Surface-engineered robust hybrid lipid nanocapsules: A next-generation nanoplatform for mRNA delivery

Abstract

Hybrid lipid nanocapsules (hLNCs) represent a promising platform for mRNA delivery with the added benefit of ambient-temperature stabilization. Conventional hLNCs use polyethyleneimine (PEI) to facilitate mRNA binding, cellular uptake, and endosomal escape. However, PEI’s high charge density is associated with cytotoxicity, nonspecific protein binding, and innate immune activation, features that may limit utility in chronic or repeat-dose applications. To overcome these limitations, we engineered the hLNC surface with either L-histidine (hLNC-Hist) or short-chain polyethylene glycol (hLNC-PEG) to improve biocompatibility. Both modifications reduced serum protein binding and enhanced cytocompatibility. hLNC-Hist preserved mRNA transfection in vitro and improved in vivo expression compared to unmodified hLNCs. In contrast, hLNC-PEG significantly reduced transfection efficiency. Notably, hLNC-Hist did not induce IgM or IgG responses and suppressed circulating pro-inflammatory cytokines following systemic administration – features that stand in contrast to the acute immune activation and anti-PEG antibody formation commonly observed with lipid nanoparticles (LNPs). Although hLNC-Hist did not achieve quite the peak transfection efficiency as LNPs in some models, hLNC-Hist did not exhibit the immunogenicity and reactogenicity that can limit repeated LNP dosing. hLNC-Hist offers a compelling alternative with an improved safety-efficacy profile for chronic mRNA delivery. Importantly, hLNC-Hist retained the ability to stabilize mRNA under ambient and elevated temperatures when embedded in sugar glass, enabling potential cold-chain-free deployment. These findings position hLNC-Hist as an immunologically silent, biocompatible, and thermally stable mRNA delivery vehicle. Its suitability for repeated administration makes it particularly attractive for emerging mRNA therapies requiring chronic dosing, including cancer immunotherapy, protein replacement, and autoimmune modulation.

Introduction

The mRNA vaccine is emerging as a novel modality in cancer therapy, showing considerable promise in reshaping the approach to addressing malignancies [[1], [2], [3], [4]]. Chen et al. reported prevention and tumor burden of B16F10-OVA tumors using mRNA encoding OVA antigen eliciting a robust CD8+ T cell response [5]. Similarly, Persano et al. reported anti-tumor activity of mRNA-encoded OVA antigen by activating dendritic and T cells [6].

The mRNA tumor vaccines are most promising for combating tumors because they can be explicitly designed to target unique mutational signatures or tumor antigens expressed/overexpressed by cancerous cells, triggering strong antitumor T and B cell responses. Moreover, mRNA vaccines have the potential to be individualized against patient-specific unique mutational signatures. As a result, many mRNA vaccines encoding tumor antigens have been registered for clinical trials. For example, Sahin et al. tested melanoma FixVac (BNT111), a liposomal RNA (RNA-LPX) vaccine, which targets four non-mutated tumor-associated antigens (Lipo-MERIT trial, ClinicalTrials.gov identifier NCT02410733) [7]. In addition, a personalized mRNA vaccine (mRNA-5671) encoding KRAS driver mutations (G12C, G12D, G12V and G13C) has been developed for the treatment of pancreatic cancer in combination with pembrolizumab and registered for clinical trial (NCT03480152) [8].

Harnessing the body’s innate molecular machinery to synthesize therapeutic proteins, mRNA therapies offer unparalleled precision and versatility. However, translating this promise into therapeutic reality hinges on achieving an optimal balance between the stability and efficient delivery of mRNA cargo [9]. Various nanoparticles, such as lipid nanoparticles, lipo-polyplex, virus-like nanoparticles, biomimetic nanoparticles, polymer-based nanoparticles, inorganic-based nanoparticles, exosome-based nanoparticles, etc., have been investigated for mRNA delivery [2,[10], [11], [12], [13], [14]]. However, challenges remain with mRNA formulation’s instability and achieving efficient delivery [[15], [16], [17], [18]]. Foremost in clinical deployment among mRNA delivery nanocarriers, lipid nanoparticles (LNPs) consist of phospholipids, an ionizable lipid, cholesterol, and PEGylated lipid, which have shown promising results in delivering mRNA therapeutics [19]. Despite their promising results in mRNA delivery, mRNA-loaded LNPs require ultra-cold storage/or cold storage, leading to costly deployment logistics [20]. Moreover, PEGylated lipids in LNPs may lead to immunogenicity and associated concerns about the efficacy of the delivered mRNA therapeutics [[21], [22], [23]], especially for chronic treatments such as cancer vaccines and protein replacement therapies. These challenges drive researchers to explore alternative delivery methods. To mitigate the immune response of LNPs, Gong et al. introduce antioxidant ionizable lipid, C-a16, which exhibits reduced immunogenicity and improved overall efficacy of LNP in mRNA delivery [24]. Li et al. synthesized an array of ionizable lipids to enhance the effectiveness of LNPs [25]. Ideally, a nanocarrier would stabilize and deliver the mRNA cargo effectively while circumventing the immune response.
We recently developed a series of robust hybrid lipid nanocapsules (hLNCs) for mRNA delivery, addressing the challenge of mRNA stability at ambient and elevated temperatures [26]. The hLNCs are composed of a mixture of polymer (oleic acid-polyethyleneimine (OA-PEI) conjugate), lipids (triglyceride and phospholipid), and non-ionic surfactant. The hLNCs we previously developed incorporate PEI on their surface to facilitate mRNA complexation. However, the free cationic charge of PEI can interact with cellular membranes or organelles, potentially leading to cytotoxic effects. Here, we have functionalized the hLNC surface with L-histidine (hLNC-Hist) or PEG (hLNC-PEG) to separate the PEI from the cellular environment. The choice of L-histidine as a surface modification was driven by its versatility and inherent pH-responsive properties. L-histidine, a natural amino acid, offers a combination of biocompatibility and environmental sensitivity [27]. At neutral pH, it remains neutral, minimizing unwanted protein adsorption and opsonization, which is essential for evading immune detection. Yet, in a slightly acidic microenvironment, L-histidine becomes cationic, making the nanocapsules suitable for endosomal escape [28,29]. PEG was chosen as a comparison for its well-known masking ability.

In this study, we aim to develop and evaluate surface-functionalized hybrid lipid nanocapsules (hLNCs) as efficient carriers for mRNA delivery. Specifically, we investigate L-histidine- (hLNC-Hist) and polyethylene glycol- (hLNC-PEG) functionalized nanocarriers to modulate surface charge, enhance biocompatibility, and minimize non-specific protein adsorption. We further assess their cellular uptake in the presence or absence of serum, transfection efficiency, immune response induction, and the stability of encapsulated mRNA at elevated temperatures.

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Materials

Labrafac Lipophile WL-1349 and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were graciously provided by Gattefosse Germany and Lipoid Germany, respectively. Kolliphore HS15 (Polyethylene glycol (15)-hydroxystearate), Oleic acid, Dulbecco’s modified Eagle’s medium (DMEM), RPMI medium, penicillin/streptomycin solution, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide (MTT), trypsin-EDTA (1×), polyvinyl alcohol (PVA), were procured from Sigma Aldrich (St. Louis, MO, USA). Polyethyleneimine (PEI) (average Mw ∼1.8 kDa) was acquired from Alfa Aesar, USA. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), DiD’ solid; (1,1’-Dioctadecyl-3,3,3′,3’-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt), Methyl-PEG-NHS-Ester (mPEG(12)-NHS) and N-Hydroxysuccinimide (NHS) were sourced from ThermoFisher Scientific (Suwanee, GA, USA). Ethidium bromide (EtBr) and Hoechst 33342 were obtained from Invitrogen (ThermoFisher Scientific, Suwanee, GA, USA). Agarose molecular biology grade was purchased from IBI Scientific. Firefly Luciferase mRNA (CleanCap® FLuc mRNA) was purchased from TriLink Biotechnologies (San Diego, California, USA). Other chemicals and solvents utilized in the study were of analytical grade and used as received.

Sunil Kumar Yadava, Anuja Tripathi, Mridula Kabilan, Julie A. Champion, Marcus T. Cicerone, Surface-engineered robust hybrid lipid nanocapsules: A next-generation nanoplatform for mRNA delivery, Journal of Controlled Release, Volume 388, Part 1, 2025, 114312, ISSN 0168-3659, https://doi.org/10.1016/j.jconrel.2025.114312.