We designed a peptide-hybrid lipid nanoparticle composed of a DNA-peptide core, a lipid bilayer shell, and an external serum-derived coating. The peptide component, derived from a histone tail sequence, was originally selected to promote DNA nuclear uptake, but mechanistic studies later revealed that its primary benefit lies in enhancing cellular uptake rather than sequence-specific intracellular interactions. To further evaluate this effect, scrambled versions of the peptide were synthesized and compared. LNPs were assembled using microfluidic mixing, with encapsulation efficiency, particle stability, and size evaluated across multiple peptide-to-DNA charge (NP) ratios. Transfection efficiency was analyzed using GFP plasmid delivery in two AT2-like models: murine MLE-12 and human A549 cells. A key part of the optimization involved testing a range of serum pre-coating conditions (0–10% serum in media), using both bovine and human serum, to reflect species specificity and define optimal coating strategies. A central challenge of local lung gene therapy is the absence of serum proteins at the delivery site; since LNP uptake is serum-dependent, we implemented a serum pre-coating strategy to "activate" LNPs prior to local administration. We evaluated multiple pre-incubation conditions using varying concentrations (0–10%) and types (bovine vs. human) of serum, and assessed the impact on both uptake and expression. Exosome-specific antibody bead capture assays and gene expression data confirmed that interactions between peptide-modified LNPs and serum exosomes were key contributors to improved transfection.
Our optimization pipeline revealed that three parameters—serum type, concentration, and incubation time—were all critical in achieving efficient uptake and high gene expression. Specifically, fined tuned NP charge ratio maintained optimal DNA encapsulation with a slight particle size increase, while a 6% serum pre-coating condition yielded the highest transfection outcomes. Only species-matched serum (e.g., human serum for A549 cells, bovine serum for MLE-12) enabled significant transfection, underscoring the importance of serum origin. Peptide-functionalized LNPs, in conjunction with serum pre-coating, achieved nearly 100% transfection in AT2 cell models and produced a 17-fold increase in gene expression relative to standard LNPs (56%). Gene expression gains were linked exosome interaction promoted by charged peptide inclusion, which we validated through transfection with exosome-depleted serum and LNP-exosome isolation using exosome-specific antibody bead capture assays. Importantly, macrophage off-target gene expression remained minimal. Preliminary studies using the bleomycin-induced fibrosis model demonstrated that peptide-enhanced LNPs can successfully deliver ACSL-1, a promising therapeutic gene, suggesting functional relevance of this delivery system in fibrotic disease contexts.
Together, our findings highlight a modular and highly tunable DNA-LNP platform for localized lung gene therapy. By dissecting and optimizing the roles of peptide incorporation, serum specificity, and EV interactions, we have constructed a system that significantly improves DNA delivery efficiency to AT2 cells while minimizing off-target effects. These insights pave the way for further translational studies using this delivery method in models of lung disease, including pulmonary fibrosis.