Biology subjects

Mitra, H.

Publications and source records attributed to Mitra, H..

6 recordsLinked to original sources

bioRxiv2026

RNA post hoc loading into empty lipid nanoparticles occurs on millisecond timescales during turbulent mixing

Lipid nanoparticles (LNPs) are conventionally produced through mixing of lipids dissolved in ethanol against a buffer containing RNA. An alternative strategy offering improved cold-chain stability involves formulating empty LNPs (eLNPs), removing ethanol, and post hoc loading (PHL) RNA into the aqueous eLNPs. The kinetics of this approach remain unknown. Here, we employ a flowthrough small-angle X-ray scattering (SAXS) setup based on a confined impinging jets (CIJ) mixer to probe PHL kinetics. We show that RNA PHL in a scalable CIJ mixer is efficient and reproducible, and that SAXS data confirms that this process concludes within [~]12 ms under favorable conditions in rapid turbulent micromixing, suggesting a diffusion-limited aggregation mechanism. Favorable conditions were identified as an acidic pH 5.5 buffer combined with turbulent CIJ mixing. In contrast, PHL performed with a neutral pH 7.4 buffer using a CIJ mixer or under laminar flow with a pH 5.5 buffer resulted in inefficient PHL.

bioengineering↗

bioRxiv2026

Anomalous diffusion of nanoparticles in semidilute hyaluronic acid solutions

Efficient drug delivery using nanoparticles (NPs) critically depends on their ability to diffuse through biological tissues to reach target cells at therapeutic concentrations. The extracellular matrix (ECM) poses a key barrier to such transport, which directly influences bio-distribution, cellular uptake, and overall therapeutic efficacy. A key regulator of this transport is hyaluronic acid/hyaluronan (HA), a major ECM polysaccharide that forms a hydrated, viscoelastic network. Increased/reduced hyaluronan concentration can elevate/decrease ECM bulk and effective viscosity. Increase in effective viscosity at the nanometer/micrometer length scales can hinder NP mobility through steric obstruction and hydrodynamic drag. There is a large variability in the HA molecular weights and concentrations, especially across age, tissue/organ, and pathological conditions. This work aims to study the diffusion of different NP types in the mixtures of HA polymers with variable molecular weights using the dynamic light scattering technique (DLS). Furthermore, we perform coarse-grained molecular dynamics (CG-MD) simulations for a model system to complement our findings from the dynamic light scattering experiments. We observe NP undergo anomalous diffusion, which is strongly dependent on the ratio of particle size/HA network mesh size, especially for higher molecular weight mixtures. This is strongly influenced by the effective viscosity, which is defined at the local environment experienced by the NPs. Our work highlights developing a simplified predictive framework coupled with simulations for a target-specific extracellular matrix environment.

biophysics↗

bioRxiv2025

Identifying differential effects from eleven mixing techniques on mRNA lipid nanoparticle physicochemistry and biological performance

Lipid nanoparticle (LNP) formulation requires a mixing step. Many studies, especially from academic groups, utilize either microfluidic mixers or hand mixing to prepare LNPs, but commercial-scale processes use turbulent-flow mixers. This discrepancy in mixing techniques has been underexplored, as LNPs made by different techniques may exhibit different performance, such that bench-scale results cannot be replicated using materials manufactured at scale. We here isolate and interrogate the effect of primary mixing. Lipid nanoparticles are produced from ten mixers (one used in two ways), holding all other formulation parameters constant, to directly compare across techniques. LNPs produced from the different mixers exhibit widely different physical properties and biological performance. Notably, manual pipetting common in academic practice yields particles that do not resemble those produces by turbulent-flow mixers. Findings are connected mechanistically to physicochemical characteristics that arise from the different flow regimes. Further establishing the relationship between mixing and LNP properties is critical. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/687311v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@79cac0org.highwire.dtl.DTLVardef@1ee037eorg.highwire.dtl.DTLVardef@12763eforg.highwire.dtl.DTLVardef@111e394_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Effects of mixing technique and ethanol removal on lipidnanoparticle physicochemical properties

Optimizing the production of lipid nanoparticle (LNP) therapeutics is necessary for drug delivery efficiency, stability, and scalability. A small but growing body of literature has begun to recognize that LNP properties (e.g., size, shape, and internal structure) depend on the flow conditions during mixing for antisolvent precipitation, in which LNPs are formulated. Here, we use different mixers, varying flow patterns (e.g., laminar or turbulent mixing) and flow rate ratios (FRR), i.e., 3:1 and 1:1, to prepare a standard LNP formulation. We then characterize the resulting formulations using small angle x-ray scattering (SAXS) to provide insights into particle shape/morphology, internal organization (L and HII phases) of yeast RNA (yRNA), and structural differences/similarities that arise from the different mixing methods. The effect of ethanol removal on the LNPs structure, formulated from each mixing technique, is also discussed. We observed the 3:1 FRR mixers outperform the 1:1 configurations in certain desired LNP physiochemical properties. The differences observed in the LNPs produced across the two configurations are discussed. Furthermore, we use computational fluid dynamics to explain the turbulent mixing schemes among the 3:1 and 1:1 mixers.

biophysics↗

bioRxiv2025

Effects of arginine on the interfacial behavior of proteins

HypothesisProtein adsorption at fluid-fluid interfaces can induce conformational rearrangements and promote aggregation. Arginine, as an amino acid is known to attenuate protein-protein interactions and lower the viscosity of protein formulations. Given its surface-active properties, we hypothesize that arginine may be effective in limiting aggregation at fluid-fluid interfaces. ExperimentsInterfacial rheology of Etanercept (Enbrel), Immunoglobulin G1 (IgG1), and Bovine Serum Albumin (BSA) was characterized across a range of concentrations, with and without L-arginine Hydrochloride. The interfacial response was examined in both linear and nonlinear regimes.{zeta} -potential was also measured for selected formulations to evaluate the relationship between electrostatic characteristics and the rheological outcomes. FindingsBased on both linear and nonlinear interfacial rheology results, arginine generally weakened protein-protein interactions; however, the effect depends on protein identity and concentration. In arginine-containing formulations, Enbrel and BSA displayed only minor variation in interfacial viscoelasticity between 0.01 and 1 mg/mL, particularly during prolonged aging; in contrast, IgG1 showed larger concentration-dependent differences. These results strengthen predictive frameworks for interfacial stability and guide the rational design of protein-amino acid biopharmaceutical formulations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/674647v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@10a0bc8org.highwire.dtl.DTLVardef@8b9be7org.highwire.dtl.DTLVardef@1a88e43org.highwire.dtl.DTLVardef@113d5a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Rheological and Lipid Characterization of Minipig and Human Skin Tissue: A Comparative Study Across Different Locations and Depths

Understanding the rheology of minipig and human skin is crucial for enhancing drug delivery methods, particularly for injections. Despite many studies on skins viscoelasticity, especially the subcutaneous layer, comparative analyses across different clinical sites are scarce, as is data on the impact of hydration or lipid levels. This study employs shear rheology and lipid analysis to evaluate viscoelasticity and lipid content across three anatomical locations --breast, belly, and neck and three different depth layers in Yucatan minipigs. It reports on how viscoelastic properties change with frequency, time, and strain, noting strain-stiffening and shear-thinning at high strain amplitudes. Human male and female abdominal tissues are also compared to minipig tissues, highlighting distinct viscoelastic traits and lipids role in them. The findings suggest the existence of species, anatomical location, tissue depth, and sex-based rheological differences. We also concluded the minipig male tissue is a more accurate model for human male subcutaneous tissue than for females.

biophysics↗