, high liposome density was achieved even with extremely dilute (in the nanomolar range) liposome solutions. This long-incubation method has been successfully employed to study the structure of an ion channel reconstituted into liposomes.
... Interestingly, previous SAXS studies indicated that lamellar lipid structures are present on the surface of DOTAP-modified LPNs [9]. The multilamellar vesicle model was proposed for the self-assembled SNALPs based on studies showing that the outer shell layer is composed of helper lipid and cationic lipid, while the nucleic acid cargo is localized between the onion-like lipid bilayers [24,25]. However, a nanostructure core model and a homogeneous core-shell model have also been proposed for such self-assembled lipid nanoparticles [26,27]. ...
Article
Rheumatoid arthritis (RA) is a common autoimmune disease, which is characterized by painful chronic inflammation in the joints, and novel safe and efficacious treatments are urgently needed. RNA interference (RNAi) therapy based on small interfering RNA (siRNA) is a promising approach for silencing specific genes involved in inflammation. However, delivery of siRNA to the target site, i.e. the cytosol of immune cells, is a challenge. Here, we designed lipid-polymer hybrid nanoparticles (LPNs) composed of lipidoid and poly(DL-lactic-co-glycolic acid) loaded with a therapeutic cargo siRNA directed against the proinflammatory cytokine tumor necrosis factor (TNF), which plays a key role in the progression of RA. We compared their efficacy and safety with reference lipidoid-based stable nucleic acid lipid particles (SNALPs) in vitro and in vivo. Cryogenic transmission electron microscopy, atomic force microscopy and small-angle X-ray scattering revealed that the mode of loading of siRNA in lamellar structures differs between the two formulations. Thus, siRNA was tightly packed in LPNs, while LPNs displayed lower adhesion than SNALPs. The LPNs mediated a higher TNF silencing effect in vitro than SNALPs in the RAW 264.7 macrophage cell line activated with lipopolysaccharide. For both types of delivery systems, macropinocytosis was involved in cellular uptake. In addition, clathrin-mediated endocytosis contributed to uptake of SNALPs. LPNs loaded with TNF siRNA mediated sequence-specific suppression of inflammation in a murine experimental arthritis model upon intra-articular administration. Hence, the present study demonstrates that LPN-mediated TNF knockdown constitutes a promising approach for arthritis therapy of TNF-mediated chronic inflammatory conditions.
... We characterized in depth the biophysical properties of the LNPs used in this study previously by cryo-electron microscopy (cryo-EM; Figure S3) and dynamic light scattering (DLS). 42 The nitrogen-to-phosphate ratio (N:P) is 3:3 and the composition of the LNP particle for the delivery vehicle used in this study is as follows: 60% cationic lipid, 38% cholesterol, and 2% polyethylene glycol (PEG). The DLS mean diameter is 96.6 nm, in the optimal range for drug delivery. ...
... We characterized in depth the biophysical properties of the LNPs used in this study previously by cryo-electron microscopy (cryo-EM; Figure S3) and dynamic light scattering (DLS). 42 The nitrogen-to-phosphate ratio (N:P) is 3:3 and the composition of the LNP particle for the delivery vehicle used in this study is as follows: 60% cationic lipid, 38% cholesterol, and 2% polyethylene glycol (PEG). The DLS mean diameter is 96.6 nm, in the optimal range for drug delivery. ...
Article
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Drug combinations can improve the control of diseases involving redundant and highly regulated pathways. Validating a multi-target therapy early in drug development remains difficult. Small interfering RNAs (siRNAs) are routinely used to selectively silence a target of interest. Owing to the ease of design and synthesis, siRNAs hold promise for combination therapies. Combining siRNAs against multiple targets remains an attractive approach to interrogating highly regulated pathways. Currently, questions remain regarding how broadly such an approach can be applied, since siRNAs have been shown to compete with one another for binding to Argonaute2 (Ago2), the protein responsible for initiating siRNA-mediated mRNA degradation. Mathematical modeling, coupled with in vitro and in vivo experiments, led us to conclude that endosomal escape kinetics had the highest impact on Ago2 depletion by competing lipid-nanoparticle (LNP)-formulated siRNAs. This, in turn, affected the level of competition observed between them. A future application of this model would be to optimize delivery of desired siRNA combinations in vitro to attenuate competition and maximize the combined therapeutic effect.
... Il est nécessaire de défocaliser de l'ordre de -6 à -10 µm les LNP afin qu'elles soient visibles dans la couche de glace (Figure 3.28). Ces valeurs sont également celles qui sont utilisées pour l'observation d'autres types de nanoparticules lipidiques [296]. Les LNP ont tout d'abord été préparées à 100 mg/mL. ...
Thesis
L'utilisation massive des nanomatériaux pose de réels enjeux sanitaires et environnementaux. C'est pourquoi ils sont désormais soumis à une réglementation qui prévoit une traçabilité de ceux-ci depuis leur fabrication jusqu'à leur distribution et l'établissement d'une fiche d'identité de la substance (composition, taille, état d'agglomération, forme, etc.). Une routine de caractérisation de nanoparticules en suspension a ainsi été développée. La Microscopie Électronique en Transmission (MET) a permis d'établir une majorité des paramètres de la fiche d'identité, en combinant à la fois imagerie et spectroscopie (analyses chimiques). La préparation, dont dépendra la qualité des observations, nécessite un développement pour chaque matériau analysé. Pour cela, trois techniques ont été mises au point : le dépôt en voie sèche qui permet une observation directe et simple, la cryogénie qui permet de fixer l'état de la suspension et l'in-situ liquide qui permet d'observer directement la suspension sans changement d'état. Les analyses MET étant locales, une comparaison avec des techniques indirectes a été effectuée par Diffusion Statique (MALS) et Dynamique (DLS) de la Lumière avec et sans fractionnement par couplage flux-force (FFF). Deux matériaux modèles ont été choisis. Le premier est une nanoémulsion de lipides stabilisés par des surfactants, servant de vecteurs à des principes actifs. Une étude de vieillissement par interaction avec des protéines a été menée et de légères variations de taille ont été obtenues. Le second matériau sélectionné est une poudre de nanoparticules de dioxyde de titane, remises en suspension, utilisée dans les crèmes solaires en tant que filtres UV. Ces particules ont été observées avant et après passage en enceinte climatique afin d'observer les effets des rayons UV sur celles-ci. Ceci a confirmé la stabilité des particules. Les protocoles de caractérisation développés au cours de cette thèse peuvent ainsi servir de supports à l'étude d'autres nanoparticules en suspension.
... It is critical to accurately characterize liposomes and drug-liposome interactions as biophysical properties of liposomes are known to influence biological activity, biodistribution, and toxicity. Among the available techniques (Dynamic Light Scattering (DLS), Size Exclusion Chromatography (SEC), Atomic Force Microscopy (AFM), and cryo-EM), cryo-EM is the most precise and direct method to determine liposome lamellarity, size, shape and ultrastructure, which may reveal clues to mechanism of action toward the clinical endpoints of efficacy and toxicity (Aissaoui et al., 2011;Al-Ahmady et al., 2016;Baxa, 2018;Bonnaud et al., 2013;Crawford et al., 2011; All rights reserved. No reuse allowed without permission. ...
Preprint
Full-text available
Liposomes are widely used as delivery systems in pharmaceutical, cosmetics and food industries, as well as a system for structural and functional study of membrane proteins. To accurately characterize liposomes, cryo-Electron Microscopy (cryo-EM) has been employed as it is the most precise and direct method to determine liposome lamellarity, size, shape and ultrastructure. However, its use is limited by the number of liposomes that can be trapped in the thin layer of ice that spans holes in the perforated carbon film on EM grids. We report a long-incubation method for increasing the density of liposomes in holes. By increasing the incubation time, high liposome density was achieved even with extremely dilute (in the nanomolar range) liposome solutions. This long-incubation method has been successfully employed to study the structure of an ion channel reconstituted into liposomes. This method will also be useful for preparing other biological macromolecules / assemblies for structural studies using cryo-EM.
... Transmission Electron Microscopy (TEM) (Pyrz and Buttrey 2008), Scanning Electron Microscopy (SEM) (Goldstein et al. 2014), and cryoEM (Crawford et al. 2011) are commonly used to study the shape of nanoparticles. The chemical identity of a nanoparticle dictates which of these techniques should be applied. ...
Article
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The increasing nanomedicine usage has raised concerns about their possible impact on human health. Present evaluation strategies for nanomaterials rely on a case-by-case hazard assessment. They take into account material properties, biological interactions, and toxicological responses. Authorities have also emphasized that exposure route and intended use should be considered in the safety assessment of nanotherapeutics. In contrast to an individual assessment of nanomaterial hazards, we propose in the present work a novel and unique evaluation strategy designed to uncover potential adverse effects of such materials. We specifically focus on spherical engineered nanoparticles used as parenterally administered nanomedicines. Standardized assay protocols from the US Nanotechnology Characterization Laboratory as well as the EU Nanomedicine Characterisation Laboratory can be used for experimental data generation. We focus on both cellular uptake and intracellular persistence as main indicators for nanoparticle hazard potentials. Based on existing regulatory specifications defined by authorities such as the European Medicines Agency and the United States Food and Drug Administration, we provide a robust framework for application-oriented classification paired with intuitive decision making. The Hazard Evaluation Strategy (HES) for injectable nanoparticles is a three-tiered concept covering physicochemical characterization, nanoparticle (bio)interactions, and hazard assessment. It is cost-effective and can assist in the design and optimization of nanoparticles intended for therapeutic use. Furthermore, this concept is designed to be adaptable for alternative exposure and application scenarios. To the knowledge of the authors, the HES is unique in its methodology based on exclusion criteria. It is the first hazard evaluation strategy designed for nanotherapeutics.
... 33, [40][41][42][43][44][45][46][47] Formulations also consist of a phospholipid and cholesterol to maintain structural integrity and an outer lipid that is decorated with polyethylene glycol (PEG). 40,41,48,49 and extends in vivo circulation time. [50][51][52] Recently, LNPs have succeeded in clinical trials to deliver small interfering RNA for treatment of metabolic disorders (patisiran) and are frontrunners in clinical development of mRNA vaccines (mRNA-1440) without noted toxic effects. ...
Article
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The promise of gene therapy for the treatment of cystic fibrosis has yet to be fully clinically realized despite years of effort toward correcting the underlying genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR). mRNA therapy via nanoparticle delivery represents a powerful technology for the transfer of genetic material to cells with large, widespread populations, such as airway epithelia. We deployed a clinically relevant lipid-based nanoparticle (LNP) for packaging and delivery of large chemically modified CFTR mRNA (cmCFTR) to patient-derived bronchial epithelial cells, resulting in an increase in membrane-localized CFTR and rescue of its primary function as a chloride channel. Furthermore, nasal application of LNP-cmCFTR restored CFTR-mediated chloride secretion to conductive airway epithelia in CFTR knockout mice for at least 14 days. On day 3 post-transfection, CFTR activity peaked, recovering up to 55% of the net chloride efflux characteristic of healthy mice. This magnitude of response is superior to liposomal CFTR DNA delivery and is comparable with outcomes observed in the currently approved drug ivacaftor. LNP-cmRNA-based systems represent a powerful platform technology for correction of cystic fibrosis and other monogenic disorders. The use of lipid nanoparticles to deliver therapeutic mRNA is an increasingly attractive gene therapy modality. Here, Robinson et al. demonstrate that lipid nanoparticle transfection of mRNA can repair the central defect in cystic fibrosis, chloride transport, both in vitro and in vivo. The authors discuss why this technology is a promising treatment option for monogenic disorders such as cystic fibrosis.
... Recently, we modified an attractive positively charged LNP, which is a lipid-based particle with diameter ~67 nm formed by mixing an ethanol stream containing the lipid mixture with an aqueous stream containing the siRNA. [18][19][20] LNP has been linked with bone formation and used for anabolic therapy, 16,21 but it has never been used in the treatment of cartilage diseases. Indeed, the LNP-siRNA system showed an exciting and promising silencing effect in vitro. ...
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Background Cartilage degeneration affects millions of people but preventing its degeneration is a big challenge. Although RNA interference (RNAi) has been used in human trials via silencing specific genes, the cartilage RNAi has not been possible to date because the cartilage is an avascular and very dense tissue with very low permeability. Purpose The objective of this study was to develop and validate a novel lipid nanoparticle (LNP)-siRNA delivery system that can prevent cartilage degeneration by knocking down specific genes. Methods LNP transfection efficiency was evaluated in vitro and ex vivo. Indian Hedgehog (Ihh) has been correlated with cartilage degeneration. The in vivo effects of LNP-Ihh siRNA complexes on cartilage degeneration were evaluated in a rat model of surgery-induced osteoarthritis (OA). Results In vitro, 100% of chondrocytes were transfected with siRNA in the LNP-siRNA group. In accordance with the cell culture results, red positive signals could be detected even in the deep layer of cartilage tissue cultures treated by LNP-beacon. In vivo data showed that LNP is specific for cartilage, since positive signals were detected by fluorescence molecular tomography and confocal microscopy in joint cartilage injected with LNP-beacon, but not on the surface of the synovium. In the rat model of OA, intraarticular injection of LNP-Ihh siRNA attenuated OA progression, and PCR results showed LNP-Ihh siRNA exerted a positive impact on anabolic metabolism and negative impact on catabolic metabolism. Conclusion This study demonstrates that our LNP-RNAi delivery system has a significantly chondroprotective effect that attenuates cartilage degeneration and holds great promise as a powerful tool for treatment of cartilage diseases by knocking down specific genes.
... 7C1 was synthesized by reacting C 15 epoxide-terminated lipids with PEI 600 at a 14:1 molar ratio, and was formulated with C 14 PEG 2000 to produce nanoparticles with a diameter between 35 and 60 nm that were stable in PBS solution for at least 40 days ( Fig. 1c-e, Supplementary Fig. 1g-i). Particles formed multilamellar vesicles rather than periodic aqueous compartments containing siRNA that make up stable nucleic acid lipid particle formulations 19 (Fig. 1d, Supplementary Fig. 1j). Because particle charge at different pH can affect delivery by modifying interactions with serum proteins, the zeta potential of 7C1 formulated with siRNA at blood physiological pH (7.4) and pKa were measured 6 (Fig. 1f). ...
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... However, the mean sizes of the liposomal complexes estimated through DLS typical measurements were relatively higher than those obtained from TEM micrographs, which is likely a consequence of the enhanced scattering from the minimal number of aggregated vesicles that are presented, even at the high dilutions used in DLS measurements. The conflicting mean particle size estimated by DLS and TEM have also been reported in the recent literature [24,25]. ...
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... The morphology of the developed complexes was observed by cryo-TEM (Tecnai G2 F20 Cryo-TEM, FEI Company, Hillsboro, Oregon, USA) (30). The specimen was applied to holey carbon affixed to the grid (Quantifoil Micro Tools GmbH, Jena, Germany) and rapidly cooled to−170°C. ...
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... 7C1 was synthesized by reacting C 15 epoxide-terminated lipids with PEI 600 at a 14:1 molar ratio, and was formulated with C 14 PEG 2000 to produce nanoparticles with a diameter between 35 and 60 nm that were stable in PBS solution for at least 40 days ( Fig. 1c-e, Supplementary Fig. 1g-i). Particles formed multilamellar vesicles rather than periodic aqueous compartments containing siRNA that make up stable nucleic acid lipid particle formulations 19 (Fig. 1d, Supplementary Fig. 1j). Because particle charge at different pH can affect delivery by modifying interactions with serum proteins, the zeta potential of 7C1 formulated with siRNA at blood physiological pH (7.4) and pKa were measured 6 (Fig. 1f). ...
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... They are composed of an ionizable amino lipid (e.g., heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA [14]), a phosphatidylcholine (1,2-distearoyl-sn-glycero-3phosphocholine, DSPC), cholesterol and a coat lipid (polyethylene glycol-dimyristolglycerol, PEG-DMG) at molar ratio of 50:10:38.5:1.5 ( Figure 1). CryoTEM revealed that LNP systems formed by mixing an ethanol stream containing the lipid mixture with an aqueous stream containing the siRNA, by either a T-tube [20] or microfluidic-based mixing [21,22], have an electron-dense core instead of the less dense aqueous core characteristic of vesicular structures [21,23]. Indeed, the LNP siRNA systems have an interior lipid core containing siRNA complexed with ionizable cationic lipid, as shown by the absence of 31 P NMR signal from free phosphorothioate in the siRNA and the complete protection of siRNA degradation by external RNases [21]. ...
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Technological advances in both siRNA (small interfering RNA) and whole genome sequencing have demonstrated great potential in translating genetic information into siRNA-based drugs to halt the synthesis of most disease-causing proteins. Despite its powerful promises as a drug, siRNA requires a sophisticated delivery vehicle because of its rapid degradation in the circulation, inefficient accumulation in target tissues and inability to cross cell membranes to access the cytoplasm where it functions. Lipid nanoparticle (LNP) containing ionizable amino lipids is the leading delivery technology for siRNA, with five products in clinical trials and more in the pipeline. Here, we focus on the technological advances behind these potent systems for siRNA-mediated gene silencing.
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Thesis
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Due to the global epidemic outbreak in recent years, membrane research and membrane-derived products have been of increasingly wide interest for medical applications. Currently, a new but important development direction of membranes in medicine goes beyond the separation function of the membrane itself to realize multifunctional integration. With the introduction of additional functions such as scaffolding, responsiveness, and sensing, membranes have exhibited excellent performance in the areas of tissue engineering, drug delivery and disease diagnosis. From this perspective, we will review the recent progress made by membranes in the medical field and emphasize the principles of function integration and separation. Possible challenges will be proposed, and future development directions for medicine-related membranes will be discussed.
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Short peptides reflecting major histocompatibility complex (MHC) class I (MHC‐I) epitopes frequently lack sufficient immunogenicity to induce robust antigen (Ag)‐specific CD8+ T cell responses. In the current work, it is demonstrated that position‐scanning peptide libraries themselves can serve as improved immunogens, inducing Ag‐specific CD8+ T cells with greater frequency and function than the wild‐type epitope. The approach involves displaying the entire position‐scanning library onto immunogenic nanoliposomes. Each library contains the MHC‐I epitope with a single randomized position. When a recently identified MHC‐I epitope in the glycoprotein gp70 envelope protein of murine leukemia virus (MuLV) is assessed, only one of the eight positional libraries tested, randomized at amino acid position 5 (Pos5), shows enhanced induction of Ag‐specific CD8+ T cells. A second MHC‐I epitope from gp70 is assessed in the same manner and shows, in contrast, multiple positional libraries (Pos1, Pos3, Pos5, and Pos8) as well as the library mixture give rise to enhanced CD8+ T cell responses. The library mixture Pos1‐3‐5‐8 induces a more diverse epitope‐specific T‐cell repertoire with superior antitumor efficacy compared to an established single mutation mimotope (AH1‐A5). These data show that positional peptide libraries can serve as immunogens for improving CD8+ T‐cell responses against endogenously expressed MHC‐I epitopes. Short peptides usually lack sufficient immunogenicity to induce robust antigen (Ag)‐specific CD8+ T‐cell responses. With a next‐generation vaccine adjuvant, it is demonstrated that position‐scanning peptide libraries of tumor antigens themselves can serve as improved immunogens to induce Ag‐specific CD8+ T cells with greater frequency and function compared to the wild‐type epitope.
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Thesis
Cancer immunotherapy has advanced rapidly over the past decade leading to clinical approval of immune checkpoint blockade and adoptive cell transfer therapies. Further efforts into development of therapeutic vaccines had generated promising results in pre-clinical and clinical studies. Here, we demonstrate novel methodology for preparation of cell membranes into nano-sized vesicles and the development of characterization methods via nanoparticle flow cytometry. Cancer cell membranes from murine melanoma cell line expressing model antigen, ovalbumin, were used for generation of PEGylated vehicles (PEG-NPs), which efficiently delivered endogenous membrane-associated cancer antigens to the draining lymph nodes after subcutaneous administration. PEG-NPs were efficiently taken up by dendritic cells and, when dosed with a potent adjuvant, led to antigen-specific T cell activation and proliferation approximately 4-fold greater than treatment with traditional freeze-thaw lysates. In combination with immune checkpoint blockade (anti-PD-1 treatment), our vaccination approach led to therapeutic cure of 63% of mice and persistent memory responses rejecting additional tumor rechallenge. We further utilized our nanoparticle platform by using adjuvant-matured dendritic cells (DCs) generating MPLA-activated dendritic cell membrane vesicles ((MPLA)DC-MVs). This preparation led to nanoparticles carrying T cell activation ligands (CD80 and CD86) and promoted their proliferation activation in vitro compared to antigen peptide alone, as demonstrated by 2-fold increase in proliferation and 5- to 8-fold increase in live cell numbers and expression of CD25 activation marker. In addition, (MPLA)DC-MVs, but not unstimulated DC-MVs, resulted in activation of immature dendritic cells in vitro, indicated by 2- and 1.3-fold greater expression of CD40 and CD80, respectively. Administration of this formulation in vivo together with OVA peptide epitope led to 2-fold enhanced expansion and maintenance of antigen-specific T cells compared to peptide alone in mice that received adoptive cell transfer or had established OVA-expressing tumors. These studies had demonstrated the use for cell membranes in immunotherapy as vaccine vehicles, but further characterization and optimization could allow for improved efficacy, prompting us to adopt flow cytometry methods aimed at nanoparticle analysis. The technique was established by analysis of lipid-based synthetic formulations focused on demonstrating effective fluorescence detection and separation of individual particle populations. Proof of concept studies were used to confirm presence of ovalbumin on membrane-derived vesicles with antibody staining. Finally, we had utilized this technique to examine antigen display on hepatitis virus C vaccine formulation in order to determine if broadly neutralizing antibodies can bind efficiently and thus if they can be raised in mice immunized with these formulations. Our studies demonstrate that similar levels of broadly neutralizing antibody binding to nanoparticles translate to similar level of protection against cross-strain viral challenge. Taken together, this work has generated a foundation for further research into the use of cell membranes as nanoparticles for immunotherapeutic approaches and techniques necessary for their characterization.
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Here we show how dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) spectroscopy experiments permit the atomic level structural characterization of loaded and empty lipid nanoparticles (LNPs). The LNPs used here were synthesized by microfluidic mixing technique and are composed of ionizable cationic lipid (DLin-MC3-DMA), a phospholipid (DSPC), cholesterol and PEG (DMPE-PEG 2000), as well as encapsulated cargoes which are either phosphorothioated-siRNA (50 or 100%) or mRNA. We show that LNPs form physically stable complexes with bioactive drug siRNA for a period of 94 days. Relayed DNP experiments are performed to study 1H-1H spin diffusion and to determine the spatial location of the various components of the LNP by studying the enhancement factors as a function of polarization time. We observe a striking feature of LNPs in the presence and in the absence of encapsulating siRNA or mRNA by comparing our experimental results to numerical spin diffusion modelling. We observe that LNPs form a layered structure and we detect that DSPC and DMPE-PEG 2000 lipids form a surface rich layer in the presence (or absence) of the cargoes, and that the cholesterol and ionizable cationic lipid are embedded in the core. Furthermore, relayed DNP 31P solid-state NMR experiments allow the location of the cargo encapsulated in the LNPs to be determined. Based on the results we propose a new structural model for the LNPs which features a homogeneous core with a tendency for layering of DSPC and DMPE-PEG at the surface.
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The biological activities of fatty acids (FAs) can differ with size even for lipids of similar compositions. The aim of this study was to develop size-controlled FA particles and to evaluate their toxicity as a function of size. Well-stabilized nano- and microscale linoleic acid (LA) were fabricated based on specific physical factors. Then, resulting LAs were characterized by size distribution, surface charge, assembly structure, composition, and serum effects. The sizes of the nano- (LAnano) and microscale (LAmicro) LAs, determined by electron microscopy, were 109 nm and 12 μm, respectively. LAnano, a multilamellar structure as determined by cryo-electron microscopy, was rapidly internalized into cells via free fatty acid receptor 3. After internalization, LAnano, but not LAmicro, induced nuclear translocation of fatty acid binding protein 4 (FABP4). Translocation of FABP4 into the nucleus then induced expression of the FA metabolism-related genes InsR and AdipoR1. Their expression was significantly increased in the presence of only LAnano. Cytotoxicity was also significantly increased in cells treated with LAnano, but not LAmicro, as indicated by the endoplasmic reticulum stress markers CHOP and GRP78. Therefore, our results demonstrated that FAs with the same composition but varying in size can cause different cellular responses.
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Microfluidic devices are mircoscale fluidic circuits used to manipulate liquids at the nanoliter scale. The ability to control the mixing of fluids and the continuous nature of the process make it apt for solvent/antisolvent precipitation of drug-delivery nanoparticles. This review describes the use of numerous microfluidic designs for the formulation and production of lipid nanoparticles, liposomes and polymer nanoparticles to encapsulate and deliver small molecule or genetic payloads. The advantages of microfluidics are illustrated through examples from literature comparing conventional processes such as beaker and T-tube mixing to microfluidic approaches. Particular emphasis is placed on examples of microfluidic nanoparticle formulations that have been tested in vitro and in vivo. Fine control of process parameters afforded by microfluidics, allows unprecedented optimization of nanoparticle quality and encapsulation efficiency. Automation improves the reproducibility and optimization of formulations. Furthermore, the continuous nature of the microfluidic process is inherently scalable, allowing optimization at low volumes, which is advantageous with scarce or costly materials, as well as scale-up through process parallelization. Given these advantages, microfluidics is poised to become the new paradigm for nanomedicine formulation and production.
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Cryo‐transmission electron microscopy (cryo‐TEM or cryo‐EM) and cryo‐electron tomography (cryo‐ET) offer robust and powerful ways to visualize nanoparticles. These techniques involve imaging of the sample in a frozen‐hydrated state, allowing visualization of nanoparticles essentially as they exist in solution. Cryo‐TEM grid preparation can be performed with the sample in aqueous solvents or in various organic and ionic solvents. Two‐dimensional (2D) cryo‐TEM provides a direct way to visualize the polydispersity within a nanoparticle preparation. Fourier transforms of cryo‐TEM images can confirm the structural periodicity within a sample. While measurement of specimen parameters can be performed with 2D TEM images, determination of a three‐dimensional (3D) structure often facilitates more spatially accurate quantization. 3D structures can be determined in one of two ways. If the nanoparticle has a homogeneous structure, then 2D projection images of different particles can be averaged using a computational process referred to as single particle reconstruction. Alternatively, if the nanoparticle has a heterogeneous structure, then a structure can be generated by cryo‐ET. This involves collecting a tilt‐series of 2D projection images for a defined region of the grid, which can be used to generate a 3D tomogram. Occasionally it is advantageous to calculate both a single particle reconstruction, to reveal the regular portions of a nanoparticle structure, and a cryo‐electron tomogram, to reveal the irregular features. A sampling of 2D cryo‐TEM images and 3D structures are presented for protein based, DNA based, lipid based, and polymer based nanoparticles. WIREs Nanomed Nanobiotechnol 2017, 9:e1417. doi: 10.1002/wnan.1417 This article is categorized under: • Therapeutic Approaches and Drug Discovery > Emerging Technologies • Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
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RNAi technology is currently experiencing a revival due to remarkable improvements in efficacy and viability through oligonucleotide chemical manipulations and/or via their packaging into nano-scale carriers. At present there is no FDA approved system for siRNA technology in humans. The design of the next generation of siRNA carriers requires a deep understanding of how nanoparticle's physicochemical properties truly impart biological stability and efficiency. For example, we now know that nanoparticles need to be sterically stabilized in order to meet adequate biodistribution profiles. At present, targeting, uptake, and in particular endosomal escape are amongst the most critical challenges impairing RNAi technologies. The disruption of endosomes encompasses membrane transformations (for example pore formation) that cost significant elastic energy. Nanoparticle size and shape have been identified as relevant parameters impacting tissue accumulation and cellular uptake. In this paper we demonstrate that the internal structure of lipid-based particles offers a different handle to promote endosomal membrane topological disruptions that enhance siRNA delivery. Specifically, we designed sterically stabilized lipid-based particles that differ from traditional liposomal systems by displaying highly ordered bicontinuous cubic internal structures that can be loaded with large amounts of siRNA. This system differs from traditional siRNA-containing liposomes (lipoplexes) as the particle-endosomal membrane interactions are controlled by elasticity energetics and not by electrostatics. The resulting "PEGylated-cuboplex" has the ability to deliver siRNA and specifically knockdown genes with efficiencies that surpass those achieved by traditional lipoplex systems.
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The self-assembly of oppositely charged biomacromolecules has been extensively studied due its pertinence in the design of functional nanomaterials. Using cryo electronic microscopy (cryo-EM), optical light scattering and fluorescence microscopy, we investigated the structure and phase behavior of PEGylated (PEG: polyethylene-glycol) cationic liposome-DNA nanoparticles (CL-DNA NPs) as a function of DNA length, topology (linear and circular) and ρchg (the molar charge ratio of cationic lipid to anionic DNA). Although all NPs studied here showed a lamellar internal nanostructure, NPs formed with short (~ 2 kbps), linear, polydisperse DNA were defect-rich and contained smaller domains. Unexpectedly, we found distinctly different equilibrium structures away from the isoelectric point. At ρchg > 1, in the excess cationic lipid regime, thread-like micelles rich in PEG-lipid were found to coexist with NPs, cationic liposomes and spherical micelles. At high concentrations these PEGylated thread-like micelles formed a well-ordered, patterned morphology with highly uniform inter-micellar spacing. At ρchg < 1, in the excess DNA regime and with no added salt, individual NPs were tethered together via long, linear DNA (48 kbps λ-DNA) into a biopolymer-mediated floc. Our results provide insight on what equilibrium nanostructures can form when oppositely charged macromolecules self-assemble in aqueous media. Self-assembled, well-ordered thread-like micelles and tethered nanoparticles may have a broad range of applications in bionanotechnology, including nanoscale lithograpy and the development of lipid-based multi-functional nanoparticle networks. .
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This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.
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For several decades extensive research has been conducted into the development of fusogenic lipid nanoparticles (LNPs) capable of introducing large, charged molecules into the cytoplasm of target cells. The majority of this work has focused on cationic LNPs encapsulating nucleic acids ranging from small oligonucleotides to large plasmid constructs thousands of bases long. However, since the introduction of siRNA payloads this quest for a non-viral, intracellular delivery systems has advanced significantly. Of particular importance was the demonstration that LNPs containing ionizable, dialkylamino lipids, enable potent hepatic gene silencing across species including humans. This review focuses on the evolution of this delivery system, summarizes the promising data now emerging from clinical trials and considers future directions for the platform.
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Small non-coding RNA (ncRNA) therapeutics make use of small ncRNA effectors for desired therapeutic purposes that are essentially short (10-20 kD) RNA segments. These small ncRNA effectors are potentially tremendously powerful therapeutic agents, but are typically unable to reach disease target cells in vivo without the assistance of a delivery system or vector. The main focus of this review is the use of lipid-based nanoparticles (LNPs) for the functional delivery of small ncRNA effectors in vivo. LNPs appear to be amongst the most effective delivery systems currently available for this purpose. Moreover, studies on LNP-mediated delivery in vivo are leading to the emergence of useful biophysical parameters and physical organic chemistry rules that provide a framework for understanding LNP-mediated in vivo delivery behaviors and outcomes. These same parameters and rules should also suggest ways and means to develop next generations of LNPs with genuine utility and long-term clinical viability.
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RNA interference (RNAi) therapeutics appear to offer substantial opportunities for future therapy. However, post-administration RNAi effectors are typically unable to reach disease target cells in vivo without the assistance of a delivery system or vector. The main focus of this review is on lipid-based nanoparticle (LNP) delivery systems in current research and development that have at least been shown to act as effective delivery systems for functional delivery of RNAi effectors to disease target cells in vivo. The potential utility of these LNP delivery systems is growing rapidly, and LNPs are emerging as the preferred synthetic delivery systems in preclinical studies and current nonviral RNAi effector clinical trials. Moreover, studies on LNP-mediated delivery in vivo are leading to the emergence of useful biophysical parameters and physical organic chemistry rules that provide a framework for understanding in vivo delivery behaviors and outcomes. These same parameters and rules should also suggest ways and means to develop next generations of LNPs with genuine utility and long-term clinical viability.
Article
The characterization of liposomes was undertaken using in-situ microfluidic transmission electron microscopy. Liposomes were imaged without contrast enhancement processing or cryogenic treatment, allowing for observation of functional liposomes in an aqueous environment. The stability and quality of the liposome structures observed were found to be highly dependent on the surface and liposome chemistries within the liquid cell. The successful imaging of liposomes suggests potential for extension of in-situ microfluidic TEM to a wide variety of other biological and soft matter systems and processes.
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A primary consideration when developing lipid nanoparticle (LNP) based small interfering RNA (siRNA) therapeutics is formulation polydispersity or heterogeneity. The level of heterogeneity of physicochemical properties within a pharmaceutical batch could greatly affect the bioperformance, quality, and ability of a manufacturer to consistently control and reproduce the formulations. This article studied the heterogeneity in the size, composition, and in vitro performance of siRNA containing LNPs, by conducting preparative scale fractionation using a sephacryl S-1000 based size-exclusion chromatography (SEC) method. Eight LNPs with size in the range of 60 ~ 190 nm were first evaluated by the SEC method for size polydispersity characterization, and it was found that LNPs in the range of 60~ 150 nm could be well-resolved. Two LNPs (LNP A and LNP B) with similar bulk properties were fractionated and fractions were studied in-depth for potential presence of polydispersity in size, composition, in vitro silencing, as well as cytotoxicity. LNP A was deemed to be monodisperse following results of a semi-preparative SEC fractionation that showed similar size, chemical composition, in vitro silencing activity, and cytotoxicity across the fractions. Therefore, LNP A represents a relatively homogeneous formulation and offers less challenge in its pharmaceutical development. In contrast, LNP B fractions were shown to be significantly more polydisperse in size distribution. Interestingly, LNP B SEC fractions also exhibited profound compositional variations (e.g., 5 fold difference in N/P ratio and 3 fold difference in lipid composition) along with up to 40 fold differences in the in vitro silencing activity. The impact of LNP size and formulation composition on in vitro performance is also discussed. The present results demonstrate the complexity and potential for presence of heterogeneity in LNP-based siRNA drug products. This underscores the need for tools that yield a detailed characterization of LNP formulations. This capability in tandem with the pursuit of improved formulation and process design can lead to more facile development of LNP-based siRNA pharmaceuticals of higher quality.
Article
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.
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For the purposes of object or defect identification required in industrial vision applications, the operations of mathematical morphology are more useful than the convolution operations employed in signal processing because the morphological operators relate directly to shape. The tutorial provided in this paper reviews both binary morphology and gray scale morphology, covering the operations of dilation, erosion, opening, and closing and their relations. Examples are given for each morphological concept and explanations are given for many of their interrelationships.
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The aim of this study was to determine the minimal number of particles required for assessing a reliable particle size distribution from images acquired using microscopy. The proposed methodology used the one-sample Kolmogorov–Smirnov statistic to choose an a priori number of particles and the two-sample Kolmogorov–Smirnov test to validate the sub-sampling procedure inherent to microscopic preparation. The methodology was applied to number-based particle size distributions of starch granules.
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Cryo-transmission electron microscopy, c-TEM, has during the last 10 years contributed significantly to the understanding of the numerous, and often complex, structures formed by amphiphilic molecules in dilute aqueous solutions. In particular, the method has evolved as an important tool for the investigation of liposomes. In this review, we discuss and show examples of how the technique has been utilised to gain new information on the form and structure of liposomes, as well as on the morphological changes taking place upon encapsulation of drugs or interactions with surfactants, DNA and other polyelectrolytes. Several examples where c-TEM has been successfully employed to visualise related amphiphilic structures, such as thread-like micelles and particles of reversed phases, are also presented. In addition, we discuss recent developments concerning sample preparation and interpretation of images, as well as possible artefacts and their origin.
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Despite recent progress, systemic delivery remains the major hurdle for development of safe and effective small inhibitory RNA (siRNA)-based therapeutics. Encapsulation of siRNA into liposomes is a promising option to overcome obstacles such as low stability in serum and inefficient internalization by target cells. However, a major liability of liposomes is the potential to induce an acute inflammatory response, thereby increasing the risk of numerous adverse effects. In this study, we characterized a liposomal siRNA delivery vehicle, LNP201, which is capable of silencing an mRNA target in mouse liver by over 80%. The biodistribution profile, efficacy after single and multiple doses, mechanism of action, and inflammatory toxicity are characterized for LNP201. Furthermore, we demonstrate that the glucocorticoid receptor (GR) agonist dexamethasone (Dex) inhibits LNP201-induced cytokine release, inflammatory gene induction, and mitogen-activated protein kinase (MAPK) phosphorylation in multiple tissues. These data present a possible clinical strategy for increasing the safety profile of siRNA-based drugs while maintaining the potency of gene silencing.
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In the 10 years that have passed since the Nobel prize-winning discovery of RNA interference (RNAi), billions of dollars have been invested in the therapeutic application of gene silencing in humans. Today, there are promising data from ongoing clinical trials for the treatment of age-related macular degeneration and respiratory syncytial virus. Despite these early successes, however, the widespread use of RNAi therapeutics for disease prevention and treatment requires the development of clinically suitable, safe and effective drug delivery vehicles. Here, we provide an update on the progress of RNAi therapeutics and highlight novel synthetic materials for the encapsulation and intracellular delivery of nucleic acids.
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Improvements in the synthesis, deprotection and purification of oligoribonucleotides are described. These advances allow for reduced synthesis and deprotection times, while improving product yield. Coupling times are reduced by half using 5-ethylthio-1H-tetrazole (S-ethyltetrazole) as the activator. Base and 2'-O-t-butyldimethylsilyl deprotection with methylamine (MA) and anhydrous triethylamine/hydrogen fluoride in N-methylpyrrolidinone (TEA.HF/NMP), respectively, requires a fraction of the time necessitated by current standard methods. In addition, the ease of oligoribonucleotide purification and analysis have been significantly enhanced using anion exchange chromatography. These new methods improve the yield and quality of the oligoribonucleotides synthesized. Hammerhead ribozymes synthesized utilizing the described methods exhibited no diminution in catalytic activity.
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Cationic lipid-mediated gene transfer and delivery still attract great attention of many gene therapy laboratories. From the point of view of the most important characteristics of lipoplex particles, e.g. its charge and size, we reviewed recent studies available. In general, the paper deals with non-viral systems of gene transfer into eukaryotic cell based on various lipids. Having usually less efficiency in gene transfer, lipid-based gene transfer vehicles (lipoplexes/genosomes) are characterized with certain advantages even over viral ones: they are less toxic and immunogenic, could be targetable and are easy for large-scale production, a size of transferred DNA being quite high. Conditions of DNA condensation during interactions with lipids are described. Results of the studies of mechanism of DNA-lipid complex interactions with the cell membrane and their transport into the nucleus are summarized. Dependence of efficiency of gene transfer on lipoplex structure and physical-chemical properties is reviewed. Advantages and disadvantages of different macromolecule complexes from the point of view of transfection efficiency, possibility of use in vivo, cytotoxicity and targeted gene transfer in certain organs and tissues are also discussed. Results of transfection of different cells using neutral, anion and cation liposomes are reviewed. The conclusion reached was that efficiency and specificity of gene transfer may grow considerably when mixed macromolecule lipid systems including polycations and glycolipids are used.
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In this study, small liposomes obtained by high-pressure homogenization were fractionated according to their particle sizes by size exclusion chromatography (SEC). The subfractions were analyzed by photon correlation spectroscopy (PCS) as well as enzymatic phosphatidylcholine (PC) assay for their particle sizes and lipid contents, respectively. For small egg PC-liposomes, a size range of 15 nm to 60 nm was found, with 80% of the vesicles being smaller than 30 nm in size. This is in contradiction to a mean size of 85 +/- 32 nm as indicated by PCS without fractionation. The PCS technique appears to underestimate very small particles below 30 nm if (few) bigger particles are present. The PCS particle size analysis of unfractionated hydrogenated egg PC/cholesterol-liposomes (2:1, mole/mole) by PCS did not yield any significant results. On fractionation, however, a particle size range of 40 nm to 120 nm was determined in a reproducible manner. Our results indicate that the combination of size exclusion fractionation with subsequent photon correlation spectroscopic particle size analysis and enzymatic PC assay can give both more detailed and more reliable insight into the particle size distribution of small liposomes than PCS alone.
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The aim of the current study was to investigate the ability of a fixed-angle routine photon correlation spectrometer (PCS) to resolve bimodal size distributions. The focus was on dispersions consisting of a majority of smaller and a minority of bigger particles. Monodisperse latex beads of sizes from 21 to 269 nm were measured first as single-size dispersions and then with various binary blends. For single-size dispersions, the mean diameters obtained were as indicated by the manufacturer, except for 21- and 34-nm particles, which were somewhat smaller. PCS analysis of blends of 21 + 102-nm and 34 + 102-nm particles resulted in bimodal distributions with particle diameters of the 2 peaks in the expected magnitude down to critical blending ratios of 0.002% and 0.08% of bigger particles, respectively. At these ratios, PCS results became inconsistent, and an increased number of monomodal results and/or high residuals were seen. For 21 + 102-nm blends, at even smaller ratios (0.001%), more consistent results were obtained again with predominantly monomodal distributions in the size range of the smaller particles (ie, the bigger particles were neglected). PCS analysis of blends of 21 + 269-nm particles yielded bimodal distributions with diameters within the expected magnitude as long as the content of bigger particles did not exceed 0.005%. Above this ratio, predominantly monomodal results with mean diameters in the magnitude of the bigger particles were obtained (ie, the smaller particles were neglected). In conclusion, a routine PCS instrument can resolve bimodal size distributions of colloidal dispersions only at certain ratios of the 2 subpopulations. Both low and high ratios lead to 1 of the 2 subpopulations being neglected.
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Evidence suggests that uremic toxins such as hippurate (HA), indoleacetate (IA), indoxyl sulfate (IS), and 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF) promote the progression of renal failure by damaging tubular cells via rat organic anion transporter 1 (rOat1) and rOat3 on the basolateral membrane of the proximal tubules. The purpose of the current study is to evaluate the in vivo transport mechanism responsible for their renal uptake. We investigated the uremic toxins transport mechanism using the abdominal aorta injection technique [i.e., kidney uptake index (KUI) method], assuming minimal mixing of the bolus with serum protein from circulating serum. Maximum mixing was estimated to be 5.8% of rat serum by measuring estrone sulfate extraction after addition of 0-90% rat serum to the arterial injection solution. Saturable renal uptake of p-aminohippurate (PAH, K(m) = 408 microM) and benzylpenicillin (PCG, K(m) = 346 microM) was observed, respectively. The uptake of PAH and PCG was inhibited in a dose-dependent manner by unlabeled PCG (IC(50) = 47.3 mM) and PAH (IC(50) = 512 microM), respectively, suggesting that different transporters are responsible for their uptake. A number of uremic toxins inhibited the renal uptake of PAH and PCG. Excess PAH, which could inhibit rOat1 and rOat3, completely inhibited the saturable uptake of IA, IS, and CMPF by the kidney, and by 85% for HA uptake. PCG inhibited the total saturable uptake of HA, IA, IS, and CMPF by 10%, 10%, 45%, and 65%, respectively, at the concentration selective for rOat3. rOat1 could be the primary mediator of the renal uptake of HA and IA, accounting for approximately 75% and 90% of their transport, respectively. rOat1 and rOat3 contributed equally to the renal uptake of IS. rOat3 could account for about 65% of the uptake of CMPF under in vivo physiologic conditions. These results suggest that rOat1 and rOat3 play an important role in the renal uptake of uremic toxins and the induction of their nephrotoxicity.
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This study evaluated the effect of the imaginary component of the refractive index on laser diffraction particle size data for pharmaceutical samples. Excipient particles 1-5 microm in diameter (irregular morphology) were measured by laser diffraction. Optical parameters were obtained and verified based on comparison of calculated vs. actual particle volume fraction. Inappropriate imaginary components of the refractive index can lead to inaccurate results, including false peaks in the size distribution. For laser diffraction measurements, obtaining appropriate or "effective" imaginary components of the refractive index was not always straightforward. When the recommended criteria such as the concentration match and the fit of the scattering data gave similar results for very different calculated size distributions, a supplemental technique, microscopy with image analysis, was used to decide between the alternatives. Use of effective optical parameters produced a good match between laser diffraction data and microscopy/image analysis data. The imaginary component of the refractive index can have a major impact on particle size results calculated from laser diffraction data. When performed properly, laser diffraction and microscopy with image analysis can yield comparable results.
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The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA >1.0 log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.
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Polymeric drug-loaded nanoparticles have been extensively studied in the field of drug delivery. Biodistribution depends on the physicochemical properties of particles, especially size. The global message from the literature is that small particles have an enhanced ability to reach their target. The present review highlights the difficulties in validating the data from biodistribution studies without accurate particle size determination.
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It has been recently reported that under certain conditions the size distribution of vesicles being formed upon addition of fresh surfactant to an aqueous solution is strongly affected by the presence of preformed and narrowly distributed vesicles. In particular, the final size distribution is strongly biased toward the size distribution of the initial vesicles (the so-called “matrix effect”). On the basis of a novel experimental approach, we present here an investigation of the matrix effect and the corresponding fission processes of oleic acid/oleate vesicles and mixed POPC/oleic acid/oleate vesicles (POPC = 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). This novel approach is based on two complementary techniques:  the use of cryotransmission electron microscopy (cryo-TEM) and the entrapment of ferritin which can be visualized by cryo-TEM in the initial preformed POPC liposomes. Addition of micellar oleate to an equimolar amount of 100 nm extruded POPC liposomes results in the formation of mixed vesicles with a new size and ferritin distribution. This can be determined by cryo-TEM, and two main findings have thus been obtained. On one hand, the matrix effect has been substantiated; i.e., the size distribution of the preformed liposomes strongly affects the final size distribution. However, and surprisingly, the final suspension contains a large amount of vesicles with a diameter between 20 and 40 nm, i.e., significantly smaller than the preformed ones. The fact that these small vesicles were not present in the initial population of preformed vesicles and the fact that some of them contain ferritin molecules, brings one to the conclusion that they have derived from fission processes of larger ferritin-containing vesicles. More generally, this cryo-TEM-based investigation also sheds light on the basic properties of oleic acid/oleate vesicles formed by spontaneous vesiculation, for example, the most probable size (main peak around 40−60 nm) and the surprisingly small unilamellarity (of the order of 1.02).
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It is often necessary to estimate the properties of particle size distributions from limited samples taken from large populations. When the distributions are broad, and higher order moments required, as in the case of volume based particle size distributions, the inferred parameters d3,50 (volume median diameter) and GSD (geometric standard deviation) can have high intrinsic errors not immediately obvious to the measuring scientist. We show that there is a critical number of particles, Ncrit, which must be counted or else the error may blow up catastrophically. Ncrit is very sensitive to the width of the distribution, and is approximately proportional to GSD11 We develop formulae to estimate the random sampling error inherent in measured values of the d3,50 and GSD for the log-normal distribution; compare the predictions to a typical experimental particle size measurement; and then generalize to the median of any arbitrary moment, dr, 50.
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A nonparametric and unsupervised method of automatic threshold selection for picture segmentation is presented. An otpimal threshold is selected by the discriminant criterion, namely, so as the maximize the separability of the resultant classes in gray levels. The procedure is very simple, utilizing only the zeroth- and first-order cumulative moments of the gray-level histogram. It is strightforward to extend the method to multithreshold problems. Several experimental results are also presented to support the validity of the method.
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Nanotechnology could be defined as the technology that has allowed for the control, manipulation, study, and manufacture of structures and devices in the "nanometer" size range. These nano-sized objects, e.g., "nanoparticles", take on novel properties and functions that differ markedly from those seen from items made of identical materials. The small size, customized surface, improved solubility, and multi-functionality of nanoparticles will continue to open many doors and create new biomedical applications. Indeed, the novel properties of nanoparticles offer the ability to interact with complex cellular functions in new ways. This rapidly growing field requires cross-disciplinary research and provides opportunities to design and develop multifunctional devices that can target, diagnose, and treat devastating diseases such as cancer. This article presents an overview of nanotechnology for the biologist and discusses the attributes of our novel XPclad((c)) nanoparticle formulation that has shown efficacy in treating solid tumors, single dose vaccination, and oral delivery of therapeutic proteins.
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Liposomes are highly versatile structures for research, therapeutic, and analytical applications. In order to assess the quality of liposomes and obtain quantitative measures that allow comparison between different batches of liposomes, various parameters should be monitored. For liposomes used in analytical and bioanalytical applications, the main characteristics include the average diameter and degree of size polydispersity; encapsulation efficiency; the ratio of phospholipids to encapsulant concentration; lamellarity determination. A detailed description of today's most commonly used methods and of novel techniques for the quantification of these aspects is presented in this report citing 182 references. Their advantages and limitations are discussed where appropriate in order to provide the reader with an understanding of the current state of the art assessment of liposome quality.
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The discovery and application of oligonucleotide technologies have already changed our fundamental understanding of biology and created a wealth of possibilities for development of new therapeutic entities. A major advantage of oligonucleotide-based technologies is the potential to develop specific therapeutic antagonists to approach targets previously considered undruggable with a traditional small-molecule approach. Although there is great potential for this technology in the development of new therapeutic agents, there are significant issues to be overcome before this technology can be readily translated into therapeutic use.Clinical Pharmacology & Therapeutics (2008); 84, 5, 628-632 doi:10.1038/clpt.2008.174
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Solid lipid nanoparticles (SLN) introduced in 1991 represent an alternative carrier system to traditional colloidal carriers, such as emulsions, liposomes and polymeric micro- and nanoparticles. SLN combine advantages of the traditional systems but avoid some of their major disadvantages. This paper reviews the present state of the art regarding production techniques for SLN, drug incorporation, loading capacity and drug release, especially focusing on drug release mechanisms. Relevant issues for the introduction of SLN to the pharmaceutical market, such as status of excipients, toxicity/tolerability aspects and sterilization and long-term stability including industrial large scale production are also discussed. The potential of SLN to be exploited for the different administration routes is highlighted. References of the most relevant literature published by various research groups around the world are provided.
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A thin aqueous film of suspended lipid vesicles?micelles is the object of choice for vitrification and subsequent study by cryoelectron microscopy. Just prior to vitrification, a thin film (compare with a soap film) is vulnerable to heat and mass exchange. Preparation of thin films in a temperature- and humidity-controlled environment is essential to prevent osmotic and temperature-induced alterations of the lipid structure, as will be explained in this chapter. Further automation of the preparative procedure by automatic blotting and PC control over the timing of critical steps (including vitrification) may further assist in the reproducible throughput of high-quality specimens. By cryotomography, taking a tilt series under low-dose conditions, a three-dimensional reconstruction of the specimen can be analyzed.
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Nanotechnology, or systems/device manufacture at the molecular level, is a multidisciplinary scientific field undergoing explosive development. The genesis of nanotechnology can be traced to the promise of revolutionary advances across medicine, communications, genomics and robotics. Without doubt one of the greatest values of nanotechnology will be in the development of new and effective medical treatments (i.e., nanomedicine). This review focuses on the potential of nanomedicine as it specifically relates to (1) the development of nanoparticles for enabling and improving the targeted delivery of therapeutic agents; (2) developing novel and more effective diagnostic and screening techniques to extend the limits of molecular diagnostics providing point-of-care diagnosis and more personalized medicine.
Non-uniform illumination correction in transmission electron microscopy
  • T Tasdizen
  • E Jurruss
  • R T Whitaker
Tasdizen, T., Jurruss, E., Whitaker, R.T., 2008. Non-uniform illumination correction in transmission electron microscopy. In: MICCAI Workshop on Microscopic Image Analysis with Applications in Biology.
Nanomedicine: Targeted Nanoparticles for the Delivery of Biosen-sors and Therapeutic Genes Challenges and opportunities for local and systemic delivery of siRNA and antisense oligonucleotides
  • T W Prow
Prow, T.W., 2004. Nanomedicine: Targeted Nanoparticles for the Delivery of Biosen-sors and Therapeutic Genes. Ph.D. Thesis. University of Texas, Medical Branch, TX. Sepp-Lorenzino, L., Ruddy, M.K., 2008. Challenges and opportunities for local and systemic delivery of siRNA and antisense oligonucleotides. Clin. Pharmacol. Ther. 84, 628–632.