field flow fractionation instruments  (Waters Corporation)

Journal: bioRxiv

Article Title: Capsid Restructuring Activates Semi-Conservative dsRNA Transcription in Cystovirus ɸ6

doi: 10.1101/2025.07.23.666269

Figure Lengend Snippet: ( A ) Two three-dimensional cryo-EM class averages are shown for the polar regions extracted from transcription-arrested double-layered particles (taDLPs). ( B ) Same as (A), but the class averages have been determined from transcription-arrested single-layered particles (taSLPs). The cryo-EM averages have been filtered to local resolution. ( C ) Asymmetrical flow field flow fractionation of P2, P7 and their 1:1 mixture. Molar mass (g/mol; solid lines) and relative UV signal at 280 nm (dotted line) are shown. ( D ) Agarose gel electrophoresis analysis of in vitro ssRNA packaging, replication and transcription assay after a 90-min incubation with standard self-assembled SLP (aSLP) and SLPs with low amount of incorporated P7 (aSLP lowP7 ) or P2 (aSLP lowP2 ). The mobility of the synthesized 33 P-labelled dsRNA (S, M, and L) and ssRNA transcripts (s and m) are indicated on the left of the autoradiogram, and the mean copy numbers of P2 and P7 in the SLPs with standard deviations are below. ( E ) Quantitation of dsRNA band intensities from the autoradiogram in (D). ( F ) Quantitation of time-dependent accumulation of ssRNA transcripts by the aSLPs. Error bars represent standard deviation of the mean. Calculation of the number of transcripts takes into account the semi-conservative nature of the process. See also Supplemental Figures 2 .

Article Snippet: AF4 experiments were performed using EclipseTM NEON (Wyatt Technology, Dernbach, Germany) field flow fractionation system ( ) at 22°C using a long analytical channel, a 400-μm spacer (Wyatt Technology), a 10-kD regenerated cellulose membrane (Wyatt Technology) and 10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.1 mM EDTA as the mobile phase.

Techniques: Cryo-EM Sample Prep, Field Flow Fractionation, Agarose Gel Electrophoresis, In Vitro, Transcription Assay, Incubation, Synthesized, Quantitation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Disc-Toroid Hybrid Lipid Nanoparticles for Efficient Drug Encapsulation and Subcutaneous Delivery

doi: 10.1101/2025.07.20.665764

Figure Lengend Snippet: Impact of LNP composition on stability, dispersity and overall size. (A1): Dynamic light scattering (DLS) in batch determined intensity-based particle size (diameter) of various LNP composition formulations as a function of time over 18 months confirms stability at 4°C. (A2): Polydispersity index (PDI) of various LNP composition formulations as a function of time, determined using DLS. (A3): Zeta (ζ) potential values for the various LNP formulations. (B1): AF4-MD analysis of the size distribution of LNP without drug incorporation (LNP 5 ) and with drug incorporation (LNP 5 -Q and LNP 5 -DHA) are compared. (B2): AF4-MD based comparison of two LNP formulations with the same polysorbate 40 surfactant, but different cores. LNP 1 -T40, contains a mixture of carnauba wax and red palm oil, whereas LNP 1 -CW-T40, consist only of a carnauba wax core. (B3): AF4-MD based comparison of two LNP formulations with the same TPGS surfactant, but different lipid cores. LNP 1 -TPGS, contains a mixture of carnauba wax and red palm oil, whereas LNP 1 -CW-TPGS, consist only of a carnauba wax core. (C1): AF4-MD based evaluation of the average R h and R g for different LNP composition formulations after separation. (C2): The Guinier plot from SAXS in batch evaluate the global R g for four different concentrations of LNP 1 -CW-TPGS in water. (C3): The Guinier plot from SAXS in batch evaluate the global R g for four different concentrations of LNP 5 in water.

Article Snippet: AF4 measurements were performed with an Eclipse Neon Asymmetric flow field-flow fractionation (AF4) system (Wyatt Technologies Corp., Germany).

Techniques: Comparison

Journal: bioRxiv

Article Title: Disc-Toroid Hybrid Lipid Nanoparticles for Efficient Drug Encapsulation and Subcutaneous Delivery

doi: 10.1101/2025.07.20.665764

Figure Lengend Snippet: Shape and internal composition evaluation. ( A1 ) Cryo-TEM image of LNP5 (Unloaded LNP with carnauba wax and red palm oil as lipid core, with both TPGS and Polysorbate 40 as surfactants), ( A1.1 ) the average mean particle size distribution, and ( A1.2 ) the average particle thickness distribution. ( A2 ) Zoomed in cryo-TEM image of LNP1-CW-TPGS (Unloaded LNP with carnauba wax only lipid core and TPGS as surfactant) to provide a clearer view of the observed shape. ( B ) Shape parameter Rg/Rh determined at peak height for the various compositions of LNP formulation using AF4-MD shows values corresponding to spherical shapes. ( C ): WAXS plots of LNP5, LNP5-Q, LNP5-DHA and LNP1-CW-TPGS. ( D ) SAXS data and modelling by a core-shell bicelle for LNP1-CW-TPGS in water at a concentration of 11.02 mg.mL-1 based on lipid fraction. The geometry parameters correspond to the values described in the sketch under (E) with D = 37 nm; d = 9 nm; h = 10 nm; H = 17 nm; m = 3.5 nm. For the calculation, the SLD for water (core) and lipid (internal toroid) were applied. ( E ) Schematic representation of the particle shape corresponding to a disk-toroid hybrid based on structural insights gained from the experimental data using cryo-TEM and SAXS: the internal lipid toroid is stabilised by a monolayer of surfactant while the water core is stabilised by a double layer of surfactant at the interface between the core and aqueous particle environment.

Article Snippet: AF4 measurements were performed with an Eclipse Neon Asymmetric flow field-flow fractionation (AF4) system (Wyatt Technologies Corp., Germany).

Techniques: Formulation, Concentration Assay

Journal: bioRxiv

Article Title: Disc-Toroid Hybrid Lipid Nanoparticles for Efficient Drug Encapsulation and Subcutaneous Delivery

doi: 10.1101/2025.07.20.665764

Figure Lengend Snippet: Drug encapsulation and quantification. ( A ) A schematic representation illustrating two alternative approaches for the indirect determination of the encapsulation efficiency of LNP, using AF4 coupled to either a UV-Vis detector or a fluorescence detector. A collection of small drug molecules filtered through the membrane of the AF4 channel is quantified using a pre-calibrated UV-Vis detector. The large drug-loaded particles separated along the channel are detected using the fluorescence detector. ( B1 ) Elution profile showing the UV-Vis signal of quinine, with the cross-flow coupled to the UV-Vis detector. Wavelength set at 250 nm. ( B2 ) The calibration curve is generated by injecting several concentrations of quinine, enabling the quantification of quinine present in the cross-flow waste. Wavelength set at 250 nm. ( B3 ) Elution profile showing the UV-Vis signal of LNP5 and LNP5-Q, respectively. The cross-flow is coupled to the UV-Vis detector, set to a wavelength of 250 nm. C1) and C2) Fluorescence detection of the LNPs shows that the quinine is located in the particle due to increased intensity of the fluorescence signal at the same elution volume as the particle. The concentration of the particles is kept the same; thus, the increasing absorption intensity and broader absorption wavelength after excitation at 250 and 350 nm are the result of the quinine in the loaded LNP5-Q-B4 compared to the pure LNP5-WS-B4.

Article Snippet: AF4 measurements were performed with an Eclipse Neon Asymmetric flow field-flow fractionation (AF4) system (Wyatt Technologies Corp., Germany).

Techniques: Encapsulation, Fluorescence, Membrane, Generated, Concentration Assay

Journal: bioRxiv

Article Title: Solution biophysics identifies lipid nanoparticle non-sphericity, polydispersity, and dependence on internal ordering for efficacious mRNA delivery

doi: 10.1101/2024.12.19.629496

Figure Lengend Snippet:

Article Snippet: Samples were injected undiluted in triplicates on an Eclipse TM field flow fractionation (FFF) instrument using a 350 µm fixed-height short channel with dilution control module and a 10 kDa regenerated cellulose membrane (Wyatt Technology, LLC.), connected to a 1260 Infinity II HPLC system with a G1310B isocratic pump and a G1329B autosampler (Agilent Technologies, Inc.).

Techniques: Field Flow Fractionation, Multi-Angle Light Scattering