This work was supported by the Austrian Science Fund Project P29110-B21, the &#8220;Hochschuljubil&#228;umsstiftung der Stadt Wien zur F&#246;rderung der Wissenschaft&#8221; Project H-3065/2011, the European Fund for Regional Development (EFRE, IWB2020), the Federal State of Upper Austria, and the &#8220;Land O&#214; Basisfinanzierung&#8221;.

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The authors have nothing to disclose.

Here, the isolation of lipoprotein particle fractions from human blood and the determination of their individual miRNA content is described step-by-step. It is critical to work in an RNase-free environment while handling isolated and synthesized miRNA&#8212;particle-embedded miRNA is obviously sheltered from enzymatic degradation. As the miRNA/particle ratio of native lipoprotein particles is rather low, artificial enrichment with miRNA is required to study the holo particle uptake of cells. Thereby, the reconstitution of HDL particles as described previously7 is modified to incorporate miRNA strands. Additionally, the separation of the lipid and protein fraction during this procedure allows scientists to examine lipid- and protein-associated components of the lipoprotein particle19. In a similar way, the labeling procedure of LDL particles is adapted. Interestingly, the addition of spermine&#8212;a natural stabilizer of nucleotides&#8212;did not influence the miRNA/particle ratio. It should be noted that, in principle, the method allows the infolding of other substances than miRNA within a lipoprotein particle. Of course, there is a limit with regard to the physical size of the substance based on the overall size of HDL (diameter: 5-12 nm) and LDL particles (diameter: 18-25 nm).
Concerning the quality control of reconstituted/labeled lipoprotein particles, HS-AFM is an applicable method to characterize HDL/LDL particles at the single particle level. In comparison to EM, it allows for shorter preparation times and near-physiological conditions (wet, room temperature).
Due to its inherent sensitivity and amplification, qPCR is the method of choice to detect low miRNA concentrations. Alternatively, single-molecule sensitive fluorescence microscopy, which is able to detect even individual molecules, would not be suitable due to the low concentrations of, for instance, fluorescently labeled miRNA strands per particle. Thus, the ratio of miRNA strands per native lipoprotein particle has been found to be 10-8. Artificial enrichment increases the ratio by a factor of 10,000, which facilitates the estimation of the cellular lipoprotein uptake rate (no significant difference is detected using native lipoprotein particles 19). The high sensitivity of qPCR makes it possible to determine this uptake rate by measuring the number of miRNA strands after the incubation time and the miRNA/particle ratio. It should be noted that the calculated value ignores cellular degradation and release of miRNA and, thus, represents at least a lower limit for the lipoprotein particle uptake ratio.
In the future, the method can be adapted to transfer pharmaceutical substances (notably also lipophilic ones) in cells and correlate their biological effect to the intracellular concentration (determined via the uptake rate of lipoprotein particles).

Lipoprotein particles are composed of a monolayer of amphiphilic lipids and cholesterol enclosing a core of cholesteryl esters and triglyceride fats. The whole particle is stabilized by membrane-embedded apolipoproteins, which define the particle&#8217;s biological functionality. Lipoprotein particles can be distinguished according to their respective increasing density and, thus, decreasing size, namely as very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), LDL, and HDL particles. Despite the transportation of water-insoluble components in the bloodstream, it has been demonstrated that HDL particles carry noncoding strands of miRNA1,2. Micro-RNAs are a class of short (usually two dozen nucleotides) RNA strands, which degrade intracellularly complementary mRNA strands and, thereby, alter the expression profile of certain proteins3,4,5,6. Furthermore, alterations of the miRNA profile have been found in a variety of diseases and, thus, the profile is applicable as a biomarker for diagnosis and prognosis. The extracellular transport of miRNAs between cells via lipoprotein particles may serve as an additional mechanism for intercellular mRNA level modulation. To quantitatively estimate the biological effect, knowledge regarding the absolute and relative miRNA content of lipoprotein particles is needed.
Quantitative real-time PCR is a suitable and relatively quick method to gain this information. Hence, the relative quantification (RQ) value can be calculated, and relative differences between different samples and lipoprotein fractions are estimable. Multiwell microfluidic array cards are a fast and easy-to-use method to determine the relative presence (equates to the RQ value) of miRNAs in a sample. Multiwell microfluidic array cards consist of 96 or 384 individual reaction chambers for individual qPCR reactions embedded in a microfluidic device. Each chamber contains the required hydrolysis probe and specific qPCR primers for one individual miRNA. The advantages are a short handling time due to standardization, a simple workflow, and a reduced number of pipetting steps. Moreover, the required sample volume is minimized. In contrast to the relative quantification, the absolute miRNA content requires a comparison of qPCR sample results with standard curves of known absolute numbers of miRNA strands. It should be noted that, due to their relatively low miRNA content, standard and, moreover, even single-molecule sensitive imaging techniques are not feasible&#8212;the artificial enrichment of lipoprotein particles with miRNA is inevitable to study cellular lipoprotein particle interaction and miRNA transfer. Regarding this, delipidation of the HDL particle followed with subsequent relipidation7 allows the incorporation of and, thus, enrichment with miRNA strands. Similar enrichment of LDL particles with miRNA is not feasible due to the hydrophobicity of the apoB-100 protein, which is the main constituent of the LDL particle. However, by addition of the polar solvent dimethyl sulfoxide (DMSO), which is able to penetrate lipid membranes, LDL particles can be artificially loaded with miRNA strands as well.
High-speed atomic force microscopy (HS-AFM) is a powerful tool for the characterization of biological specimens offering subnanometer spatial and subsecond temporal resolution8. Hence, it is a well-suited technique for the quality control of modified lipoprotein particles as native/reconstituted/labeled lipoprotein particles can be imaged under a near-physiological environment.
Here, a qPCR-based protocol is presented step-by-step to determine the absolute/relative miRNA content of lipoprotein particles and cell samples, which allows an estimation of the cellular lipoprotein particle uptake rate. Moreover, a method for the enrichment of lipoprotein particles with miRNA is demonstrated. This method may be adapted for the general manipulation of lipoprotein content and, hence, demonstrates the applicability of lipoprotein particles as targets for drug delivery.

<table><tbody><tr><td>Ultracentrifuge</td><td>Beckman</td><td>Optima L-100 XP</td><td>Lipoprotein isolation</td></tr><tr><td>Ultracentrifuge rotor</td><td>Beckman</td><td>TI 55.2</td><td>Lipoprotein isolation</td></tr><tr><td>Vacutainer (EDTA)</td><td>Becton Dickinson</td><td>366643</td><td>Lipoprotein isolation</td></tr><tr><td>Kaliumbromid</td><td>Sigma Aldrich</td><td>2110</td><td>Lipoprotein isolation</td></tr><tr><td>Ultracentrifuge tubes</td><td>Beckman</td><td>342414</td><td>Lipoprotein isolation</td></tr><tr><td>Ultracentrifuge tube sealer</td><td>Beckman</td><td>342428</td><td>Lipoprotein isolation</td></tr><tr><td>Sodiumchlorid</td><td>Carl Roth GmbH</td><td>P029.2</td><td>Lipoprotein isolation</td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>D/0650/50</td><td>Lipoprotein isolation</td></tr><tr><td>Dialysis tubes, MWCO 12 - 14k</td><td>Spectra</td><td>132700</td><td>Lipoprotein isolation</td></tr><tr><td>synthetic miRNA</td><td>microSynth</td><td>-</td><td>synthetic miRNA</td></tr><tr><td>TRIS buffer pH 7</td><td>Ambion</td><td>AM9850G</td><td>synthetic miRNA</td></tr><tr><td>TRIS buffer pH 8</td><td>Ambion</td><td>AM9855G</td><td>synthetic miRNA</td></tr><tr><td>sterile tubes</td><td>Carl Roth GmbH</td><td>ENE8.1</td><td>synthetic miRNA</td></tr><tr><td>Photometer</td><td>Eppendorf</td><td>Eppendorf Biophotometer</td><td>Bradford assay</td></tr><tr><td>Cuvette</td><td>Carl Roth GmbH</td><td>XK20</td><td>Bradford assay</td></tr><tr><td>Coomassie G-250 Stain</td><td>BioRad GmbH</td><td>161-0786</td><td>Bradford assay</td></tr><tr><td>Sodiumchlorid</td><td>Carl Roth GmbH</td><td>3957.1</td><td>Delipitation</td></tr><tr><td>EDTA</td><td>Fluka Analytical</td><td>03690-100ML</td><td>Delipitation</td></tr><tr><td>TRIS buffer pH 8.0</td><td>Ambion</td><td>AM9855G</td><td>Delipitation</td></tr><tr><td>Ethanol 100%</td><td>AustrAlco</td><td>Ethanol Absolut 99.9%</td><td>Delipitation</td></tr><tr><td>Diethyl ether</td><td>Carl Roth GmbH</td><td>3942.1</td><td>Delipitation</td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>Multifuge X3R</td><td>Delipitation</td></tr><tr><td>Chloroform</td><td>Carl Roth GmbH</td><td>3313.1</td><td>Reconstitution</td></tr><tr><td>Methanol</td><td>Carl Roth GmbH</td><td>4627.1</td><td>Reconstitution</td></tr><tr><td>Phosphatidylcholine</td><td>Sigma Aldrich GmbH</td><td>P3556-25mg</td><td>Reconstitution</td></tr><tr><td>Cholesterol oleate</td><td>Sigma Aldrich GmbH</td><td>C-9253-250mg</td><td>Reconstitution</td></tr><tr><td>cholesterol</td><td>Sigma Aldrich GmbH</td><td>C8667-500mg</td><td>Reconstitution</td></tr><tr><td>glass tubes</td><td>Carl Roth GmbH</td><td>K226.1</td><td>Reconstitution</td></tr><tr><td>spermine</td><td>Sigma Aldrich GmbH</td><td>S3256-1G</td><td>Reconstitution</td></tr><tr><td>Thermomixer</td><td>Eppendorf</td><td>Thermomixer comfort</td><td>Reconstitution</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma Aldrich GmbH</td><td>3097-25G</td><td>Reconstitution</td></tr><tr><td>Magnetic stir bar</td><td>Carl Roth GmbH</td><td>0955.2</td><td>Reconstitution</td></tr><tr><td>100&amp;micro;L micropipettes</td><td>Carl Roth GmbH</td><td>A762.1</td><td>Reconstitution</td></tr><tr><td>PBS</td><td>Carl Roth GmbH</td><td>9143.2</td><td>Dialysis</td></tr><tr><td>Amberlite XAD-2 beads</td><td>Sigma Aldrich GmbH</td><td>10357</td><td>Dialysis</td></tr><tr><td>Slide-A-Lyzer casette (cut-off 20 kDa)</td><td>Thermo Scientific</td><td>66003</td><td>Dialysis</td></tr><tr><td>Syringe</td><td>Braun</td><td>9161406V</td><td>Dialysis</td></tr><tr><td>Syringe needle</td><td>Braun</td><td>465 76 83</td><td>Dialysis</td></tr><tr><td>EGTA</td><td>Carl Roth GmbH</td><td>3054.2</td><td>Labeling of LDL particles</td></tr><tr><td>DMSO</td><td>life technologies</td><td>D12345</td><td>Labeling of LDL particles</td></tr><tr><td>Atomic Force Microscope</td><td>RIBM</td><td>SS-NEX</td><td>Quality control of reconstituted/labeled lipoprotein particles</td></tr><tr><td>Muscovite Mica (V-1 Grade)</td><td>Christine Gr&amp;ouml;pl</td><td>G250-1/V1</td><td>Quality control of reconstituted/labeled lipoprotein particles</td></tr><tr><td>AFM Cantilever</td><td>Nanoworld</td><td>USC-F1.2-k0.15</td><td>Quality control of reconstituted/labeled lipoprotein particles</td></tr><tr><td>Gwyddion 2.49</td><td>Czech Metrology Institute</td><td>http://gwyddion.net</td><td>Quality control of reconstituted/labeled lipoprotein particles</td></tr><tr><td>Chamber slides, Nunc Lab-Tek</td><td>VWR</td><td>734-2122</td><td>Cell culture</td></tr><tr><td>HBSS</td><td>Carl Roth GmbH</td><td>9117.1</td><td>Cell culture</td></tr><tr><td>Cell counter</td><td>Omni Life Science GmbH</td><td>Casy</td><td>Cell culture</td></tr><tr><td>miRNeasy Mini Kit</td><td>QIAGEN</td><td>217004</td><td>miRNA extraction</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5415R</td><td>miRNA extraction</td></tr><tr><td>20G needle</td><td>Braun</td><td>Sterican 465 75 19</td><td>miRNA extraction</td></tr><tr><td>5ml syringe</td><td>Becton Dickinson</td><td>309050</td><td>miRNA extraction</td></tr><tr><td>PCR cabinet</td><td>Esco</td><td>PCR-3A1</td><td>Reverse Transcription</td></tr><tr><td>TaqMan Reverse Transcription Kit</td><td>Thermo Scientific</td><td>4366596</td><td>Reverse Transcription</td></tr><tr><td>Rnase-free water</td><td>Carl Roth GmbH</td><td>T143</td><td>Reverse Transcription</td></tr><tr><td>0.2 ml tubes</td><td>Brand</td><td>781305</td><td>Reverse Transcription</td></tr><tr><td>Centrifuge</td><td>Carl Roth GmbH</td><td>Microcentrifuge AL</td><td>Reverse Transcription</td></tr><tr><td>Thermocycler</td><td>Sensoquest</td><td>Labcycler</td><td>Reverse Transcription</td></tr><tr><td>TaqMan Primer</td><td>Thermo Scientific</td><td>-</td><td>qPCR</td></tr><tr><td>iTaq Universal probe supermix</td><td>BioRad GmbH</td><td>1725131</td><td>qPCR</td></tr><tr><td>PCR machine</td><td>Corbett</td><td>Rotor-Gene RG-6000</td><td>qPCR</td></tr><tr><td>TaqMan MicroRNA Reverse Transcription Kit</td><td>Applied Biosystems</td><td>4366596</td><td>TaqMan Array</td></tr><tr><td>Megaplex RT Primers, Human Pool A v2.1</td><td>Applied Biosystems</td><td>4399966</td><td>TaqMan Array</td></tr><tr><td>TaqMan PreAmp Master Mix</td><td>Applied Biosystems</td><td>4391128</td><td>TaqMan Array</td></tr><tr><td>Megaplex PreAmp Primers, Human Pool A v2.1</td><td>Applied Biosystems</td><td>4399933</td><td>TaqMan Array</td></tr><tr><td>TaqMan Universal Master Mix II, no UNG</td><td>Applied Biosystems</td><td>4440040</td><td>TaqMan Array</td></tr><tr><td>TaqMan Advanced miRNA Human A Cards</td><td>Applied Biosystems</td><td>A31805</td><td>TaqMan Array</td></tr><tr><td>Nuclease-free water</td><td>Thermo Scientific</td><td>R0581</td><td>TaqMan Array</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; (25mM)</td><td>Thermo Scientific</td><td>R0971</td><td>TaqMan Array</td></tr><tr><td>TE, pH 8.0</td><td>Invitrogen</td><td>AM9849</td><td>TaqMan Array</td></tr><tr><td>7900HT Fast Real-Time PCR System</td><td>Applied Biosystems</td><td>4351405</td><td>TaqMan Array</td></tr><tr><td>TaqMan Array Card Sealer</td><td>Applied Biosystems</td><td>-</td><td>TaqMan Array</td></tr></tbody></table>

enrichment of native lipoprotein particles with microrna and subsequent determination of their absolute/relative microrna content and their cellular transfer rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Watch this Scientific Journal Video about Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer 

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

enrichment-native-lipoprotein-particles-with-microrna-subsequent

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Biochemistry

8.7K Views.  Medical University Vienna. Lipoprotein deeltjes zijn inheemse transportvoertuigen. De verrijking van vreemde stoffen vergemakkelijkt het gebruik ervan als spoorvervoerder. Specifieke etikettering van moleculen of hele deeltjes maakt het mogelijk om de cellulaire opname te meten.De twee verrijkingsmethoden zijn snel en gebruiksvriendelijk en kunnen worden aangepast voor een breed scala aan stoffen. Het aantonen van de procedure zal Markus Axmann en Andreas Karner, twee postdocs van mijn lab. Om te beginnen met de...

Video: Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3&#39; UTR of the target mRNA is mediated by a 2&#8211;8 nucleotide seed sequence at the 5&#39; end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3&#39; UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3&#39; UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

This report describes a fast and reliable method for validating mRNA targets of cellular miRNAs. The method uses synthetic biotinylated Locked Nucleic Acid (LNA)-based miRNA mimics to capture target mRNA. Subsequently, streptavidin-coated magnetic beads are employed to pulldown the target mRNA for quantification by qPCR polymerase chain reaction.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3&#39; ...

Genetics

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

Immunology and Infection

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Bioengineering

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and Pfizer/BioNTech. These vaccines have demonstrated the efficacy of mRNA-LNP therapeutics and opened the door for future clinical applications. In mRNA-LNP systems, the LNPs serve as delivery platforms that protect the mRNA cargo from degradation by nucleases and mediate their intracellular delivery. The LNPs are typically composed of four components: an ionizable lipid, a phospholipid, cholesterol, and a lipid-anchored polyethylene glycol (PEG) conjugate (lipid-PEG). Here, LNPs encapsulating mRNA encoding firefly luciferase are formulated by microfluidic mixing of the organic phase containing LNP lipid components and the aqueous phase containing mRNA. These mRNA-LNPs are then tested in vitro to evaluate their transfection efficiency in HepG2 cells using a bioluminescent plate-based assay. Additionally, mRNA-LNPs are evaluated in vivo in C57BL/6 mice following an intravenous injection via the lateral tail vein. Whole-body bioluminescence imaging is performed by using an in vivo imaging system. Representative results are shown for the mRNA-LNP characteristics, their transfection efficiency in HepG2 cells, and the total luminescent flux in C57BL/6 mice.

Testing the In Vitro and In Vivo Efficiency of mRNA-Lipid Nanoparticles Formulated by Microfluidic Mixing

Here, a protocol for formulating lipid nanoparticles (LNPs) that encapsulate mRNA encoding firefly luciferase is presented. These LNPs were tested for their potency in vitro in HepG2 cells and in vivo in C57BL/6 mice.

Lipid nanoparticles (LNPs) have attracted widespread attention recently with the successful development of the COVID-19 mRNA vaccines by Moderna and ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Biology

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

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