asymmetrical flow field-flow fractionation af4  (Postnova Analytics)

Journal: bioRxiv

Article Title: Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

doi: 10.64898/2026.03.31.715590

Figure Lengend Snippet: ( a ) SAXS profiles of NT LNPs and tLNPs, showing the characteristic Bragg peak feature associated with internal lipid–RNA organization. ( b ) Fitting of the Bragg peak feature using a multiple-Lorentz model where red is the first-order Bragg peak fit, green represents higher-order particle disorder and blue is the cumulative fit of the two features. (c) In-line AF4-UV-Vis contour maps (200–300 nm) depicting wavelength-resolved absorbance of LNPs during separation. The 260 nm absorbance signal is characteristic of encapsulated RNA, enabling identification of RNA-containing LNP populations across the AF4 elution profile. Overlapping spectral features indicate the presence of multiple co-eluting populations with distinct compositional profiles, motivating subsequent chemometric deconvolution. ( d ) Absorbance at 260 nm (RNA-associated signal) and 280 nm (protein-associated signal) from ( c ) plotted against the corresponding 260:280 ratio for each LNP formulation. Deviations in the 260:280 ratio across elution time indicate heterogeneity in RNA and protein content, suggesting the presence of compositionally distinct subpopulations that cannot be resolved by bulk measurements alone. These data were further subjected to chemometric analysis (see Supplemental Figure 6). The determined ( e ) R h profiles derived from in-line DLS and ( f ) molar mass profiles derived from MALS analysis for NT LNPs (beige) and tLNPs (colors) overlaid with UV fractograms from in-line AF4 separation. ( g ) Peak 260:280 ratios from ( d ), shown for comparison across LNP groups. ( h ) In-line DLS R h and MALS-derived ( i ) mass, ( j ) radius of gyration, and ( k ) polydispersity plotted for comparison across LNP groups. Measurements are reported mean ± standard error for ( h–k).

Article Snippet: 100 μL of LNP at a particle concentration of ∼10 particles were injected and eluted isocratically from an Eclipse TM NEON field flow fractionation (AF4) instrument with dilution control module using a variable-height short channel with a 275 μm spacer and a 10 kDa regenerated cellulose membrane (Wyatt Technology), connected to a 1260 Infinity II HPLC system with a G711A quaternary pump and a G7167A multisampler (Agilent Technologies).

Techniques: Formulation, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

doi: 10.64898/2026.03.31.715590

Figure Lengend Snippet: ( a ) SAXS elution profiles of NT LNPs and tLNPs. Dotted boxes indicate the main LNP elution region, expanded in ( b ). Evolving factor analysis (EFA) ( b ) applied to AF4-SAXS data, highlighting frames corresponding to discrete evolving species (C1–C3) within each formulation. The resolved components correspond to distinct LNP subpopulations that differ in size and internal organization and a third component (when present) reflecting a minor population of highly heterogeneous or higher-mass particles. The temporal evolution of EFA-selected frames across the elution peak indicates that these components arise from partially overlapping populations that co-elute but differ in hydrodynamic size and composition. ( c ) Singular value decomposition (SVD) of SAXS datasets, indicating the number of independent signals present in each formulation. The presence of multiple significant singular values indicates that the SAXS signal cannot be described by a single particle population, necessitating decomposition into independent scattering components. (d) Volatility of ratio (V r ) analysis used to assess the statistical uniqueness of deconvoluted component subpopulations relative to the ensemble-averaged population. ( e-h ) Regularized alternating least squares (REGALS) deconvolutions of SAXS data yields component-resolved scattering profiles. Shown are the ( e ) ensemble-averaged SAXS profile over the elution peak, and the REGALS-derived scattering profiles for the ( f ) C1, ( g ) C2, and ( h ) C3 subpopulations. These structural components are consistent with compositionally distinct populations inferred from UV-based chemometric analysis, linking RNA-associated heterogeneity to differences in particle size and morphology. Together, these analyses demonstrate that the ensemble SAXS signal arises from multiple, structurally distinct tLNP subpopulations that can be resolved and interpreted through chemometric decomposition.

Article Snippet: 100 μL of LNP at a particle concentration of ∼10 particles were injected and eluted isocratically from an Eclipse TM NEON field flow fractionation (AF4) instrument with dilution control module using a variable-height short channel with a 275 μm spacer and a 10 kDa regenerated cellulose membrane (Wyatt Technology), connected to a 1260 Infinity II HPLC system with a G711A quaternary pump and a G7167A multisampler (Agilent Technologies).

Techniques: Formulation, Single Particle, Derivative Assay

Journal: bioRxiv

Article Title: Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

doi: 10.64898/2026.03.31.715590

Figure Lengend Snippet: ( a ) Guinier analyses with corresponding residuals for SVD-resolved C1 and C2 components of NT LNPs and tLNPs, with the exception of F(ab’) 2 tLNPs, where only C3 is shown. White regions indicate components for which Guinier analysis failed due to large size (q min R g > 1.3). ( b ) P(r) analyses normalized by I(0) for the average profile and individual components (C1–C3) for NT LNPs and tLNPs. ( c ) Radius of gyration (R g ) and ( d ) maximum dimension (D max ) of the average profile and individual components derived from GNOM analysis for NT LNPs and tLNPs. Blank regions denote populations where GNOM analysis was invalid (q min D max > 4). ( e ) LNP shape factor calculated as D max / R g , where values of ∼2.58 and ∼3.0 correspond to spherical and prolate ellipsoid geometries, respectively. DENSS ab initio electron density reconstructions from the AF4-UV-DLS-MALS-SAXS profiles for ( f ) NT LNPs, ( g ) nanobody tLNPs, ( h ) DAPRin tLNPs, ( i ), F(ab’) 2 tLNPs, and ( j ) antibody tLNPs.

Article Snippet: 100 μL of LNP at a particle concentration of ∼10 particles were injected and eluted isocratically from an Eclipse TM NEON field flow fractionation (AF4) instrument with dilution control module using a variable-height short channel with a 275 μm spacer and a 10 kDa regenerated cellulose membrane (Wyatt Technology), connected to a 1260 Infinity II HPLC system with a G711A quaternary pump and a G7167A multisampler (Agilent Technologies).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

doi: 10.64898/2026.03.31.715590

Figure Lengend Snippet: Targeted mRNA delivery to the placenta is driven by tLNP structural subspecies. ( a-d ) DiR-labeled NT LNPs and tLNPs containing mCherry mRNA were incubated with placental BeWo b30 trophoblasts at a dose of 150 ng of mRNA per 150,000 cells. After ( a ) 1 h, ( b ) 4 h, and ( c ) 24 h, cellular accumulation was quantified. After ( d ) 24 h, mCherry expression was also quantified. Normalized DiR and mCherry MFI was calculated by normalizing to cells treated with NT LNPs. ( e–m ) NT LNPs and tLNPs containing FLuc mRNA were administered intravenously via retroorbital injection into pregnant and nonpregnant mice at a dose of 12 µg mRNA per mouse. After 6 h, mice were euthanized, and major organs were dissected. For pregnant mice, luminescence imaging of ( e ) livers and spleens and ( f ) placentas and fetuses were performed via an in vivo imaging system (IVIS). Luminescence from ( e-f ) was quantified via region of interest (ROI) analysis to obtain luminescence flux in the ( g ) liver, ( h ) spleen, ( i ) placentas, and ( j ) fetuses of pregnant mice. For nonpregnant mice, luminescence imaging of ( k ) livers and spleens was performed. Luminescence from ( k ) was quantified via region of interest (ROI) analysis to obtain luminescence flux in the ( m ) liver and ( m ) spleen of nonpregnant mice. Signal is reported mean ± SD from n = 3 biological replicates for ( a–d ) and n = 4 biological replicates for ( e–m). One-way ANOVA with post hoc Student’s t-tests using the Holm–Sídak correction for multiple comparisons was used to compare fluorescence in for ( a–d ) and luminescence in ( g–h, l–m ) across treatment groups. Nested one-way ANOVA with post hoc Student’s t-tests using the Holm–Sídak correction for multiple comparisons was used to compare luminescence in ( i–j ) across treatment groups. ( n–q ) Spearman correlations for ( n ) placental, ( o ) pregnant hepatic, and ( p ) nonpregnant hepatic luminescence values using the physicochemical parameters from traditional characterization methods, static SAXS analyses, and AF4-UV-DLS-MALS-SAXS analyses. ( q ) Heatmap representing the entire dataset. For Spearman correlation graphs, dotted lines represent r = –0.6 and 0.6.

Article Snippet: 100 μL of LNP at a particle concentration of ∼10 particles were injected and eluted isocratically from an Eclipse TM NEON field flow fractionation (AF4) instrument with dilution control module using a variable-height short channel with a 275 μm spacer and a 10 kDa regenerated cellulose membrane (Wyatt Technology), connected to a 1260 Infinity II HPLC system with a G711A quaternary pump and a G7167A multisampler (Agilent Technologies).

Techniques: Labeling, Incubation, Expressing, Injection, Imaging, In Vivo Imaging, Fluorescence

Journal: bioRxiv

Article Title: Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

doi: 10.64898/2026.03.31.715590

Figure Lengend Snippet: ( a-b ) NT LNPs and tLNPs containing FLuc mRNA were administered intravenously via retroorbital injection into pregnant and nonpregnant mice at a dose of 12 µg mRNA per mouse. After 6 h, mice were euthanized, and serum was collected. Serum levels of C3a, TNF, IFN-γ, IL-6, ALT, and AST were quantified in ( a ) pregnant and ( b ) nonpregnant mice via ELISA. Measurements are reported mean ± SD from n = 3–4 biological replicates. One-way ANOVA with post hoc Student’s t-tests using the Holm–Sídak correction for multiple comparisons was used to compare cytokine levels across treatment groups. ( c–e ) Spearman correlations for ( c ) TNF, ( d ) IFN-γ, and ( e ) IL-6 serum levels in pregnant (top) and nonpregnant (bottom) mice using the physicochemical parameters from traditional characterization methods, static SAXS analyses, and AF4-UV-DLS-MALS-SAXS analyses. ( f ) Heatmap representing the entire dataset. For Spearman correlation graphs, dotted lines represent r = –0.6 and 0.6.

Article Snippet: 100 μL of LNP at a particle concentration of ∼10 particles were injected and eluted isocratically from an Eclipse TM NEON field flow fractionation (AF4) instrument with dilution control module using a variable-height short channel with a 275 μm spacer and a 10 kDa regenerated cellulose membrane (Wyatt Technology), connected to a 1260 Infinity II HPLC system with a G711A quaternary pump and a G7167A multisampler (Agilent Technologies).

Techniques: Injection, Enzyme-linked Immunosorbent Assay

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