Apolipoprotein Fusion Enables Spontaneous Functionalization of mRNA Lipid Nanoparticles with Antibody for Targeted Cancer Therapy

Abstract

Introduction: The clinical success of mRNA@lipid nanoparticle (LNP) vaccines, especially during the COVID-19 pandemic, has highlighted the broad therapeutic potential of mRNA for various diseases. While LNPs provide effective protection against enzymatic degradation and facilitate cellular uptake of mRNA, limitations such as nonspecific tissue distribution and unintended liver accumulation have constrained their clinical utility. Passive targeting approaches often rely on the enhanced permeability and retention (EPR) effect and can fall short in achieving precise cell specificity. Although antibody-mediated active targeting can address this issue, existing chemical conjugation strategies typically involve multi-step reactions, toxic reagents, and PEG detachment problems, prompting the need for simpler, more stable alternatives. In response, we developed a chemical-free platform by fusing an apolipoprotein (ApoA1) directly to an antibody―termed “Grab Antibody” (GrAb). Owing to the strong lipid-binding affinity of ApoA1, GrAb spontaneously assembles into a stable protein corona on the LNP surface, maintaining appropriate antibody orientation. Using trastuzumab as a model antibody, we demonstrate that HER2-targeted GrAb-LNPs can efficiently deliver p53 mRNA to HER2-overexpressing tumors, yielding significant antitumor effects with minimal off-target toxicity. Results: A 30-minute incubation at 37°C allowed GrAb to form a robust protein corona on the LNP surface (Fig. 1A). Modulating the lipid-to-protein (LP) molar ratio from 500:1 to 100,000:1 revealed a drop in mRNA encapsulation efficiency at higher GrAb levels, but negligible leakage once the ratio exceeded 10,000:1 (Fig. 1B). Size-exclusion chromatography (SEC) showed that GrAb strictly co-eluted with LNP fractions (Fig. 1C), and fluorescence measurements confirmed high incorporation of Cy5-labeled antibody (Fig. 1D). Cryo-TEM images of nude LNPs and trastuzumab GrAb-conjugated LNPs (HerLNPs) appeared similarly spherical (Fig. 1E). In HER2- overexpressing SK-OV-3 cells, HerLNPs exhibited selective binding and increased DiD fluorescence signals (Fig. 1F), which were abolished when HER2 receptors were pre-blocked (Fig. 1G). Intracellular uptake of Cy5-labeled mRNA was notably higher with HerLNPs (Fig. 1H), leading to greater p53 protein expression in vitro (Fig. 1I). In vivo, intravenous injection of DiD-labeled Fluc mRNA@HerLNPs led to superior tumor accumulation and reduced liver uptake, as shown by near-infrared fluorescence imaging (Fig. 1J, 1K, 1L). Repeated dosing with p53@HerLNPs significantly suppressed tumor growth (Fig. 1M, 1N) and induced marked apoptosis (Fig. 1O) in an SK-OV-3 xenograft model. Importantly, treated mice maintained stable body weights (Fig. 1P) and showed normal levels of liver enzymes (Fig. 1Q), indicating minimal systemic toxicity. Histological examination confirmed the absence of hepatic damage in HerLNP-treated animals, whereas mice receiving p53@ConLNPs displayed evident liver injury (Fig. 1R). Together, these findings validate GrAb-LNPs as an effective, targeted mRNA delivery platform offering enhanced therapeutic efficacy and reduced off-target effects in cancer treatment.