Background: Lipid nanoparticles (LNPs) represent one of the most effective non-viral vectors for nucleic acid delivery and have demonstrated clinical success in siRNA therapies and mRNA vaccines. While considerable research has focused on optimizing ionizable lipids and helper lipids, the impact of PEGylated lipid content on LNP-mediated mRNA delivery, especially in terms of in vitro transfection efficiency and in vivo performance, remains insufficiently understood. Methods: In this study, LNPs were formulated using a self-synthesized ionizable lipid and varying molar ratios of DMG-PEG2000. Nanoparticles were prepared via nanoprecipitation, and their physicochemical properties, mRNA encapsulation efficiency, cellular uptake, and transfection efficiency were evaluated in HeLa and DC2.4 cells. In vivo delivery efficiency and organ distribution were assessed in mice following intravenous administration. Results: The PEGylated lipid content exerted a significant influence on both the in vitro and in vivo performance of LNPs. A bell-shaped relationship between PEG content and transfection efficiency was observed: 1.5% DMG-PEG2000 yielded optimal mRNA transfection in vitro, while 5% DMG-PEG2000 resulted in the highest transgene expression in vivo. This discrepancy in optimal PEG content may be attributed to the trade-off between cellular uptake and systemic circulation: lower PEG levels enhance cellular internalization, whereas higher PEG levels improve stability and in vivo bioavailability at the expense of cellular entry. Furthermore, varying the PEG-lipid content enabled the partial modulation of organ distribution, offering a formulation-based strategy to influence biodistribution without altering the ionizable lipid structure. Conclusions: This study highlights the critical role of PEGylated lipid content in balancing nanoparticle stability, cellular uptake, and in vivo delivery performance. Our findings provide valuable mechanistic insights and suggest a straightforward formulation-based strategy to optimize LNP/mRNA systems for therapeutic applications.

Hydrolyzed polyacrylamide stabilized oil-in-water emulsions are highly persistent because the polymer strengthens both continuous-phase rheology and the oil–water interfacial film, making demulsification difficult in polymer-flooding produced liquids. Here, an hydrolyzed polyacrylamide degrading bacterium, Delftia lacustris EPDB-8, was isolated, and its ability to destabilize hydrolyzed polyacrylamide-containing emulsions was investigated from molecular, bulk rheological, and interfacial perspectives. EPDB-8 effectively degraded HPAM, causing marked reductions in total organic carbon, total nitrogen, absolute zeta potential, and polymer molecular weight, with an approximately 63-fold decrease after 7 days. SEM, FT-IR, and GPC analyses showed that biodegradation proceeded through deamidation and random chain scission, collapsing the polymer network and generating low-molecular-weight fragments. Driven by bacterial hydrolyzed polyacrylamide degradation, these structural alterations disrupted the viscoelastic composite interfacial film formed by hydrolyzed polyacrylamide and indigenous surface-active species, directly causing emulsion stabilization to shift from polymer-assisted viscous and steric protection to a less effective asphaltene-dominated interfacial structure and thereby accelerating droplet aggregation, coalescence, and phase separation. Although bacterial cells exerted a transient particle-assisted interfacial effect, long-term emulsion stability remained governed by polymer integrity. This study establishes a mechanistic link between hydrolyzed polyacrylamide biodegradation and the rheological and interfacial evolution governing emulsion breakdown, providing a cost-effective and environmentally benign biological strategy for demulsification and treatment of polymer-flooding produced water. These findings offer practical guidance for the design of microbial-based produced-water treatment systems and contribute to the sustainable management of oilfield wastewater generated during enhanced oil recovery operations.