Manufacturing Parameters, Physicochemical Characteristics, Protein-Biofluid Interactions,and Biological Performance of RNA-Loaded Lipid Nanoparticles: A Systematic Review

Background: Ribonucleic acid (RNA)-loaded lipid nanoparticles (LNPs) are established platforms for the delivery of messenger RNA (mRNA), small interfering RNA (siRNA), and other functional RNA molecules. Their biological performance is determined by interconnected factors extending from manufacturing and particle assembly to physicochemical characteristics, interactions with biological fluids, cellular trafficking, and in-vivo function. This systematic review aimed to integrate the available evidence on manufacturing parameters, physicochemical characteristics, protein-corona interactions, and biological performance of RNA-loaded LNPs. Methods: This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA 2020) guidelines. PubMed, Scopus, and Web of Science were searched from inception to 12 July 2026 without language or publication-date restrictions. The first 50 pages of Google Scholar and the reference lists of relevant articles were additionally screened. Eligible experimental studies investigated RNA-loaded LNPs and contributed evidence to at least one prespecified domain: manufacturing/process-related physicochemical characteristics, primary in-vitro biological delivery, downstream or in-vivo functional outcomes, or protein-corona and serum/protein interactions. Methodological quality was assessed using the quantitative Standard Quality Assessment Criteria for Evaluating Primary Research Papers from a Variety of Fields (QualSyst). Owing to substantial methodological and experimental heterogeneity, the evidence was synthesized systematically without meta-analysis. Results: A total of 45 unique parent studies were included. Thirty studies evaluated manufacturing/process-related physicochemical outcomes, 26 investigated primary in-vitro biological delivery, 37 assessed downstream or in-vivo functional outcomes, and 17 examined protein-corona or serum/protein interactions, with substantial overlap among evidence domains. Manufacturing variables, including mixer architecture, total flow rate, flow-rate ratio, scale-up conditions, dilution, and post-processing, were associated with changes in particle size, polydispersity, encapsulation efficiency, surface characteristics, and particle structure; however, the direction and magnitude of these effects varied among formulations and platforms. Similar conventional physicochemical characteristics did not consistently correspond to equivalent biological performance, and greater cellular association or uptake was not invariably accompanied by greater productive RNA expression or functional activity. Protein-corona studies demonstrated formulation- and biofluid-dependent interactions that were associated with changes in cellular uptake, intracellular trafficking, biodistribution, and functional RNA delivery. Apolipoprotein E was a frequently investigated mediator, particularly in hepatic delivery, although multiple other protein classes were also implicated. Conclusions: The available evidence indicates that RNA-LNP performance should be considered as the product of an interconnected relationship among manufacturing conditions, physicochemical and structural characteristics, biological-fluid interactions, and subsequent cellular and in-vivo function. Manufacturing effects are highly dependent on formulation and platform context, while conventional physicochemical attributes alone are insufficient to predict biological performance. Protein-corona formation represents an additional context-dependent component of LNP biological identity. Greater methodological standardization and integrated process–physicochemical–biological studies are required to enable more reliable cross-study comparisons and future quantitative synthesis.