Engineered internal architecture of core-shell lipid nanoparticles promotes efficient mRNA endosomal release

Domain: neuroscience
Published: 2026-01-30
Editorial outcome: minor_revision

Abstract
Messenger RNA (mRNA) therapeutics rely on lipid nanoparticles (LNPs) for delivery, yet inefficient endosomal escape remains a major bottleneck, with only a small fraction of internalized cargo reaching the cytoplasm. Conventional LNPs encapsulate mRNA in amorphous lipid cores, where partial charge neutralization and lack of structural order limit protonation-driven membrane disruption. Here, we present an architectural strategy that engineers LNP internal structure using ionizable lipid–coated gold nanoparticles (IC-AuNPs) as rigid, pH-responsive cores. The Au cores template the formation of radially ordered core–shell architectures that stabilize particles at physiological pH while amplifying charge segregation and curvature stress under acidic endosomal conditions. As a result, Au-LNPs achieve a twofold increase in endosomal escape and ~100-fold greater cytoplasmic mRNA diffusion compared to conventional LNPs. Functionally, Au-LNPs enhance mRNA expression in vitro, increases in vivo protein production up to sevenfold, boost antibody responses to SARS-CoV-2 vaccines, and improve therapeutic efficacy in a triple-negative breast cancer model.

Introduction
Messenger RNA (mRNA)-based therapeutics are poised to transform preventive and curative medicine, offering promising solutions for a broad arrange of diseases, including infectious disorders, cancers, and genetic conditions1–3. Central to the clinical success of mRNA therapies is the development of efficient, safe, and versatile delivery systems that can protect the mRNA cargo, navigate biological barriers, and ultimately release mRNA into the cytoplasm for effective translation4–7. Among the various non-viral vectors, lipid nanoparticles (LNPs) have emerged as the leading platform for mRNA encapsulation and delivery, exemplified by the success of LNP-formulated mRNA vaccines against COVID-198–12.

Despite these advances, a critical bottleneck in mRNA delivery remains the inefficient escape of LNPs-encapsulated mRNA from endosomes into the cytosol, with current cytoplasmic delivery rates estimated at around 2%5,13,14. Once internalized, LNPs become trapped in the endo-lysosomal system, where mRNA is prone to degradation if not promptly released15,16. Over the past decade, researchers have explored a wide range of strategies to enhance endosomal escape17,18. Chemical modifications of ionizable lipids, incorporation of pH-responsive groups, and careful tuning of lipid compositions have all expanded the design toolkit for LNPs and yielded valuable improvements19–21. Nevertheless, achieving robust and reliable endosomal escape remains an ongoing challenge, underscoring the need to explore new design principles that move beyond established paradigms.

Conventional LNPs encapsulate mRNA in an amorphous mixture of ionizable lipids, helper lipids, cholesterol, and polyethyleneglycol (PEG)–lipids1. Upon exposure to the acidic endosomal environment, ionizable lipids become protonated and can, in principle, promote membrane fusion by forming non-bilayer hexagonal (H_II) phases22. In practice, however, the effective charge density of ionizable lipids is partially neutralized by mRNA within the LNP core, attenuating their membrane-disruptive capacity23. Moreover, the lack of structural order within conventional LNPs may limit the extent and coordination of these phase transitions, resulting in suboptimal cytosolic release5. These intrinsic architectural constraints highlight the need for alternative design principles that address not only lipid chemistry but also the internal organization of LNPs.

In this work, we present a distinct architectural approach that shifts the focus from solely chemical modifications to the internal structure of LNPs. Specifically, we leverage ionizable lipid–coated gold nanoparticles (IC-AuNPs) as rigid, pH-responsive cores that template the formation of radially ordered core–shell architectures. This design stabilizes nanoparticles under physiological conditions (pH 7.4), preserving their integrity during circulation, while amplifying membrane-disruptive forces in the acidic endosomal environment (pH~5.5–6.0) through enhanced charge segregation and curvature stress (Fig. 1). By systematically optimizing this architecture, we achieved a twofold increase in endosomal escape efficiency and a ~100-fold enhancement in cytoplasmic mRNA diffusion compared to conventional LNPs. The resulting Au-LNPs exhibited markedly higher mRNA expression across multiple cell types in vitro and up to a fivefold increase in protein expression in vivo. Functionally, Au-LNPs enhanced the immunogenicity of a SARS-CoV-2 spike mRNA vaccine and improved therapeutic efficacy in a triple-negative breast cancer model. Importantly, gold was used here as a proof-of-concept scaffold; the underlying design principle is generalizable to other chemically compatible cores. By shifting the focus from lipid chemistry alone to the internal architecture of LNPs, this work establishes a new paradigm for overcoming the endosomal escape bottleneck and expands the design toolkit for next-generation mRNA therapeutics.

Results
Design and physicochemical characterization of gold-lipid nanoparticles
AuNPs were selected as a proof-of-concept core material primarily due to their chemical versatility, including high surface reactivity for versatile modifications and precise size control (Fig. 2a). To explore whether internal architecture could enhance mRNA delivery, we synthesized IC-AuNPs as structural cores for LNP assembly. AuNPs were first PEGylated and then coated with ionizable lipids such as MC3 or SM-102 via phase transfer mediated by hydrophobic forces, yielding hydrophobic IC-AuNPs that readily integrated into organic solvents (Fig. 2a, Methods). Successful coating was confirmed by Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR), which revealed diminished PEG-specific peaks alongside the emergence of MC3 signatures (Supplementary Fig. 1a, b).

For LNP assembly, both conventional LNPs and Au-LNPs were prepared from the same pre-mixed lipid master solution (Methods), ensuring identical lipid composition across groups. The critical difference lay in the assembly sequence. In conventional LNPs, mRNA was directly mixed with the lipid solution, resulting in amorphous lipid–mRNA condensates. In contrast, for Au-LNPs, mRNA was first condensed onto IC-AuNPs to form a stable mRNA–Au core complex. This condensation step was confirmed by gel electrophoresis, which showed complete retardation of mRNA migration and no detectable free mRNA in solution (Supplementary Fig. S1c), indicating efficient binding to the IC-AuNPs. The resulting mRNA–Au complexes were then encapsulated by the lipid master mix to generate ordered core–shell nanoparticles. This two-step assembly process positioned mRNA and ionizable lipids radially around the Au core, establishing a distinct internal architecture compared with conventional LNPs.

Transmission electron microscopy (TEM) visualized ~13nm Au cores (Fig. 2b) coated with MC3 (Au-MC3) and surrounded by a 2.5nm lipid layer, confirming successful core–shell formation (Fig. 2c, Methods). When assembled into Au-LNPs, these MC3-coated cores yielded nanoparticles with an average dry-state diameter of 42.37±0.25nm (Fig. 2d and Supplementary Fig. S1d). In contrast, dynamic light scattering (DLS) indicated a hydrated hydrodynamic size of ~70.2nm (Fig. 2e, “Methods”), while preliminary cryo-EM measurements confirmed a hydrated size of 69.32±0.34nm with intact spherical morphology (Supplementary Fig. S1e, Methods). The smaller values from TEM are consistent with the expected shrinkage of lipid nanoparticles during negative staining and drying, as noted in previous LNP studies24. Importantly, TEM of conventional LNPs was also included for direct comparison (Supplementary Fig. S1f). To confirm that lipid coating is generalizable, we extended the phase transfer method to three different ionizable/cationic lipids and verified successful attachment to the AuNPs surface in all cases (Supplementary Fig. S1g). Similar core–shell morphology was also observed when ALC-0315 or DOTAP were used in place of MC3 (Supplementary Fig. S1h), demonstrating that the architecture is reproducible across different lipid chemistries and not an artifact of a single lipid.

Notably, Au-LNPs exhibited significantly higher uniformity than conventional LNPs, with no oversized particles detected by DLS and a markedly lower polydispersity index (PDI) of 0.073 compared to 0.128 for LNPs (Fig. 2e). Mechanistically, the absence of larger particles in Au-LNPs can be attributed to enhanced hydrophobic free energy around the gold core, which promotes tight lipid assembly and prevents the formation of poorly hydrated lipid aggregates that otherwise yield oversized structures.

We further investigated the impact of IC-AuNPs incorporation on LNP structural stability through molecular dynamics (MD) simulations and membrane fluidity assays. MD simulations revealed that Au-LNPs exhibited a significantly lower assembly enthalpy (–354.3kJ/mol) compared to conventional LNPs (–210.7kJ/mol), indicating that the two-step templated process yields thermodynamically more stable structures (Supplementary Fig S1i). Membrane viscosity was assessed using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), which showed higher fluorescence intensity at acidic pH in Au-LNPs, suggesting an increased presence of hydrophobic domains25. These findings support the conclusion that Au-MC3 increases hydrophobic free energy, thereby promoting lipid encapsulation around the mRNA–Au complex and stabilizing Au-LNPs assembly (Fig. 2f).

Liquid Chromatography–Mass Spectrometry (LC-MS) analysis confirmed that ionizable lipid content was indistinguishable between LNPs and Au-LNPs formulations (Supplementary Fig. 1j, k, “Methods”), demonstrating that incorporation of the Au core does not alter overall lipid composition and ruling out formulation bias as a confounding variable. To validate the functional contribution of the Au core, we characterized the Zeta potential of Au-MC3 compared with free MC3 lipids across a range of pH values (Methods). Both exhibited characteristic pH-dependent charge shifts, but Au-MC3 showed a significantly larger change in surface potential (Supplementary Fig. 1l), indicating that the gold core amplifies the protonation-driven responsiveness of ionizable lipids.

Together, these results establish that incorporating IC-AuNPs as scaffolds drive the formation of stable, structurally ordered Au-LNPs, while maintaining identical lipid composition compared to conventional LNPs, thereby isolating internal architecture as the key variable underlying their distinct behavior.

AuNP size dictates assembly stability and delivery efficiency
We next investigated how the size of the AuNPs core influences LNP assembly and mRNA delivery. IC-AuNPs with diameters of 13, 18, 25, and 50nm were synthesized and coated with MC3, then incorporated into LNPs using the same lipid master solution as above. TEM and DLS analysis revealed that 13nm Au cores produced monodisperse Au-LNPs with narrow size distributions, whereas larger cores disrupted lipid organization and yielded broader distributions with oversized aggregates (Fig. 2e and Supplementary Fig 1l–n, “Methods”).

Functional studies using luciferase (Fluc) mRNA demonstrated a strong correlation between Au core size, assembly stability, and transfection efficiency. Au-LNPs with 13nm cores enhanced luciferase expression by approximately threefold relative to conventional LNPs, whereas 18nm cores provided only a modest 1.5-fold increase. By contrast, 25nm and 50nm cores showed no improvement or even reduced expression efficiency (Supplementary Fig. S1m). These findings indicate that only small Au cores are compatible with ordered LNPs assembly, while larger cores occupy excessive internal volume and destabilize lipid packing.

Consistent with these observations, DLS confirmed that only 13nm Au-MC3 cores produced stable nanoparticles with uniform size distributions, whereas larger cores resulted in heterogeneous particles with higher polydispersity (Supplementary Fig. 1n). Together, these results identify ~13nm as an optimal Au core size for templating ordered Au-LNPs, directly linking nanoscale architecture to functional mRNA delivery efficiency.

Enhanced pH-responsive membrane disruption by Au-LNPs
Efficient endosomal escape requires ionizable lipids to become protonated in acidic compartments and destabilize the endosomal membrane. Having established that Au-LNPs exhibit increased thermodynamic stability and amplified pH responsiveness, we next assessed their membrane fusion potential under endosomal-like conditions. We first employed a sulforhodamine B (SRB)–loaded liposome assay as a widely used proxy for pH-dependent membrane fusion of lipid nanoparticles (Methods)26. Both Au-LNPs and conventional LNPs showed minimal leakage at physiological pH (7.4) and robust activity near the endosomal pH (~5.5), consistent with ionizable lipid protonation. Across the acidic range, Au-LNPs consistently induced higher leakage than conventional LNPs, with a significant enhancement at pH 5.5 (Fig. 2g).

To provide a more biologically relevant validation, we complemented the SRB assay with a pH-dependent hemolysis assay using red blood cells (RBCs, Methods). In line with the SRB results, Au-LNPs triggered greater membrane disruption at acidic pH (65% hemolysis at pH 5.5 vs. ~57% for conventional LNPs; Supplementary Fig. 2a), while remaining inert at neutral pH. Together, these assays confirm that Au-LNPs possess superior pH-triggered membrane-lytic activity compared with conventional LNPs.

To mechanistically link this enhanced activity to lipid chemistry, we measured the Zeta potential of Au-MC3 versus free MC3 lipids across a pH gradient. Both systems exhibited the expected protonation-dependent charge shift, but Au-MC3 underwent a substantially larger change in surface potential (Supplementary Fig. S1l). This finding supports the model that the Au core amplifies protonation-driven charge segregation, thereby increasing the effective charge density of ionizable lipids at the particle–membrane interface. The enhanced membrane destabilization near pH 5.5 is also consistent with prior reports showing that protonated ionizable lipids promote non-bilayer hexagonal (H_II) phases, which are highly disruptive to membranes5,22. Together, these results demonstrate that Au-LNPs not only stabilize nanoparticle architecture under physiological conditions but also potentiate the pH-triggered membrane-disruptive behavior of ionizable lipids under acidic conditions, a critical prerequisite for efficient cytosolic mRNA release.

In vitro assessment of mRNA expression efficiency of Au-LNPs
We conducted a series of in vitro experiments that analyzed mRNA expression in cells to evaluate the actual mRNA delivery efficiency of Au-LNPs and the TEM imaging of endosome escape of based on unique electrodensity of Au-LNPs (Methods). We first assessed the impact of different lipids coatings on Au-LNPs by using Fluc as a reporter gene. HeLa and 293 cells were transfected with Au-LNPs, each functionalized with different ionizable/cationic lipids, and containing Fluc-mRNA. Following transfection, luciferase activity of cell lysate was measured to quantify mRNA expression. Among the tested coatings, Au-MC3 demonstrated the most significant enhancement in mRNA expression compared to conventional LNPs (Fig. 3a). This enhancement is possibly attributed to the favorable zeta potential shift of Au-MC3 under acidic conditions, which facilitates better interaction between the nanoparticles and the cell membrane, leading to improved endosome escape and subsequent mRNA translation (Fig. 3b). The toxicity of Au-LNPs with MC3 were verified by cell-counting-kit-8 (CCK8) assay (Methods), showing no statistically significant difference between the Au-LNPs treated and LNPs treated HeLa cells (Fig. 3c). MC3 was selected as surface modification of IC-AuNPs for subsequent research.

The interaction between Au-MC3 and mRNA was characterized using gel electrophoresis, isothermal titration calorimetry (ITC), and DLS (Supplementary Figs. 1c and 2b, c). Gel electrophoresis demonstrated that Au-MC3 efficiently retarded mRNA migration, confirming strong binding between the cationic MC3-coated cores and the anionic mRNA. ITC further revealed an exothermic binding profile with favorable enthalpy, consistent with electrostatic interactions and hydrogen bonding driving mRNA condensation on the Au-MC3 surface. Correspondingly, DLS analysis showed an increase in hydrodynamic diameter upon mRNA loading, indicating the formation of stable Au–mRNA complexes. Together, these results confirm that Au-MC3 cores act as effective nucleation sites for mRNA condensation, enabling subsequent lipid encapsulation into ordered core–shell Au-LNPs.

We then assessed the optimal dosage for maximal FLuc-mRNA expression delivered by LNPs and Au-LNPs (Fig. 3d and Supplementary Fig. 2d). In both systems, luciferase intensity increased as the mRNA dosage increased. However, in the conventional LNPs group, protein expression peaked at 0.5 μg/mL; beyond this dose, at 1 μg/mL, protein expression decreased. Au-LNPs showed continued increase without peaking up to 2 μg/mL, possibly attributable to enhanced escape enabling higher dose tolerance without saturation/degradation (Supplementary Fig. S2d). The improved expression efficiency mRNA delivered by Au-LNPs may be attributed to improved endosomal escape efficiency.

To further evaluate the mRNA delivery performance of Au-LNPs, we employed confocal microscopy (CLSM) and flow cytometry to measure GFP-mRNA expression in HeLa and 293 cells using both conventional LNPs and Au-LNPs delivery systems at two different dosages (Methods). As expected, cells treated with Au-LNPs exhibited substantially higher GFP transfection efficiency and mean GFP expression intensity compared to those treated with conventional LNPs at both high and low dosages (Fig. 3e, f and Supplementary Fig. 2e, f). Notably, at the low dosage, Au-LNPs demonstrated dominant superiority, achieving a transfection efficiency of 91.4% compared to 6.72% with conventional LNPs. This enhanced performance could be due to improved endosomal escape efficiency. Similar trends were observed using Fluc as a reporter gene, confirmed across five different cell types and with three LNPs formulations (Supplementary Fig. 2g–l).

Mechanistic insights into Au-LNP–mediated endosomal escape
To elucidate the endosomal escape mechanisms of Au-LNPs compared to conventional LNPs, we employed CLSM and TEM (Fig. 4, Methods). Using Cy5-labeled mRNA, we quantified endosomal escape by measuring the cytosolic diffusion area of the Cy5 signal and calculating the endosomal escape efficiency (Fig. 4a–d and Supplementary Fig. 3a–e, Methods). After 6 h of incubation, a substantial portion of Cy5-mRNA delivered by Au-LNPs reached the cytoplasm, as evidenced by diffuse Cy5 fluorescence (Fig. 4c). Notably, the cytoplasmic diffusion area of Cy5-mRNA in Au-LNP–treated cells was 100-fold greater than that in cells treated with conventional LNPs (Fig. 4d)13, and the endosomal escape efficiency of Au-LNPs was twice as high (Supplementary Fig. S3d. To assess endosomal maturation, we quantified colocalization between Cy5-mRNA (green) and LysoTracker (Red, Methods) via CLSM. Reduced overlap (~50% lower; Supplementary Fig. S3e) across doses (0.25–1 μg/mL) and times (4–6 h) in Au-LNP-treated cells indicates less mRNA in acidified compartments, implying Au-LNPs reduced endosomal acidification (lower LysoTracker colocalization; Fig. 4a–c and Supplementary Fig. S3e), favoring early escape at pH~5.5–6.0 where MC3 is protonated, while avoiding progression to highly degradative lysosomes (pH~4.5). This balance enhances cytoplasmic release and protects the mRNA cargo. This aligns with enhanced escape (Supplementary Fig. S3d). This observation, consistent with our membrane disruption studies under different pH conditions, support the notion that Au-LNPs inhibit endosome acidification and maturation, thereby maintaining conditions more favorable for mRNA release.

TEM provided additional insights into how Au-LNPs influence endosomal morphology (Fig. 4e–h). Au-LNPs exhibit favorable stable characteristics within biological environments, as demonstrated in Fig. 2f, g. The high electron density and unique nanostructure of Au-LNPs enabled clear visualization27–30. Au-LNPs were found in vesicles ranging from 0.2 to 5 µm in diameter, encompassing early endosomes (EE, 0.1–0.5 µm) and micropinosomes (MS, 0.5–5 µm). Both LNPs- and Au-LNPs-treated cells displayed more large vesicles (>1 µm) than the negative control (Supplementary Fig. S3f), a hallmark of disrupted trafficking between early and late endosomes/lysosomes31. Importantly, Au-LNP–treated cells contained significantly more enlarged vesicles than LNP-treated cells (Fig. 4f), suggesting a stronger membrane disruption capability. We observed that these enlarged vesicles often fused with existing MS (Fig. 4g, h), and in some cases, vesicles with partially compromised membranes also underwent fusion. The exact cause of these membrane defects—whether membrane fusion events, proton sponge effects, or other factors—remains unclear. Moreover, Au-LNPs tended to cluster near damaged areas of partially disrupted vesicles, possibly due to osmotic pressure gradients (Fig. 4g, h).

To further investigate whether Au-LNPs modulate endosomal maturation, we examined the expression and localization of canonical endosomal markers32,33. Confocal immunofluorescence staining revealed that LNP-treated cells exhibited strong colocalization of mRNA with the late endosomal/lysosomal marker LAMP1, whereas Au-LNPs–treated cells showed markedly reduced LAMP1 signal, with mRNA distributed more diffusely in the cytoplasm (Fig. 5a). In contrast, no significant differences were observed in the early endosomal marker RAB5 between LNPs- and Au-LNPs–treated groups (Fig. 5b). Quantification confirmed that LAMP1 intensity was significantly reduced in Au-LNP–treated cells, while RAB5 intensity remained unchanged (Fig. 5c).

These observations were corroborated by Western blot and qPCR analyses. LAMP1 protein and mRNA levels were consistently decreased following Au-LNP treatment at both 4 and 6 h, whereas RAB5 expression showed no significant differences compared with conventional LNPs (Fig. 5d, e). Together, these results indicate that Au-LNPs selectively interfere with late endosomal maturation without altering early endosome formation. By attenuating progression to LAMP1 degradative compartments, Au-LNPs create a temporal window that favors endosomal escape of mRNA at the early-to-late transition stage, consistent with the enhanced cytosolic release observed in our colocalization and TEM studies.

Collectively, our findings reveal fundamental differences between Au-LNPs and conventional LNPs in promoting endosomal escape (Fig. 1). Previous research has shown that ionizable lipids in acidic endo-lysosomal compartments become positively charged and interact with the negatively charged endosomal membrane, encouraging the formation of a metastable hexagonal (H_II) phase that facilitates mRNA release15,16. However, in conventional LNPs, the uniform distribution of ionizable lipids and mRNA can diminish the lipids’ effective charge density, as they partially neutralize each other’s charges, reducing their membrane-disruptive potential. By contrast, Au-LNPs employ a pH-sensitive core that becomes strongly positively charged at low pH. This positively charged core attracts negatively charged mRNA, drawing it closer to the core region, while simultaneously repelling positively charged ionizable lipids and pushing them toward the endosomal membrane. This strategic segregation ensures that the ionizable lipids maintain a higher effective charge density and more readily induce the H_II phase. Consequently, membrane destabilization is enhanced, enabling more efficient mRNA release into the cytoplasm. Once mRNA diffuses into the cytosol, the local environment reverts to near-neutral pH (~7), decreasing the core’s positive charge and weakening its attraction to mRNA. Freed from the core’s electrostatic constraints, the mRNA diffuses more readily into the cytoplasm. Thus, the pH-sensitive core in Au-LNPs serves a dual function: directing ionizable lipids toward the endosomal membrane to promote escape and orchestrating a charge-driven redistribution of mRNA and lipids for more efficient cytosolic release. This mechanistic model explains the superior endosomal escape efficiency and mRNA diffusion achieved by Au-LNPs, demonstrating how architectural engineering and charge responsiveness can overcome the intrinsic limitations of conventional LNPs. By offering a deeper understanding of the interplay between nanoparticle structure, lipid chemistry, and endosomal microenvironment, our findings provide a blueprint for future design strategies aimed at enhancing mRNA delivery efficacy.

Au-LNPs enhanced mRNA delivery across administration routes and organ targeting compatibility
We then applied Au-LNPs in mouse models to evaluate its in vivo potential (Methods). Mice were administered Au-LNPs or conventional LNPs prepared via both ethanol injection and microfluidic methods. In vivo chemiluminescence imaging, reflecting mRNA expression levels, revealed that mice treated with Au-LNPs exhibited significantly higher signals, showing up to a fivefold increase compared to those treated with conventional LNPs (Fig. 6a–c). This result aligns with our in vitro findings, highlighting the robustness of the Au-LNP system in enhancing mRNA expression across diverse biological environments. Notably, the chemiluminescence intensity from microfluidic preparations was an order of magnitude higher than that from ethanol injection, consistent with our nanoflow cytometry results (Fig. 6b, c).

We also demonstrated that Au-LNPs are effective across different administration routes. As shown in Fig. 5d, intramuscular administration of Au-LNPs led to a 7-fold increase in luminescence intensity compared to conventional LNPs. We investigated mRNA expression and organ distribution from both Au-LNPs and LNPs. Due to the similar surface properties of Au-LNPs and LNPs, we expected comparable biodistribution profiles across major organs such as the liver and spleen (Supplementary Fig. S4a–c). This similarity in biodistribution suggest that Au-LNPs can maintain the organ-targeting characteristics of LNPs while significantly enhancing mRNA delivery and expression.

As IC-AuNPs do not influence the native chemistries of lipids in LNPs, our promoting mRNA endosome escape structure design can incorporate lipids with different chemistries to achieve targeted organ delivery while still enhancing mRNA delivery efficiency. We incorporated the Selective Organ Targeting (SORT) system—known for its affinity to particular organs—to allow preferential accumulation of Au-LNPs in the liver or lungs, respectively. This level of control over organ targeting was further confirmed by enhanced mRNA expression in the lungs, as indicated by increased chemiluminescence in those regions (Fig. 6e and Supplementary Fig. S4d)34,35. These findings suggest that IC-AuNPs-assisted enhance endosome escape not only enhances delivery efficiency across different preparation methods but also improves the efficacy of various LNPs formulation to achieve specific organ-targeted delivery.

Enhanced immunogenicity of Au-LNPs in SARS-CoV-2 spike-mRNA vaccines
The potential of Au-LNPs in enhancing the efficacy of preventive vaccines was thoroughly investigated using Spike-mRNA for SARS-CoV-2 as a model antigen (Methods). To assess the immune response induced by the Au-LNPs-based vaccine, we measured the geometric titer of antibodies produced following immunization. The results were promising, demonstrating that Au-LNPs were significantly more effective in eliciting a robust immune response compared to conventional LNPs. Specifically, after the initial immunization, the geometric titer of antibodies in the group vaccinated with Au-LNPs was double that observed in the group receiving the conventional LNPs-based vaccine (Fig. 7a, b). This remarkable increase in antibody titers is likely attributable to the enhanced mRNA endosome escape and expression facilitated by the Au-LNPs system. The increased efficiency in structure design and subsequent antigen production likely led to a more potent activation of the immune system, resulting in a stronger antibody response.

The enhanced immune response observed after the initial dose was further amplified following a booster immunization. In the Au-LNPs group, the geometric antibody titer increased by an additional 68% compared to the conventional LNPs group after the booster dose (Fig. 7c), further highlighting the superior efficacy of Au-LNPs in vaccine applications. This suggests that the Au-LNPs platform not only enhances the primary immune response but also significantly boosts the secondary response, which is critical for achieving long-lasting immunity. In addition to the overall increase in antibody titers, the quality and functionality of the antibodies produced were also significantly improved with Au-LNPs. Virus neutralization assays were conducted to evaluate the effectiveness of the antibodies in preventing viral entry into host cells. The results showed a 46% increase in the half-maximal inhibitory concentration (IC50) for the antibodies generated by the Au-LNPs-based vaccine compared to those from the conventional LNPs group (Fig. 7d, f). This higher IC50 value indicates that the antibodies induced by the Au-LNPs vaccine were more effective at neutralizing the virus, providing stronger protection against SARS-CoV-2 infection.

Au-LNPs in therapeutic mRNA vaccines efficacy in triple-negative breast cancer model
After confirming the excellent performance of Au-LNPs in preventive vaccines, we aimed to investigate the potential application of Au-LNPs in therapeutic vaccines (Methods). The triple-negative breast cancer (TNBC) is chosen as therapeutic mRNA vaccines model for its aggressive nature and lack of targeted therapies, making it one of the most challenging forms of cancer to treat. The Wilms’ tumor 1 gene (WT1) focally amplifying in TNBC is chosen as mRNA coding region, which induces T cells to recognize tumor antigen through vaccination, activating an immune response to produce tumor antibodies36–41. Seven days after the 1×10^6 4T1-Fluc tumor cells orthotopic injection, the mice were randomly divided into three groups: phosphate-buffered saline (PBS), WT1-mRNA loaded by LNPs, or Au-LNPs. The mice were intraperitoneally injected three times with different groups every 2 days (Fig. 8a). The TNBC volume growth rate and survival rate are evaluation indicators. The results were encouraging, demonstrating that Au-LNPs significantly enhanced the efficacy of the WT1-mRNA vaccine in reducing tumor burden and extending survival time. Compared to PBS group and conventional LNPs group, all mice of the Au-LNPs group survived for 30 days (Fig. 8b, c).

In addition to the significant reduction in tumor volume and extension in survival rate, the bioluminescence intensity, used as an indicator of tumor activity, was markedly lower in mice treated with Au-LNPs compared to those treated with conventional LNPs (Fig. 8d, e). Bioluminescence imaging provides a non-invasive way to monitor the metabolic activity and growth of tumors in vivo. The lower bioluminescence intensity observed in the Au-LNPs-treated group indicates not only a reduction in tumor size but also a decrease in the overall metabolic activity of the tumor cells. This reduction in metabolic activity suggests that the tumor cells are less viable and less capable of sustaining growth and proliferation, which could lead to improved long-term outcomes for patients. Specifically, Au-LNPs treatment led to reductions of 87% in tumor size and 86% in tumor weight in comparison with PBS group at 16 days (Fig. 8f and Supplementary Fig. S5a). This substantial decrease in tumor size and tumor weights suggests that the enhanced mRNA delivery and expression capabilities of Au-LNPs can be effectively harnessed to improve the outcomes of cancer immunotherapies.

The enhanced efficacy of Au-LNPs in reducing both tumor size and metabolic activity highlights their potential to improve therapeutic outcomes in cancer treatment, particularly in difficult-to-treat cancers such as TNBC. The ability of Au-LNPs to deliver mRNA more effectively, promote stronger antigen expression, and stimulate DC cell maturation may lead to more robust and sustained immune responses against cancer cells (Fig. 8g). This could translate into better clinical outcomes, including prolonged survival and potentially lower recurrence rates for patients with aggressive cancers. Moreover, the success of the Au-LNPs in this TNBC model suggests that this platform could be broadly applicable to other types of cancers, especially those that are similarly challenging to treat. By enabling more efficient mRNA delivery and enhancing immune activation, Au-LNPs have the potential to become a key component in the development of next-generation cancer vaccines and immunotherapies.

Biocompatibility and biosafety evaluation of Au-LNPs
Ensuring the biocompatibility and biosafety of new nanomaterials is critical for their success for their translation into clinical applications. The biocompatibility and biosafety of Au-LNPs were assessed through a series of in vivo tests, including weight monitoring, cytokine assays, and histopathological examination of major organs (Methods). Weight monitoring and cytokine assays indicated that Au-LNPs were well-tolerated in mice, with no significant adverse effects observed during study (Supplementary Fig. S5b, c). The stability in body weight and the absence of abnormal cytokine levels suggest that Au-LNPs do not elicit significant inflammatory or toxic responses, which is a promising indicator of their safety profile.

Hematoxylin and eosin (H&E) staining of major organs further supported the favorable biosafety profile of Au-LNPs (Methods). The histopathological analysis revealed no apparent damage or inflammation in key organs such as the liver, kidneys, and lungs, confirming that Au-LNPs are bio-tolerant and do not cause significant tissue damage compared to conventional LNPs (Supplementary Fig. S5d, Fig. S6). Metabolism in vivo was conducted using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Results indicated that 48 h post-administration, the levels of Au elements in vivo significantly decreased and returned to baseline, exhibiting no significant difference from the PBS control group (Supplementary Fig. S7). These findings are critical as they demonstrate that the inclusion of gold nanoparticles in the LNPs system does not compromise the safety of the formulation, making Au-LNPs a viable option for clinical use.

In this study, we introduced a novel structural design strategy for LNPs that leverages an engineered core-shell architecture to significantly improve mRNA delivery. By incorporating a representative MC3-AuNP core, we created a pH-sensitive environment within the LNPs that enhances their stability at physiological pH and facilitates more effective membrane disruption under the acidic conditions of endosomes. Comprehensive characterization, including molecular dynamics simulations, membrane fluidity measurements, and pH-responsive membrane disruption assays, demonstrated that this internal architecture optimization yields thermodynamically favorable assemblies and promotes hexagonal-phase lipid arrangements conducive to efficient endosomal escape.

Compared to conventional LNPs, our Au-LNPs achieved a twofold increase in endosomal escape efficiency and a 100-fold enhancement in cytoplasmic mRNA diffusion, ultimately leading to higher mRNA expression in both in vitro and in vivo models. This improved performance was further validated in applications ranging from enhanced immunogenicity with SARS-CoV-2 spike-mRNA vaccines to therapeutic efficacy in a triple-negative breast cancer model. Notably, while the biodistribution and clearance data indicate that Au cores in our formulation do not cause long-term retention under the conditions tested, suggesting that gold itself is not a safety concern in this context. Nevertheless, for eventual clinical translation, the same architectural principle can be readily extended to FDA-approved or biodegradable core materials that offer well-established safety profiles. Thus, while Au-LNPs validate the concept, future work will focus on adapting this internal core–shell strategy to clinically preferred materials to accelerate translational potential.

Together, our findings thus establish a new conceptual framework for designing LNPs, shifting the focus from solely chemical modifications to architectural optimization. By doing so, we provide a versatile and scalable route to overcoming the longstanding bottleneck of endosomal escape, thereby advancing the clinical potential of mRNA therapeutics and vaccines.

Methods
Materials
A complete list of reagents, suppliers, and catalog numbers is provided in Supplementary Table S1. Key materials include: HAuCl₄ (Sinoreagent, Cat. 10010711), sodium citrate (Sinoreagent, Cat. 10019418), SH-PEG (Aladdin, Cat. T164400-200mg), DLin-MC3-DMA (MC3, AVT, Cat. 1224606-06-7), SM-102 (AVT, Cat. 2089251-47-6), ALC-0315 (AVT, Cat. 2036272-55-4), DSPC (Avanti, Cat. 850365C), cholesterol (Merck, Cat. 1.04675), DMG-PEG2000 (Avanti, Cat. 880151P), DOTAP (Qiyue, Cat. Q-0362051), DOPC (Psaitong, Cat. D10336), Cy5-mRNA (BiosynRNA, Cat. BR1RP015), and other reagents as listed. All water was nuclease-free, and consumables were endotoxin-free.

Synthesis of AuNPs
AuNPs were synthesized by the Turkevich method. HAuCl₄ (1mM, 30mL) was brought to vigorous boil, followed by injection of sodium citrate (1% w/v; 0.90mL for ~13nm or 0.45mL for ~18nm particles under 700rpm/min). The solution color changed from pale yellow to deep red within 10min. For larger AuNPs (25 and 50nm), a seeded growth protocol using 18nm AuNPs as seeds was employed. All glassware was cleaned with aqua regia (HCl:HNO₃ = 3:1) prior to use.
For 25nm/50nm AuNPs:
The as-prepared 18nm/25nm AuNPs (28.4mL) served as the seed solution. The temperature of the seed solution was adjusted to 90°C (or your specific temp) under continuous stirring (400rpm/min). To promote growth, 1mL of sodium citrate (1% w/v) was added, followed by the injection of 600μL of HAuCl₄ (25mM). The mixture was maintained at 90°C for 30min to ensure complete reduction of the gold precursor. A slight color shift was observed, indicating particle growth.

PEGylation of AuNPs (Au-PEG)
AuNPs (30mL) without purification were incubated with SH-PEG (10mg/mL in ethanol; 500μL under 400rpm/min) at pH 8.0 for 12h at room temperature. Particles were pelleted by centrifugation (11,000×g, 20min), washed ×3 with ddH₂O, and resuspended for storage at 4°C.

Ionizable lipid coating (IC-AuNPs)
Au-PEG (50μL, 1mg/mL, aqueous) was added onto MC3 in chloroform (200μL, 25mg/mL). After vortexing (3000rpm, 10min), the AuNPs transferred to the organic phase, confirming adsorption. To remove the organic solvent and unbound lipids, the Au-MC3 nanoparticles were precipitated by centrifugation (11,000×g, 20min). The supernatant containing chloroform and excess lipids was carefully discarded. The nanoparticle pellet was then washed once with ethanol to remove residual organic solvent, centrifuged again, and finally resuspended in 50mM acetic acid buffer (pH 4.0). Identical procedures were used for SM-102, ALC-0315, or DOTAP formulation coating.

mRNA synthesis
GFP, Fluc, Spike, and WT1 mRNAs were transcribed in vitro (EasyCap T7 kit, Vazyme) and purified with VAHTS RNA Clean Beads. Products were dissolved in RNase-free water. Cy5-labeled mRNA was obtained from BiosynRNA (BR1RP015).

LNP and Au-LNP formulation
LNPs and Au-LNPs were prepared from the same lipid master mix (SM-102: DSPC: cholesterol: DMG-PEG2000 = 50:10:38.5:1.5 mol%). Lipids were dissolved in ethanol (22.1mg/mL).
Au-MC3 nanoparticles, suspended in 50mM acetic acid buffer (pH 4.0), were aliquoted to achieve a fixed mass ratio of 2:1 (Au-MC3: mRNA). Subsequently, the calculated amount of mRNA was added directly to the Au-MC3 suspension. The mixture was incubated at 4°C for 10min in a static state.
LNPs: aqueous phase = mRNA in 50mM acetic acid (pH 4.0)
Au-LNPs: aqueous phase = mRNA condensed onto Au-MC3 cores in 50mM acetic acid (pH 4.0).
Formulations were generated using a CD-01 microfluidic chip (INano) at total flow rate 12mL/min (aqueous: organic ratio 3:1) at room temperature. Suspensions were dialyzed overnight against PBS (10kDa MWCO), sterile-filtered (0.22μm), and stored at 4°C.

Encapsulation efficiency
Encapsulation efficiency was measured by RiboGreen assay. LNPs/Au-LNPs (10μL) were added to PBS (90μL) or PBS+1% Triton X-100 (90μL). Fluorescence (Ex 485nm, Em 525nm) was measured, and efficiency calculated:

Physicochemical characterization
DLS/ζ: Zetasizer Pro (Malvern, Zetasize software 7.12). For ζ-potential titration, particles (10ng/mL mRNA) were measured in buffers prepared by mixing 50mM citric acid/sodium citrate.
TEM: negatively stained (phosphotungstic acid) samples imaged on JEOL JEM-2100F. >100 particles from 10 images were analyzed in ImageJ 1.51.w42.
Cryo-EM: imaged on Talos Glacios (200kV, Ceta CMOS, 57,000×, defocus –1.2 to –2.5μm).
LC-MS: The concentration of SM-102 in the supernatant, obtained after precipitating Au-MC3 particles with acetonitrile and centrifugation, was determined by LC-MS on a SCIEX Triple Quad™ 4500 (C18 column, monitoring the m/z 710.7→472.3 ESI+ mode).

Membrane disruption assays
SRB leakage: DOPC liposomes loaded with SRB (3:1 v/v, SRB:DOPC) were prepared; free dye removed by Sepharose CL-2B column. Leakage quantified by dequenching; Triton X-100=100% release. Microplate reader: Softmax Pro 5.4.1
RBC hemolysis: Whole blood (SIPEIFUB006-02) was washed ×2. 2% RBC suspensions were incubated with nanoparticles (0.5μg/mL mRNA) at varying pH for 2h at 37°C. Hemolysis quantified by A541; PBS=0%, Triton=100%.

Cell culture and transfection
HEK293 and HeLa cells were maintained in DMEM + 10% FBS + 1% P/S. Cells were transfected in Opti-MEM containing LNPs or Au-LNPs. GFP expression was measured by flow cytometry, luciferase by luminescence assay, and cytotoxicity by CCK-8 assay. The HeLa cell line (1101HUM-PUMC000011) and 293 cell line (1101HUM-PUMC000010) was obtained from the Cell Resource Center, Peking Union Medical College (which is part of the National Science and Technology Infrastructure, the National Biomedical Cell-Line Resource, NSTI-BMCR. http://cellresource.cn) These 4T1, EMT6 and JC cell lines used in this study was kindly provided by Dr. Kai Miao at University of Macau.

Confocal imaging and lysosomal colocalization
Two hundered ninety three cells were seeded in glass-bottom dishes and incubated with LNPs or Au-LNPs containing Cy5-mRNA (0.25–1 μg/mL). After 4–6h, lysosomes were stained with LysoTracker Green (75nM, 1h) and nuclei with Hoechst 33342 (1μg/mL, 10min). Live imaging was performed on Leica SP5 (Leica LASX 4.8.0.28989).
For quantification, two independent experiments with three fields each were analyzed. Colocalization (Pearson’s Correlation Coefficient) and cytosolic diffusion area were measured in Fiji (threshold 32–76).

Western Blot and qPCR
Cells treated with LNPs or Au-LNPs were lysed in RIPA buffer. Equal protein (30μg) was resolved by SDS-PAGE and blotted to PVDF. Membranes were probed for RAB5, LAMP1, and GAPDH. Bands were quantified in ImageJ (1.51.w) and normalized. Uncropped scans of the blots and gels are provided in the Supplementary Information Figs. 8 and 9.
For qPCR, RNA was extracted (Vazyme RC112-01), reverse transcribed (Vazyme R323-01), and amplified using SYBR Green (APE K1170, 7500 software v2.3). RAB5 and LAMP1 expression was normalized to GAPDH. Primer sequences are listed in Supplementary Methods.

Immunofluorescence staining
Cells were fixed (4% PFA), permeabilized (0.2% Triton X-100), blocked (5% BSA), and incubated with anti-RAB5 or anti-LAMP1 antibodies overnight at 4°C. After secondary antibody incubation, nuclei were counterstained with DAPI. Images were acquired on Leica SP5.

Cryo-EM sample preparation
Vitrification was performed using a Thermo Vitrobot at 8°C, 100% humidity43,44. Samples were blotted for 3.5s, plunge-frozen in liquid ethane, and stored in liquid nitrogen.

DPH fluorescence anisotropy
LNPs or Au-LNPs were incubated with DPH (1×10⁻⁷M) for 16h in buffers at varying pH. Fluorescence anisotropy was recorded at Ex 350nm/Em 428nm (FluoroMax Plus). Apparent microviscosity was calculated using published formulas25.

Molecular dynamics (MD) simulations
Molecular dynamics (MD) simulations were conducted using GROMACS 2021.7 with the CHARMM36 all-atom force field. Models of lipids and RNA fragments were generated using the Automated Topology Builder (ATB). Partial charges for RNA were refined using Multiwfn. Gold atom parameters were adapted from published CHARMM data.
Two systems were constructed: (i) a conventional LNP assembly (ionizable lipid + helper lipids + mRNA) and (ii) an Au-LNP assembly (Au core + MC3 coating + helper lipids + mRNA). Each system was solvated in a cubic water box and neutralized.
Energy minimization (steepest descent) was followed by equilibration under NVT and NPT. Production simulations were performed at 300K, and 1bar Long-range electrostatics were calculated using Particle Mesh Ewald (PME) with a real-space cutoff of 1.2nm. All covalent bonds involving hydrogen atoms were constrained with LINCS.
Each production runs 50,000,000 steps with timestep 0.3fs, corresponding to 15 ns per system. Enthalpy values were computed from trajectory averages. Au-LNPs exhibited lower assembly enthalpy (–354.3kJ/mol) compared to conventional LNPs (–210.7kJ/mol), consistent with enhanced thermodynamic stability observed experimentally.

Animal studies
Mice were housed in a specific pathogen-free (SPF) facility under controlled environmental conditions. The ambient temperature was maintained at 22±2°C, with a relative humidity of 40–60% and a 12-h light/12-hour dark cycle. The animals were kept in individually ventilated cages (IVC) with free access to standard rodent chow and water. All animal procedures were approved by NCNST IACUC (No. NCNST21-2208-YC01). Mice were randomized into groups. Female BALB/c mice (6–8 weeks, ~20g) were randomized into groups (n=5). Humane endpoints were defined as tumor >1500mm³ or distress. Female BALB/cAnNCrl mice (6–8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.
Bioluminescence imaging: Mice received Fluc-mRNA (2.5mg/kg) by i.m. or i.v. injection. Organs were imaged by IVIS Spectrum 24h later with Living Image 4.3.1.
Vaccination: Spike-mRNA (2.5mg/kg) was injected on days 0 and 14. ELISA plates were coated with Spike (300ng/well). Sera dilutions were 1:40–1:5120 (prime) and 1:80–1:327,680 (boost). Secondary antibody was diluted 1:2000.
Pseudovirus neutralization: Vero E6 cells were seeded at 5×10⁴ cells/well. Serum dilutions (1:20, 11-point, 2-fold) were incubated with VSV-based pseudovirus for 1–2h. Infection was quantified at 14–16h using CQ1 imaging; IC50 values were fit with a logistic regression model.
TNBC model: 4T1-Fluc cells (1×10⁶) were implanted orthotopically. Mice were treated with PBS, LNPs, or Au-LNPs encoding WT1-mRNA i.p. every 2 days. Tumor burden and survival were monitored.
ICP-MS clearance: Organs were digested in aqua regia using M6 microwave digestion and analyzed on Thermo iCAP TQ ICP-MS. Calibration used a 5ppm standard with QC every 10 samples.

Statistics and reproducibility
Statistical significance was determined using Student’s t-test (two groups) or one-/two-way ANOVA in GraphPad Prism 9.4.0.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript, NCOMMS-25-12523-T, on “Engineered Internal Architecture of Core-Shell Lipid Nanoparticles Promotes Efficient mRNA Endosomal Release”, submitted to Nat. Com., reports on a new strategy in mRNA-LNP structure engineering/design that shows a marked increase in mRNA delivery efficiency. The work presented is comprehensive, covering structural and stability characterization of the novel mRNA-loaded gold-lipid nanoparticles (Au-LNP), in vitro evaluation of mRNA expression efficiency in two cell types, in vivo testing in mouse models, biocompatibility and biosafety evaluation. Of note are the higher mRNA expression both in in vitro and in vivo models of the Au-LNPs compared to the benchmarking MC3-LNP and their superior efficacy in two mRNA therapeutic applications.\nThe work will be of notable significance in the field as mRNA endosomal escape into the cytosol has been identified as the bottleneck in LNP-mediated therapeutics (Chatterjee et al., PNAS, 2024), while organ-specific mRNA accumulation and translation is a persistent challenge for targeted delivery of mRNA drugs (Su et al., nature communications, 2024).\nMost of the conclusions and claims are supported, however I would like the authors to address the following aspects:\n- The molecular exchange of PEG on AuNPs by IL MC3, identified by the disappearance of the PEG peak at 2875 cm-1 in the FTIR spectra is not correct. The Au-PEG is in fact flat in the range of 3000-2000 cm-1.\n- The authors claim that membrane disruption by Au-LNPs “decreased as pH rose from below 4.5, then increased—peaking at pH 5.5—before declining again at more neutral pH values” while that “of traditional LNPs consistently decreased with increasing pH”. The trends are the same, but the absolute values are systematically lower for LNPs. Also figure 2 g has no measurement at physiological pH of 7.4 and the disruption looks like it’s around zero for both formulations, questioning the claim that “Notably, at physiological pH, Au-LNPs induced greater membrane disruption than conventional LNPs”.\n- The authors claim that “The enhanced membrane destabilization near pH 5.5 may be attributed to significant charge variations that promote hexagonal-phase lipid structures, which are more disruptive to membranes.” Is this based on unpublished data or on literature?\n- The structural characterization based on TEM and DLS from Figure 2 presents some inconsistencies. While the authors claim that Au-LNPs have an average diameter of 80 nm, the inset in Figure 2d is more around 45-50 nm in diameter. Is the value mentioned based on image analysis? And if so, what is the size distribution and how many TEM images were collected and analysed? Also, why was there no TEM of the traditional LNP? Finally, why haven’t the authors used cryo-EM instead of TEM as the negative-staining and then drying of LNPs might affect their original morphology.\n- For the DLS data, the authors do not mention the polydispersity index, which is a critical quality attribute for NA-based therapeutics applications.\n- How did the authors come to the conclusion that “Across various mRNA doses and time points, Au-LNPs significantly reduced endosomal acidification compared to traditional LNPs (Figures 4a–c, Figure S3)” simply by tracking “Cy5-labeled mRNA and lysotracker-labeled lysosomes following transfection to determine whether Au-LNPs alter endosomal maturation”?\n- In the Methods section the authors mention that “The lipid molar ratio of LNPs has not been adjusted”. Can the authors explain what is meant by that and why they did that, and whether the same is true also for the Au-LNPs? How can the authors be sure of the mechanistic details of the endosomal escape they present in Figure 1 if they don’t control the molar ratio in the LNPs?\n- The Methods section is poorly written and in some parts it is more like a set of instructions or a protocol for a student to follow. It is also not detailed enough for the work to be reproduced.\nThe following minor aspects need to be addressed:\n- Page 4, 1st line, “Figure 2a, Methods” has to be replaced by Figure 1.\n- Page 4, second paragraph, add (Figure 2b) after “of 13 nm”\n- Page 4, second paragraph, replace (Figure 2b,c) by Figure 2c.\n- Page 5, end of second paragraph, can the authors add a reference to substantiate the claim of the last sentence?\n- Page 5, third paragraph, can the authors add “/cationic” after ionisable on the fourth line as DOTAP is a cationic lipid?\n- Page 5, end of third paragraph, the authors refer to Hela and 293 cells in Figure 3c, while it shows data at different mRNA concentration for LNP and Au-LNP for only one cell line, Hela, based on its caption.\n- Page 6, discrepancy between the values of transfection efficiency at low dosage referred to in Figure S2c, 91.4% compared to 6.72 in figure S2c compared to 92.6% and 6.2% in main text.\n- Page 6, penultimate paragraph, “Figure S3d” referred to is actually “Figure S3e”.\n- Page, second line of last paragraph, “Figures 4e-i” referred needs to be replaced by “Figure 4e-h” as there is no figure 4i. Again towards the end of the same paragraph, “Figure 4g-i” needs to be replaced by “Figure 4g-h”.\n- Page 13, last paragraph, 3rd line, in ethanol instead of “ethonal”.\n- References 21 and 23 are the same\n- Captions of Figure 1 and that of Figure 2a need to be revised.\n- Figure 4e, remove (a) and (b) and replace with left middle and right.\n- Figure g and h, optionally replace endosome process with endosomal escape.\n- Figure 7 a caption, treatment plan instead of treatment plane.\n- In the methods section, DPH Fluorescence anisotropy missing a reference at the end of 1st paragraph on page 10.\n- In the methods section, ELISAs 1st line of 2nd paragraph is missing a reference on the preparation of pseudo-typed viruses.\n- Figure S1b, legend SH-PEG is presumably Au-PEG.\n- Figure S1f caption mentions “Firefly luciferase chemiluminescence (FLuc) assay in HeLa cells, treated for 24 h with the indicated concentrations of LNP…” while there are no concentrations on the figure itself.\n- Figure S2g, legend is ALC-0315 and not AIC-0315.\n- Figure S2g-I caption, “…cells treated with Moderna BNT and SORT” is actually Pfizer/Biontech (ALC-0315, g), Moderna (SM-102, h).\n- Figure S3e, caption refers to concentrations of 0.5, 1 and 3 μg/mL while legend is from 0.25 to 1 μg/mL.\n- Radiance scale is missing from mice bioluminescence data on Figures 5b-e, 7d and S4a-d.\n- Hoechst instead of Hochest on the CLSM images of Figures 4a-c and S3a-c.\n- Vortex instead of Vertox on Figure 2a.", "reviewer_2": "The manuscript proposes advances in the technology of LNP for RNA delivery by including a core of AuNPs. In particular, the authors propose a structural optimization of AuNP core for enhancement of RNA delivery.\nFollowing a physical-chemical characterization, the AuLNPs have been studied in terms of in vitro transfection by measuring luciferase activity HeLa and 293 cells and mRNA delivery has been investigated by confocal microscopy. The authors showed that AuLNPs have a transfection efficiency significantly superior to that achieved by LNP. TEM analysis of cells incubated with Au-LNP showed enlarged vesicles attributed to a stronger membrane disruption capability of Au-LNP. More in details, the authors hypothesize that positively charged core of AuNPs attracts negatively charged mRNA, “drawing it closer to the core region, while simultaneously repelling positively charged ionizable lipids and pushing them toward the endosomal membrane”. The superiority of the AuLNP has also been demonstrated in vivo by two different administration routes. Enhanced immunogenicity of SARS-CoV-2 Spike-mRNA Vaccines has been demonstrated; on the other hand, a significant reduction in tumor volume and extension in survival rate has been obtained.\nFirst of all, the rationale for using AuNP as core for LNP is not clear. Actually, the authors motivate the choice of the AuNP with the high electron density together with its biocompatibility. However, the electron density cannot be considered an advantage for drug delivery purposes. Moreover, according to the literature, the biocompatibility of gold cannot be considered superior to that of other biomaterials, such as lipids or other inorganic materials. Moreover, the authors affirm that a further advantage of AuNP is their versatile surface modifications. However, in this system the AuNP are the core of the core/shell particles, thus not requiring surface modifications.\nThe authors affirm that AuNP are “ideal” marker for localization of LNP by TEM; however, while AuNP are useful to study NP localization and structure, the choice of the core for drug delivery purpose in not presented. Moreover, also when AuNP are used for visualization into the cell, the visualization of AuNP does not correspond to the visualization of the LNP that, once into the cells, should lose their integrity.\nThe authors affirm that “To achieve efficient endosomal escape and mRNA capture, we require Au core that responds well to changes in pH.” However, while for ionizable lipids the mechanism of endosomal escape is well-know, any mechanism of endosomal escape is clearly described here for the AuNPs.\nThe preparation protocol of the core-shell LNP arises many concerns. The authors prepared Au-PEG-NP by reaction of PEG-SH with the preformed AuNPs. Then, they perform a ligand exchange demonstrated by change of the solubility of AuNPs from the aqueous to organic solvent. It is not clear what is the mechanism for PEGylation. Have the authors performed a chemical conjugation? If the case, mechanism of “ligand exchange” is not clear once mixed with the lipids. Finally, in the case the PEGylation is required for the core-shell NP in which lipids for the outer layer, PEGylated lipids can be used thus avoiding a previous conjugation of PEG to the AuNPs.\nThe authors prepared the IL-AuNPs by mixing the AU-PEG NPs with cationic (DOTAP) or ionizable (SM102) lipids. The Au-LNP are then prepared by mixing the IL-Au NP with the RNA, that should provide lipoplexes. Thus, it is not clear how this preparation protocol should provide core-shell LNP encapsulating RNA; on the other hand, the mechanism seems to provide lipoplexes for DOTAP, while in the case of SM102 any pH change (typically used in the PLN preparation) is used to modify the charge of the ionizable lipid thus inducing the efficient RNA encapsulation. In line with this, it is not clear what kind of interaction, hydrophobic or electrostatic, takes place among the AuNP and the lipids. Finally, MC3 is not mentioned in the preparation method.\nIn Figure 2, a core-shell structure is showed; however, it is not reported if the different lipids used, especially cationic and ionizable, provide a different assembling with the AU-NPs and, consequently different core/shell structure.\nThe authors speculate on the lipid assembly to justify results of luciferase expression. In particular, at lines 146-150 they affirm that AuNP with higher size “interferes with the optimal assembly of the lipid components”. However, they did not demonstrate what kind of assembly arrive by mixing AuNP and the lipids, as well as when also mixing the RNA.\nThe methods of the self-quenching sulforhodamine B (SRB) to study endosomal escape is questionable because suitable to study the liposomes stability, but not the stability or ensosomal escape of Au-LNP.\nThe speculation for the highest transfection efficiency of MC3-AuNP is not convincing because the authors affirm “favorable zeta potential shift of Au-MC3 under acidic Conditions”; actually, it is well known that MC3 switchs to positive zeta potential in acidic condition of the endosomes while the contribution of AuNP is not clear. Moreover, at line 218, the authors affirm that the Au-LNP reduces the endosomal acidification favoring RNA release, while the acid pH is known to be responsible of RNA release when using LNP. This explanation is too superficial and not suitable to justify the observed results.\nAlthough the manuscript reports positive finding, important concerns on rationale and methodologies make very difficult a deep evaluation of the proposed technology. The manuscript is, in my opinion, far to be suitable for publication in Nature Communication.", "reviewer_3": "In this manuscript the authors incorporated gold into lipid nanoparticles to form a core-shell LNP system for mRNA delivery. The authors then tested this system for delivery of mRNA in vitro & in vivo, finding that incorporation of gold into the nanoparticles results in higher expression of mRNA. Due to the incorporation of gold into the LNPs, microscopy-based evaluation of endosomal escape was performed, and the authors found that the increased mRNA expression may be due to increased endosomal escape as compared to traditional LNP systems. The authors then show this system provides benefits in vaccine and cancer therapeutic applications.\nOverall, the results are of high interest as they shed light on methods to increase endosomal escape of LNP systems, which is a key bottleneck to efficient delivery. However, there are some minor comments that should be addressed to suggest accepting the manuscript for publication.\nStrengths:\n1. Research is well designed and executed.\n2. Presentation of methods will permit replication.\n3. Data are unambiguous and properly analyzed.\n4. Conclusions are supported by data.\nBelow are minor comments:\n1. In figure 2g, the authors state the Au-LNPs disrupt liposomal membranes greater than traditional LNP systems. The graph seems to show single points at each pH tested, were replicates tested & is there associated statistical analysis to suggest there is a difference between the systems?\n2. On line 195-196, the authors state “in the Au-LNPs group, we did not observe a peak within our tested dosage range”. It would be insightful to include further datapoints to assess if/at what dose the protein expression peaks following Au-LNP delivery. The authors claim the increased expression efficiency of mRNA may be attributed to improving endosomal escape, but how does this explain the lack of a protein expression peak?\n3. The authors state as a conclusion “making Au-LNPs a viable option for clinical use”. Inclusion of gold nanoparticles into this system doesn’t seem to affect the safety of the formulation, but there are concerns about the use of gold nanoparticles in the clinic due to accumulation in clearance organs. Are the gold nanoparticles in this LNP system cleared from the animal? Do the authors expect the Au-LNP systems would be used with repeat dosage? In this case, further safety testing should be done."}, "author_response": "Response to Reviewers for Manuscript NCOMMS-25-12523-T\nReviewer #1 (Remarks to the Author):\nThe manuscript, NCOMMS-25-12523-T, on “Engineered Internal Architecture of\nCore-Shell Lipid Nanoparticles Promotes Efficient mRNA Endosomal Release”,\nsubmitted to Nat. Com., reports on a new strategy in mRNA-LNP structure\nengineering/design that shows a marked increase in mRNA delivery efficiency. The\nwork presented is comprehensive, covering structural and stability characterization of\nthe novel mRNA-loaded gold-lipid nanoparticles (Au-LNP), in vitro evaluation of\nmRNA expression efficiency in two cell types, in vivo testing in mouse models,\nbiocompatibility and biosafety evaluation. Of note are the higher mRNA expression\nboth in in vitro and in vivo models of the Au-LNPs compared to the benchmarking\nMC3-LNP and their superior efficacy in two mRNA therapeutic applications.\nThe work will be of notable significance in the field as mRNA endosomal escape into\nthe cytosol has been identified as the bottleneck in LNP-mediated therapeutics\n(Chatterjee et al., PNAS, 2024), while organ-specific mRNA accumulation and\ntranslation is a persistent challenge for targeted delivery of mRNA drugs (Su et al.,\nnature communications, 2024).\nMost of the conclusions and claims are supported, however I would like the authors to\naddress the following aspects:\nGeneral Response:\nWe thank the reviewer for their enthusiastic evaluation of our manuscript and for\nrecognizing the comprehensive nature of our study. We are pleased that the reviewer\nhighlights the significance of our architectural engineering approach in overcoming\nendosomal escape limitations.\n1- The molecular exchange of PEG on AuNPs by IL MC3, identified by the\ndisappearance of the PEG peak at 2875 cm-1 in the FTIR spectra is not correct. The\nAu-PEG is in fact flat in the range of 3000-2000 cm-1.\nResponse:\nWe thank the reviewer for this insightful comment and for drawing our attention\nto the ambiguity in our FTIR description. Upon careful re-examination, we agree that\nthe Au-PEG spectrum may appear relatively flat in the 3000-2000 cm⁻¹ region in the\noverlaid plot (Supplementary Figure S1a), likely due to signal normalization, low PEG\ndensity on the 13 nm AuNPs, and overlap with AuNPs and Au-MC3 traces. We also\nwant to thank the reviewer for drawing our attention to this ambiguity, and we\nacknowledge that our earlier phrasing — “molecular exchange of PEG by MC3”—was\nmisleading. In fact, our data support that PEG remains on the Au surface, while MC3\nis coated onto the PEG-modified AuNPs via hydrophobic interactions, rather than\nreplacing PEG. We have revised the manuscript text to avoid the term “exchange” and\nhave made corresponding adjustments to the schematic illustration in Figure 1 to ensure\nclarity and avoid ambiguity.\nTo strengthen clarity, we made the following changes:\nRevised Text (Line 94-102):\n“AuNPs were selected as a proof-of-concept core material primarily due to their chemical versatility,\nincluding high surface reactivity for versatile modifications and precise size control (Figure 2a). To\nexplore whether internal architecture could enhance mRNA delivery, we synthesized IC-AuNPs as\nstructural cores for LNP assembly. AuNPs were first PEGylated and then coated with ionizable\nlipids such as MC3 or SM-102 via phase transfer mediated by hydrophobic forces, yielding\nhydrophobic IC-AuNPs that readily integrated into organic solvents (Figure 2a, Methods).\nSuccessful coating was confirmed by Fourier transform infrared (FTIR) spectroscopy and nuclear\nmagnetic resonance (NMR), which revealed diminished PEG-specific peaks alongside the\nemergence of MC3 signatures (Supplementary Figure S1a–b).”\nFigure Update: We include an enlarged inset of the FTIR spectra (2800–3000 cm⁻¹)\nand the corresponding NMR (3.5–4.0 ppm) in Figure S1a and S1b, which resolves the\noverlap issue and makes the data more interpretable. We also adjusted the schematic\nillustration in Figure 1 to eliminate potential ambiguity.\nFigure S1. Characterization of AuNP modification and Au-LNP assembly.\na. FTIR spectra of IC-AuNPs blue, Au-PEG red, and bare AuNPs black.\nb. 1H NMR spectra of SH-PEG blue, Au-PEG purple, DLin-MC3-DMA red, and Au-\nMC3 black.\nc. Agarose gel electrophoresis images of IC-AuNPs and mRNA at different mass ratios.\nd. TEM analysis of Au-LNPs.\ne. Cryo-EM image of Au-LNPs.\nf. TEM image of conventional LNPs, with cryo-EM image shown in the inset at the top\nright.\ng. Photographs of phase-transfer results of Au-PEG with three ionizable lipids SM-102\nA, DLin-MC3-DMA B, and DOTAP C.\nh. TEM images of Au-LNPs of ALC-0135 and Au-LNPs of DOTAP.\ni. Molecular dynamics simulation snapshots showing a cubic box with edge length\n11.047 nm containing 9 chains of 22-nt polyA RNA and 20 chains of SM-102 lipids,\nsimulated under traditional LNPs and Au-LNPs conditions.\nj. Standard curve for LC–MS quantification of SM-102 and measured SM-102 content\nin Au-LNPs and conventional LNPs from three independent experiments.\nk. Zeta potential profiles of free MC3 molecules versus Au-MC3 nanoparticles under\nidentical pH conditions in 50 mM citrate buffer.\nl. UV–Vis absorption spectra of AuNPs of different diameters 13, 18, 25, and 50 nm.\nm. Firefly luciferase FLuc expression in HeLa cells treated for 24 h with 0.5 μg/mL\nmRNA delivered by Au-LNPs containing gold cores of different diameters 13, 18, 25,\n50 nm; conventional LNPs were used as a positive control.\nn. DLS size distributions of Au-LNPs formulated with 18, 25, and 50 nm Au cores.\nFigure 1. Schematic of the enhanced endosomal escape mechanism of Au-LNPs.\nIn conventional LNPs, the efficacy of ionizable lipids is hindered as they are neutralized\nby electrostatic interaction with the mRNA cargo. The unique internal architecture of\nour Au-LNPs resolves this issue. The positively charged gold core attracts and\nsequesters the mRNA, which in turn electrostatically repels the ionizable lipids in the\nsurrounding shell. This repulsion liberates the lipids, maximizing their availability to\ninteract with and disrupt the endosomal membrane, leading to superior cytoplasmic\nmRNA delivery.\n2- The authors claim that membrane disruption by Au-LNPs “decreased as pH rose\nfrom below 4.5, then increased—peaking at pH 5.5—before declining again at more\nneutral pH values” while that “of traditional LNPs consistently decreased with\nincreasing pH”. The trends are the same, but the absolute values are systematically\nlower for LNPs. Also figure 2 g has no measurement at physiological pH of 7.4 and\nthe disruption looks like it’s around zero for both formulations, questioning the\nclaim that “Notably, at physiological pH, Au-LNPs induced greater membrane disruption\nthan conventional LNPs”.\nResponse:\nWe thank the reviewer for carefully examining our SRB release data and for\nhighlighting the missing pH 7.4 measurement and imprecise phrasing. We acknowledge\nthat our original description overstated the distinction between Au-LNPs and\nconventional LNPs and did not sufficiently emphasize that the observed disruption\ndifferences are most relevant at endosomal pH, not physiological pH.\nWe agree that both Au-LNPs and conventional LNPs show the same general trend\nof decreasing disruption as pH increases toward neutrality, which is expected for\nionizable lipids such as SM-102 that are designed to remain neutral at physiological pH\nwhile protonating under acidic endosomal conditions. However, a key distinction is that\nAu-LNPs display a pronounced biphasic response with a peak at pH 5.5 (corresponding\nto early endosomes), whereas conventional LNPs show a more monotonic decline.\nImportantly, the absolute values for Au-LNPs are systematically higher (by ~20–30%\nat pH 5.5), highlighting their architectural advantage in endosomal escape.\nTo directly address the reviewer’s concern:\nNew Data Addition: We repeated the SRB release assay at pH 7.4 (1×PBS, n = 3\nreplicates). As anticipated, disruption was minimal for both formulations (0.371% ±\n0.171% for Au-LNPs vs. 0.396% ± 0.564% for LNPs; p = 0.9452, two-tailed t-test),\nconfirming stability at physiological pH. This new data point has been added to Figure\n2g.\nUpdated Figure\nFigure 2. Morphology and characterization of Au-LNPs.\na. Schematic representation of IC-AuNP modification and the potential Au core\nstructure.\nb. TEM image of gold nanoparticles (13 nm diameter).\nc. IC-AuNPs showing a corona-like morphology.\nd. Au-LNPs incorporating 13 nm gold nanoparticles exhibiting a “single-sided fried\negg” morphology.\ne. Average hydrodynamic sizes of LNPs and Au-LNPs measured by DLS (ethanol\ninjection method).\nf. Lipid membrane fluidity of LNPs and Au-LNPs under varying pH conditions.\ng. Disruption efficiency of artificial liposomes under varying pH conditions for LNPs\nand Au-LNPs. Statistical significance was assessed by one-way ANOVA (for >2\ngroups) or two-tailed Student’s t-test (for 2 groups). **P < 0.01, ***P < 0.001, ****P\n< 0.0001.\nRevised Text (Results, Line 182-187):\nOriginal: “For Au-LNPs, membrane disruption decreased as pH rose from below 4.5,\nthen increased—peaking at pH 5.5—before declining again at more neutral pH\nvalues. Notably, at pH, Au-LNPs induced greater membrane disruption than\nconventional LNPs.”\nRevised:“We first employed a sulforhodamine B (SRB)–loaded liposome assay as a widely used\nproxy for pH-dependent membrane fusion of lipid nanoparticles (Methods)1. Both Au-LNPs and\nconventional LNPs showed minimal leakage at physiological pH (7.4) and maximal activity near\nendosomal pH (~5.5), consistent with ionizable lipid protonation. Across the acidic range, Au-LNPs\nconsistently induced higher leakage than conventional LNPs, with a significant enhancement at pH\n5.5 (Figure. 2g).”\nReferences:\n1. Goldbach, N., et al. De novo design of monomeric helical bundles for pH-\ncontrolled membrane lysis. Protein Science 32(2023).\n3- The authors claim that “The enhanced membrane destabilization near pH 5.5 may\nbe attributed to significant charge variations that promote hexagonal-phase lipid\nstructures, which are more disruptive to membranes.” Is this based on unpublished\ndata or on literature?\nResponse:\nWe thank the reviewer for seeking clarification on this mechanistic interpretation\n(Results, page 5). This claim is not based on unpublished data but represents a\nhypothesis drawn from our observations and interpreted in light of well-established\nliterature on ionizable lipids. Specifically, our SRB release assay showed a biphasic\ndisruption pattern with a clear peak at pH5.5 for Au-LNPs, in contrast to the monotonic\ntrend observed for conventional LNPs. We interpret this in the context of published\nstudies showing that protonation of ionizable lipids at endosomal pH (~5.0–6.5) drives\ncharge variations that favor the formation of non-lamellar hexagonal (H_II) phases,\nwhich destabilize membranes and facilitate cargo release.\nFoundational studies such as Cullis & Hope (Nature, 1978) and Semple et al. (Nat.\nBiotechnol., 2010) demonstrated that ionizable lipid protonation promotes H_II\nstructures responsible for endosomal escape1,2. More recent reports have reinforced this\nconcept at endosomal-relevant pH values, including Johansson et al. (Nat. Commun.,\n2022) and Aliakbarinodehi et al. (ACS Nano, 2024), which describe pH-driven\nhexagonal-phase transitions in ionizable lipid systems3,4.\nOur contribution is to suggest that the internal core structure amplifies this well-\ndocumented mechanism by enforcing charge segregation: the core attracts negatively\ncharged mRNA while repelling protonated ionizable lipids toward the membrane\ninterface, thereby enhancing the efficiency of H_II-phase formation5,6. This\ninterpretation is fully consistent with the literature and supported by our empirical data\nbut does not constitute new unpublished mechanistic evidence.\nRevisions Made to the Manuscript:\nAdded to Introduction (Line 66-74):\n“Conventional LNPs encapsulate mRNA in an amorphous mixture of ionizable lipids,\nhelper lipids, cholesterol, and polyethylene glycol (PEG)–lipids. Upon exposure to the\nacidic endosomal environment, ionizable lipids become protonated and can, in principle,\npromote membrane fusion by forming non-bilayer hexagonal (H_II) phases. In practice,\nhowever, the effective charge density of ionizable lipids is partially neutralized by\nmRNA within the LNP core, attenuating their membrane-disruptive capacity. Moreover,\nthe lack of structural order within conventional LNPs may limit the extent and\ncoordination of these phase transitions, resulting in suboptimal cytosolic release. These\nintrinsic architectural constraints highlight the need for alternative design principles\nthat address not only lipid chemistry but also the internal organization of LNPs.”\nRevised Results (Line 199-201):\n“The enhanced membrane destabilization near pH 5.5 is also consistent with prior\nreports showing that protonated ionizable lipids promote non-bilayer hexagonal (H_II)\nphases, which are highly disruptive to membranes5,7 .”\nReferences:\n1. Cullis, P.R. & Hope, M.J. EFFECTS OF FUSOGENIC AGENT ON\nMEMBRANE STRUCTURE OF ERYTHROCYTE-GHOSTS AND\nMECHANISM OF MEMBRANE-FUSION. Nature 271, 672-674 (1978).\n2. Semple, S.C., et al. Rational design of cationic lipids for siRNA delivery.\nNature Biotechnology 28, 172-U118 (2010).\n3. Aliakbarinodehi, N., et al. Time-Resolved Inspection of Ionizable Lipid-\nFacilitated Lipid Nanoparticle Disintegration and Cargo Release at an Early\nEndosomal Membrane Mimic. Acs Nano 18, 22989-23000 (2024).\n4. Johansson, J.M., et al. Cellular and biophysical barriers to lipid nanoparticle\nmediated delivery of RNA to the cytosol. Nature Communications 16(2025).\n5. Yu, H., et al. Inverse cubic and hexagonal mesophase evolution within\nionizable lipid nanoparticles correlates with mRNA transfection in\nmacrophages. Journal of the American Chemical Society 145, 24765-24774\n(2023).\n6. Yu, H., et al. Real-time pH-dependent self-assembly of ionisable lipids from\nCOVID-19 vaccines and in situ nucleic acid complexation. Angewandte\nChemie-International Edition 62(2023).\n7. Chatterjee, S., Kon, E., Sharma, P. & Peer, D. Endosomal escape: A\nbottleneck for LNP- mediated therapeutics. Proceedings of the National\nAcademy of Sciences of the United States of America 121(2024).\n4- The structural characterization based on TEM and DLS from Figure 2 presents\nsome inconsistencies. While the authors claim that Au-LNPs have an average\ndiameter of 80 nm, the inset in Figure 2d is more around 45-50 nm in diameter. Is the\nvalue mentioned based on image analysis? And if so, what is the size distribution and\nhow many TEM images were collected and analysed? Also, why was there no TEM\nof the traditional LNP? Finally, why haven’t the authors used cryo-EM instead of\nTEM as the negative-staining and then drying of LNPs might affect their original\nmorphology.\nResponse:\nWe thank the reviewer for raising these important points regarding our structural\ncharacterization. We acknowledge that our initial description did not include sufficient\nmethodological details and have new revised the manuscript accordingly.\nBasis of the 80 nm Value and Potential Visual Discrepancy:\nThe ~80 nm diameter was derived from both DLS, which measures hydrodynamic\nsize. TEM, by contrast, measures the dried-state diameter, which is expected to appear\nsmaller due to dehydration or flattening during negative staining1. For Au-LNPs, our\nquantitative TEM analysis gave an average dry-state size of 42.16 ± 0.289 nm\n(Supplementary Figure S1d), while DLS measured ~70.2 nm in hydrated state. Thus,\nthe apparent ~45–50 nm size in Figure 2d is consistent with TEM measurements under\nnegative-stain conditions.\nFigure S1d TEM analysis of Au-LNPs\nSize Distribution and Number of Images Analyzed:\nWe analyzed 100 particles across 10 TEM images in three independent replicates\nusing ImageJ. The size distribution was approximately Gaussian, with a mean of 42.16\nnm and SD 0.289 nm. The PDI from DLS was 0.073, further supporting good\nuniformity. These details have been added to methods and Results.\nTEM of Traditional LNPs:\nWe agree with the reviewer that TEM imaging of conventional LNPs is important\nfor comparison. These data were omitted in the initial submission and have now been\nincluded as Supplementary Figure S1f. As expected, traditional LNPs displayed\nspherical morphologies but with lower contrast, consistent with the lack of an electron-\ndense Au core.\nFigure S1f. TEM image of LNPs and Cryo-EM image of LNPs in the top right corner.\nWhy TEM Instead of Cryo-EM?\nWe acknowledge that Cryo-EM provides the most accurate representation of\nhydrated LNP morphology and avoid artifacts from staining and drying. Negative-stain\nTEM was initially chosen for its speed and accessibility during optimization and is still\nwidely used as a first-pass tool in LNP characterization. To address this limitation, we\nhave obtained preliminary cryo-EM data (Supplementary Figure S1e), which confirmed\nhydrated Au-LNPs with an average diameter of 61.98 ± 9.43 nm (n=10). These values\nare consistent with our DLS measurements and demonstrate preserved spherical\nmorphology.\nRevisions Made to the Manuscript\nResults (pages Line 114-128):\n“Transmission electron microscopy (TEM) visualized ~13 nm Au cores (Figure 2b) coated with\nMC3 (Au-MC3) and surrounded by a 2.5 nm lipid layer, confirming successful core–shell formation\n(Figure 2c, Methods). When assembled into Au-LNPs, these MC3-coated cores yielded\nnanoparticles with an average dry-state diameter of 42.2 ± 0.3 nm (Figure 2d, Supplementary Figure\nS1d). In contrast, dynamic light scattering (DLS) indicated a hydrated hydrodynamic size of ~70.2\nnm (Figure 2e, Methods), while preliminary cryo-EM measurements confirmed a hydrated size of\n62.0 ± 9.4 nm with intact spherical morphology (Supplementary Figure S1e, Methods). The smaller\nvalues from TEM are consistent with the expected shrinkage of lipid nanoparticles during negative\nstaining and drying, as noted in previous LNP studies2. Importantly, TEM of conventional LNPs\nwas also included for direct comparison (Supplementary Figure S1f). To confirm that lipid coating\nis generalizable, we extended the phase transfer method to three different ionizable/cationic lipids\nand verified successful attachment to the AuNP surface in all cases (Supplementary Figure S1g).\nSimilar core–shell morphology was also observed when ALC-0315, or DOTAP were used in place\nof SM-102 (Supplementary Figure S1h), demonstrating that the architecture is reproducible across\ndifferent lipid chemistries and not an artifact of a single lipid.”\nUpdated Supplementary Figure S1\nFigure S1. Characterization of AuNP modification and Au-LNP assembly.\na. FTIR spectra of IC-AuNPs blue, Au-PEG red, and bare AuNPs black.\nb. 1H NMR spectra of SH-PEG blue, Au-PEG purple, DLin-MC3-DMA red, and Au-\nMC3 black.\nc. Agarose gel electrophoresis images of IC-AuNPs and mRNA at different mass ratios.\nd. TEM analysis of Au-LNPs.\ne. Cryo-EM image of Au-LNPs.\nf. TEM image of conventional LNPs, with cryo-EM image shown in the inset at the top\nright.\ng. Photographs of phase-transfer results of Au-PEG with three ionizable lipids SM-102\nA, DLin-MC3-DMA B, and DOTAP C.\nh. TEM images of Au-LNPs of ALC-0135 and Au-LNPs of DOTAP.\ni. Molecular dynamics simulation snapshots showing a cubic box with edge length\n11.047 nm containing 9 chains of 22-nt polyA RNA and 20 chains of SM-102 lipids,\nsimulated under traditional LNPs and Au-LNPs conditions.\nj. Standard curve for LC–MS quantification of SM-102 and measured SM-102 content\nin Au-LNPs and conventional LNPs from three independent experiments.\nk. Zeta potential profiles of free MC3 molecules versus Au-MC3 nanoparticles under\nidentical pH conditions in 50 mM citrate buffer.\nl. UV–Vis absorption spectra of AuNPs of different diameters 13, 18, 25, and 50 nm.\nm. Firefly luciferase FLuc expression in HeLa cells treated for 24 h with 0.5 μg/mL\nmRNA delivered by Au-LNPs containing gold cores of different diameters 13, 18, 25,\n50 nm; conventional LNPs were used as a positive control.\nn. DLS size distributions of Au-LNPs formulated with 18, 25, and 50 nm Au cores\nAdded Methods (Line 514 and 550-552):\n“Cryo-EM: imaged on Talos Glacios (200 kV, Ceta CMOS, 57,000×, defocus –1.2 to –2.5 μm).”\n“Cryo-EM Sample Preparation\nVitrification was performed using a Thermo Vitrobot at 8 °C, 100% humidity3,4. Samples were\nblotted for 3.5 s, plunge-frozen in liquid ethane, and stored in liquid nitrogen.”\nReferences:\n1. Ubhe, A.S. Imaging of Liposomes by Negative Staining Transmission\nElectron Microscopy and Cryogenic Transmission Electron Microscopy.\nMethods in molecular biology (Clifton, N.J.) 2622, 245-251 (2023).\n2. Jiang, A.Y., et al. Combinatorial development of nebulized mRNA delivery\nformulations for the lungs. Nature Nanotechnology 19, 364-375 (2023).\n3. Crawford, R., et al. Analysis of lipid nanoparticles by Cryo-EM for\ncharacterizing siRNA delivery vehicles. International Journal of\nPharmaceutics 403, 237-244 (2011).\n4. Kochovski, Z., Chen, G., Yuan, J. & Lu, Y. Cryo-Electron microscopy for the\nstudy of self-assembled poly(ionic liquid) nanoparticles and protein\nsupramolecular structures. Colloid and Polymer Science 298, 707-717 (2020).\n5- For the DLS data, the authors do not mention the polydispersity index, which is a\ncritical quality attribute for NA-based therapeutics applications.\nResponse:\nWe thank the reviewer for highlighting the importance of the polydispersity index\n(PDI) as a critical quality attribute for nucleic acid -based therapeutics (Figure 2e). PDI\nwas determined by DLS (Table R1, Zetasizer Nano, Malvern;) using the cumulants\nmethod, and the results are now reported in Table R1. Au-LNPs exhibited consistently\nlow PDI values (0.07305± 0.00484, n=3), reflecting high monodispersity and uniform\nassembly, consistent with our MD simulations (Supplementary Figure S1i). In contrast,\nconventional LNPs prepared by ethanol injection displayed higher PDI (0.1275 ±\n0.007228), indicating broader size distribution and occasional aggregation, in\nagreement with literature reports1,2. Importantly, both formulations fall well within the\naccepted range for mRNA therapeutics (PDI < 0.25), confirming their suitability for\nnucleic acid delivery. 2-4\nTable R1 PDI of LNPs and Au-LNPs\nLNPs Au-LNPs\n1 0.1321 0.07204\n2 0.1192 0.0688\n3 0.1313 0.07832\nRevised Text in Results (Line 129-134)\n“Notably, Au-LNPs exhibited significantly higher uniformity than conventional LNPs, with no\noversized particles detected by DLS and a markedly lower polydispersity index (PDI) of 0.073\ncompared to 0.128 for LNPs (Figure. 2e). Mechanistically, the absence of larger particles in Au-\nLNPs can be attributed to enhanced hydrophobic free energy around the gold core, which promotes\ntight lipid assembly and prevents the formation of poorly hydrated lipid aggregates that otherwise\nyield oversized structures.”\nUpdated Methods (Line 510-511):\n“DLS/ζ: Zetasizer Pro (Malvern). For ζ-potential titration, particles (10 ng/mL mRNA) were\nmeasured in buffers prepared by mixing 50 mM citric acid/sodium citrate.”\nUpdated Figure 2e: Added PDI values directly in the Figure 2e and as insets on the\nDLS traces PDI: 0.07305 (Au-LNPs), 0.1275 (LNPs).\nFigure 2. Morphology and characterization of Au-LNPs.\na, Schematic representation of IC-AuNP modification and the potential Au core\nstructure.\nb, TEM image of gold nanoparticles (13 nm diameter).\nc, IC-AuNPs showing a corona-like morphology.\nd, Au-LNPs incorporating 13 nm gold nanoparticles exhibiting a “single-sided fried\negg” morphology.\ne, Average hydrodynamic sizes of LNPs and Au-LNPs measured by DLS (ethanol\ninjection method).\nf, Lipid membrane fluidity of LNPs and Au-LNPs under varying pH conditions.\ng, Disruption efficiency of artificial liposomes under varying pH conditions for LNPs\nand Au-LNPs. Statistical significance was assessed by one-way ANOVA (for >2\ngroups) or two-tailed Student’s t-test (for 2 groups). **P < 0.01, ***P < 0.001, ****P\n< 0.0001.\nReferences:\n1. Wang, H., et al. Facile lipid nanoparticle size engineering approach via\ncontrollable fusion induced by depletion forces. Journal of Colloid and\nInterface Science 691(2025).\n2. Hassett, K.J., et al. Impact of lipid nanoparticle size on mRNA vaccine\nimmunogenicity. Journal of Controlled Release 335, 237-246 (2021).\n3. Nag, K., et al. DoE-derived continuous and robust process for manufacturing\nof pharmaceutical-grade wide-range LNPs for RNA-vaccine/drug delivery.\nScientific Reports 12(2022).\n4. Heshmati, N., Chakka, L.R.J., Zhang, Y. & Maniruzzaman, M. Fabrication of\nmRNA encapsulated lipid nanoparticles using state of the art SMART-MaGIC\ntechnology and transfection in vitro. Scientific Reports 14(2024).\n6- How did the authors come to the conclusion that “Across various mRNA doses and\ntime points, Au-LNPs significantly reduced endosomal acidification compared to\ntraditional LNPs (Figures 4a–c, Supplementary Figure S3)” simply by tracking “Cy5-\nlabeled mRNA and lysotracker-labeled lysosomes following transfection to determine\nwhether Au-LNPs alter endosomal maturation”?\nResponse:\nWe thank the reviewer for raising this important point. We recognize that our\noriginal statement was too strong, as relying solely on LysoTracker is not sufficiently\nrigorous to establish altered endosomal maturation. In our study, we used Cy5-labeled\nmRNA together with LysoTracker which is an acidotropic probe widely employed to\nvisualize organelle acidification in live cells. Quantitative confocal analysis of\ncolocalization showed that Au-LNP–delivered mRNA exhibited substantially reduced\noverlap with LysoTracker signals compared to conventional LNPs, across multiple\ndoses and time points in Supplementary Figure S3e. This indicated that less mRNA\nremained in acidified compartments, consistent with enhanced escape or attenuated\nmaturation. However, we acknowledge that this evidence was indirect.\nTo address the reviewer’s concern and provide more direct support, we performed\nadditional experiments examining canonical endosomal markers (new Figure 5.\nWestern blot, qPCR, and immunofluorescence analyses revealed that the expression of\nthe early endosome marker RAB5 was unchanged between Au-LNPs and LNPs groups,\nwhereas the late endosome/lysosome marker LAMP1 was significantly downregulated\nin Au-LNP–treated cells1-3. This result confirms that the transition from early to late\nacidified compartments was attenuated when cells were treated with Au-LNPs. The\nmolecular evidence aligns well with our original imaging data, where reduced\ncolocalization between Cy5-mRNA and LysoTracker was observed.\nAccordingly, we have revised the Results section to more accurately reflect the\nevidence. The text now states that Au-LNP–treated cells displayed reduced\ncolocalization between Cy5-mRNA and LysoTracker-labeled acidic organelles,\nsuggesting that fewer particles were retained in acidified compartments, and that this\ninterpretation is corroborated by downregulation of LAMP1 but not RAB5. Together,\nthese findings indicate that Au-LNPs attenuate endosomal maturation and acidification,\nthereby facilitating efficient endosomal escape.\nRevisions Made to the Manuscript\nRevised\nResults (Line 258-265):\n\"To assess endosomal maturation, we quantified colocalization between Cy5-mRNA (green) and\nLysoTracker (Red, Methods) via CLSM. Reduced overlap ( ~50% lower; Supplementary Figure\nS3e) across doses (0.25–1 μg/mL) and times (4–6h) in Au-LNP-treated cells indicates less mRNA\nin acidified compartments, implying Au-LNPs reduced endosomal acidification (lower LysoTracker\ncolocalization; Figures 4a–c, Supplementary Figure S3e), favoring early escape at pH ~5.5–6.0\nwhere MC3 is protonated, while avoiding progression to highly degradative lysosomes (pH ~4.5).\nThis balance enhances cytoplasmic release and protects the mRNA cargo. This aligns with enhanced\nescape (Supplementary Figure S3d).\"\nNew Paragraph added\nResults (Line 283-298):\n“To further investigate whether Au-LNPs modulate endosomal maturation, we examined the\nexpression and localization of canonical endosomal markers1,2. Confocal immunofluorescence\nstaining revealed that LNP-treated cells exhibited strong colocalization of mRNA with the late\nendosomal/lysosomal marker LAMP1, whereas Au-LNP–treated cells showed markedly reduced\nLAMP1 signal, with mRNA distributed more diffusely in the cytoplasm (Figure. 5a). In contrast,\nno significant differences were observed in the early endosomal marker RAB5 between LNPs- and\nAu-LNPs–treated groups (Figure. 5b). Quantification confirmed that LAMP1 intensity was\nsignificantly reduced in Au-LNP–treated cells, while RAB5 intensity remained unchanged (Figure.\n5c).\nThese observations were corroborated by Western blot and qPCR analyses. LAMP1 protein\nand mRNA levels were consistently decreased following Au-LNP treatment at both 4 and 6 h,\nwhereas RAB5 expression showed no significant differences compared with conventional LNPs\n(Fig. 5d–e). Together, these results indicate that Au-LNPs selectively interfere with late endosomal\nmaturation without altering early endosome formation. By attenuating progression to LAMP1\ndegradative compartments, Au-LNPs create a temporal window that favors endosomal escape of\nmRNA at the early-to-late transition stage, consistent with the enhanced cytosolic release observed\nin our colocalization and TEM studies.”\nWe have also updated the Methods section to clarify that LysoTracker provides\nan indirect readout of acidification and that our conclusions are now supported by\nboth imaging and molecular marker analyses.\nNew Figure added:\nFigure 5. Mechanistic study of Au-LNP–mediated endosomal escape via\ninhibition of endosomal maturation.\na. b. Confocal fluorescence images of 293T cells treated with 0.5 or 1 µg/mL LNPs\nor Au-LNPs. Nuclei (blue), mRNA (red), and late endosome/lysosome marker\nLAMP1 (green, a) or early endosome marker RAB5 (green, b) were immunostained.\nScale bars, 10 µm.\nc. Quantification of LAMP1 and RAB5 mean fluorescence intensity from images in a\nand b.\nd. Western blot analysis and densitometric quantification of LAMP1 and RAB5\nprotein expression after treatment with LNPs or Au-LNPs for 4 and 6 h.\ne. qPCR analysis of LAMP1 and RAB5 mRNA levels in 293T cells after treatment\nwith LNPs or Au-LNPs for 4 and 6 h.\nStatistical significance was assessed using one-way ANOVA (for >2 groups) or two-\ntailed Student’s t-test (for 2 groups). **P < 0.01, ***P < 0.001, ****P < 0.0001.\nFigure S3. Confocal and ultrastructural analysis of endosomal escape.\na. CLSM images of HeLa cells treated with 0.25 μg/mL Cy5-mRNA for 4 h.\nb. CLSM images of HeLa cells treated with 0.5 μg/mL Cy5-mRNA for 4 h.\nc. CLSM images of HeLa cells treated with 1 μg/mL Cy5-mRNA for 4 h.\nd. Quantification of endosomal escape rate in HeLa cells treated with 0.25, 0.5, and 1\nμg/mL Cy5-mRNA for 4 h or 6 h.\ne. Quantification of overlap between lysosome signal and mRNA signal.\nf. TEM image of untreated HeLa cell\nUpdated Methods (Line 529-548):\nConfocal Imaging and Lysosomal Colocalization\n293 cells were seeded in glass-bottom dishes and incubated with LNPs or Au-LNPs containing\nCy5-mRNA (0.25–1 μg/mL). After 4–6 h, lysosomes were stained with LysoTracker Green (75\nnM, 1 h) and nuclei with Hoechst 33342 (1 μg/mL, 10 min). Live imaging was performed on\nLeica SP5.\nFor quantification, two independent experiments with three fields each were analyzed.\nColocalization (Pearson's Correlation Coefficient) and cytosolic diffusion area were measured in\nFiji (threshold 32–76).\nWestern Blot and qPCR\nCells treated with LNPs or Au-LNPs were lysed in RIPA buffer. Equal protein (30 μg) was\nresolved by SDS-PAGE and blotted to PVDF. Membranes were probed for RAB5, LAMP1, and\nGAPDH. Bands were quantified in ImageJ and normalized.\nFor qPCR, RNA was extracted (Vazyme RC112-01), reverse transcribed (Vazyme R323-01), and\namplified using SYBR Green (APE K1170). RAB5 and LAMP1 expression was normalized to\nGAPDH. Primer sequences are listed in Supplementary Methods.\nImmunofluorescence Staining\nCells were fixed (4% PFA), permeabilized (0.2% Triton X-100), blocked (5% BSA), and\nincubated with anti-RAB5 or anti-LAMP1 antibodies overnight at 4 °C. After secondary antibody\nincubation, nuclei were counterstained with DAPI. Images were acquired on Leica SP5.”\nUpdated Supplementary Methods (Line 34-39)\nReal-Time Quantitative PCR (qPCR) Primer sequences\nGAPDH: F- Primer: GGAGCGAGATCCCTCCAAAAT\nR- Primer: GGCTGTTGTCATACTTCTCATGG\nLAMP1: Beyotime QH03969S\nRAB5: Beyotime QH21261S\nReferences:\n1. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature\nReviews Molecular Cell Biology 2, 107-117 (2001).\n2. Saftig, P. & Klumperman, J. Lysosome biogenesis and lysosomal membrane\nproteins: trafficking meets function. Nature Reviews Molecular Cell Biology\n10, 623-635 (2009).\n3. Sahay, G., et al. Efficiency of siRNA delivery by lipid nanoparticles is limited\nby endocytic recycling. Nature Biotechnology 31, 653-658 (2013).\n7- In the Methods section the authors mention that “The lipid molar ratio of LNPs has\nnot been adjusted”. Can the authors explain what is meant by that and why they did\nthat, and whether the same is true also for the Au-LNPs? How can the authors be\nsure of the mechanistic details of the endosomal escape they present in Figure 1 if\nthey don’t control the molar ratio in the LNPs?\nResponse:\nWe sincerely thank the reviewer for this critical and insightful suggestion. In our\ninitial protocol, both conventional LNPs and Au-LNPs were prepared from the same\nlipid master mix (SM-102:DSPC:cholesterol:DMG-PEG = 50:10:38.5:1.5 mol%), with\nidentical volumes used for synthesis. By “not adjusted,” we meant that we did not re-\noptimize or rebalance the lipid ratios specifically for Au-LNPs. This approach was\nintentional: our aim was to isolate the structural effect of incorporating the Au core,\nwhile keeping the lipid composition identical to that of standard LNPs.\nWe realized that directly verifying the lipid composition in the final purified\nnanoparticles was necessary to eliminate any ambiguity. We therefore developed a\nliquid chromatography–mass spectrometry (LC-MS) method to quantify the content of\nthe key ionizable lipid, SM-102, in both LNP and Au-LNP formulations. Samples were\nfully disrupted in acetonitrile, analyzed against a standard calibration curve, and\ncompared across three independent replicates. The results, now presented in\nSupplementary Figure S1j, show no statistically significant difference in SM-102\ncontent between the two groups. This direct validation confirms that the incorporation\nof AuNPs did not alter lipid composition during formulation.\nThese findings substantially strengthen the mechanistic interpretation presented in\nFigure 1. Our schematic was based on the well-established principle that protonated\nionizable lipids promote hexagonal (H_II) phase transitions at endosomal pH, thereby\ndriving membrane disruption. With the LC-MS data confirming that the lipid ratios\nwere identical, we can confidently attribute the enhanced endosomal escape and mRNA\nexpression of Au-LNPs to architectural effects introduced by the Au core, not to\ncompositional differences.\nTo clarify this point, we have revised the Methods (Line 495-503) to state\nexplicitly:\n“LNPs and Au-LNPs were prepared from the same lipid master mix (SM-102 : DSPC: cholesterol:\nDMG-PEG2000 = 50:10:38.5:1.5 mol%). Lipids were dissolved in ethanol (22.1 mg/mL).\nLNPs: aqueous phase = mRNA in 50 mM acetic acid (pH 4.0).\nAu-LNPs: aqueous phase = mRNA condensed onto Au-MC3 cores in 50 mM acetic acid (pH 4.0).\nFormulations were generated using a CD-01 microfluidic chip (INano) at total flow rate 12 mL/min\n(aqueous:organic ratio 3:1) at room temperature. Suspensions were dialyzed overnight against PBS\n(10 kDa MWCO), sterile-filtered (0.22 μm), and stored at 4 °C.”\nWe also revised the Results (Line 103-113 and 145-148) to note:\n“For LNP assembly, both conventional LNPs and Au-LNPs were prepared from the same pre-mixed\nlipid master solution (Methods), ensuring identical lipid composition across groups. The critical\ndifference lay in the assembly sequence. In conventional LNPs, mRNA was directly mixed with the\nlipid solution, resulting in amorphous lipid–mRNA condensates. In contrast, for Au-LNPs, for\nAu-LNPs, mRNA was first condensed onto IC-AuNPs to form a stable mRNA–Au core complex.\nThis condensation step was confirmed by gel electrophoresis, which showed complete retardation of\nmRNA migration and no detectable free mRNA in solution (Supplementary Figure S2b), indicating\nefficient binding to the IC-AuNPs. The resulting mRNA–Au complexes were then encapsulated by\nthe lipid master mix to generate ordered core–shell nanoparticles. This two-step assembly process\npositioned mRNA and ionizable lipids radially around the Au core, establishing a distinct internal\narchitecture compared with conventional LNPs.” and\n“Liquid Chromatography–Mass Spectrometry (LC-MS) analysis confirmed that ionizable lipid\ncontent was indistinguishable between LNPs and Au-LNPs formulations (Supplementary Figure.\nS1i, Methods), demonstrating that incorporation of the Au core does not alter overall lipid\ncomposition and ruling out formulation bias as a confounding variable.”\nAdded Methods (Line 515-517)\n“LC-MS: The concentration of SM-102 in the supernatant, obtained after precipitating Au-MC3\nparticles with acetonitrile and centrifugation, was determined by LC-MS on a SCIEX Triple\nQuad™ 4500 (C18 column, monitoring the m/z 710.7 → 472.3 ESI+ mode).”\nFigure S1. Characterization of AuNP modification and Au-LNP assembly.\na. FTIR spectra of IC-AuNPs blue, Au-PEG red, and bare AuNPs black.\nb. 1H NMR spectra of SH-PEG blue, Au-PEG purple, DLin-MC3-DMA red, and Au-\nMC3 black.\nc. Agarose gel electrophoresis images of IC-AuNPs and mRNA at different mass ratios.\nd. TEM analysis of Au-LNPs.\ne. Cryo-EM image of Au-LNPs.\nf. TEM image of conventional LNPs, with cryo-EM image shown in the inset at the top\nright.\ng. Photographs of phase-transfer results of Au-PEG with three ionizable lipids SM-102\nA, DLin-MC3-DMA B, and DOTAP C.\nh. TEM images of Au-LNPs of ALC-0135 and Au-LNPs of DOTAP.\ni. Molecular dynamics simulation snapshots showing a cubic box with edge length\n11.047 nm containing 9 chains of 22-nt polyA RNA and 20 chains of SM-102 lipids,\nsimulated under traditional LNPs and Au-LNPs conditions.\nj. Standard curve for LC–MS quantification of SM-102 and measured SM-102 content\nin Au-LNPs and conventional LNPs from three independent experiments.\nk. Zeta potential profiles of free MC3 molecules versus Au-MC3 nanoparticles under\nidentical pH conditions in 50 mM citrate buffer.\nl. UV–Vis absorption spectra of AuNPs of different diameters 13, 18, 25, and 50 nm.\nm. Firefly luciferase FLuc expression in HeLa cells treated for 24 h with 0.5 μg/mL\nmRNA delivered by Au-LNPs containing gold cores of different diameters 13, 18, 25,\n50 nm; conventional LNPs were used as a positive control.\nn. DLS size distributions of Au-LNPs formulated with 18, 25, and 50 nm Au cores.\n.\n8- The Methods section is poorly written and in some parts it is more like a set of\ninstructions or a protocol for a student to follow. It is also not detailed enough for the\nwork to be reproduced.\nResponse:\nWe are grateful for the reviewer's constructive feedback on the Methods section.\nTo ensure that our experiments can be fully understood and replicated, we have\ncompletely rewritten and expanded this section. Specifically, we have now included the\nsources and catalog numbers for all key reagents, specified the models and parameter\nsettings of the instruments used, and provided a more detailed description of the\nexperimental procedures and data analysis methods. We believe the revised version is\nnow clearer, more complete, and more rigorous.\nThe following minor aspects need to be addressed:\nResponse\nWe sincerely thank the reviewer for their meticulous reading of our manuscript and for\nproviding these detailed and constructive suggestions. We have carefully addressed all\nof the minor aspects raised. In several cases, the relevant paragraphs were completely\nrevised to improve clarity and accuracy; as a result, the changes may not always appear\nas simple edits in the marked-up file. We would like to assure the reviewer that all\nsuggested corrections have been implemented in the revised manuscript.\n- Page 4, 1st line, “Figure 2a, Methods” has to be replaced by Figure 1.\nThank you for pointing this out. This has been corrected to Figure 1.\n- Page 4, second paragraph, add (Figure 2b) after “of 13 nm”\nThis has been corrected as suggested in Line 114.\n- Page 4, second paragraph, replace (Figure 2b,c) by Figure 2c.\nThank you. We have made this correction in Line 116.\n- Page 5, end of second paragraph, can the authors add a reference to substantiate the\nclaim of the last sentence?\nThis is an excellent suggestion. We have now added a relevant reference to support\nthis claim in Line 201.\n- Page 5, third paragraph, can the authors add “/cationic” after ionisable on the fourth\nline as DOTAP is a cationic lipid?\nThank you for this precise suggestion. We have added\"/cationic “as requested to\naccurately describe DOTAP.\n- Page 5, end of third paragraph, the authors refer to Hela and 293 cells in Figure 3c,\nwhile it shows data at different mRNA concentration for LNP and Au-LNP for only\none cell line, Hela, based on its caption.\nWe apologize for this error in the main text. We have now corrected this oversight in\nthe revised manuscript by removing the mention of '293 cells'.\n- Page 6, discrepancy between the values of transfection efficiency at low dosage\nreferred to in Figure S2c, 91.4% compared to 6.72 in figure S2c compared to 92.6%\nand 6.2% in main text.\nThank you for spotting this inconsistency. This was a typographical error in the main\ntext. We have now corrected the values in the text to be 91.4% and 6.72%, which are\nthe correct values in Line 245-246.\n- Page 6, penultimate paragraph, “Figure S3d” referred to is actually “Figure S3e”.\nThis has been corrected to Supplementary Figure S3f.\n- Page, second line of last paragraph, “Figures 4e-i” referred needs to be replaced by\n“Figure 4e-h” as there is no figure 4i. Again towards the end of the same paragraph,\n“Figure 4g-i” needs to be replaced by “Figure 4g-h”.\nThank you for this careful observation. Both references have been corrected to\n\"Figure 4e-h\" and \"Figure 4g-h\" respectively in Line 278 and 282\n- Page 13, last paragraph, 3rd line, in ethanol instead of “ethonal”.\nThe all “ethonal” spelling has been corrected to \"ethanol\"\n- References 21 and 23 are the same\nThank you for identifying this. We have removed the duplicate reference and\nrenumbered the subsequent references accordingly.\n- Captions of Figure 1 and that of Figure 2a need to be revised.\nWe agree. We have revised the captions for Figure 1and Figure 2a to be more\ndescriptive and accurate.\n- Figure 4e, remove (a) and (b) and replace with left middle and right.\nThis is a good suggestion for clarity. We have removed the (a) and (b) labels and\nreplaced them with descriptive text in the figure captions\n- Figure g and h, optionally replace endosome process with endosomal escape.\nWe agree that \"endosomal escape\" is more precise in this context. We have updated\nthe figure captions accordingly\n- Figure 7 a caption, treatment plan instead of treatment plane.\nThis typo has been corrected to \"treatment plan\"\n- In the methods section, DPH Fluorescence anisotropy missing a reference at the end\nof 1st paragraph on page 10.\nThank you for noting this omission. We have now added the appropriate reference in\nLine 142 and Line 557.\n- In the methods section, ELISAs 1st line of 2nd paragraph is missing a reference on\nthe preparation of pseudo-typed viruses.\nWe have now included a reference for the pseudovirus preparation Methods in Line\n587-589.\n- Figure S1b, legend SH-PEG is presumably Au-PEG.\nAs suggested by the reviewer, we have revised Figure 2b. The new Figure 2g now\nincludes the critical Au-PEG control group.\n- Figure S1f caption mentions “Firefly luciferase chemiluminescence (FLuc) assay in\nHeLa cells, treated for 24 h with the indicated concentrations of LNP…” while there\nare no concentrations on the figure itself.\nWe apologize for this mission. We have now added the corresponding concentrations\nin Supplementary Figure S1m caption.\n- Figure S2g, legend is ALC-0315 and not AIC-0315.\nThis typo in the figure has been corrected to \"ALC-0315\".\n- Figure S2g-I caption, “…cells treated with Moderna BNT and SORT” is actually\nPfizer/Biontech (ALC-0315, g), Moderna (SM-102, h).\nThank you for this critical correction. We have revised the caption to accurately\nstate:\"Pfizer/BioNTech (formulated with ALC-0315, j) and Moderna (formulated with\nSM-102, k)\". The reference to SORT has been corrected as well.\n- Figure S3e, caption refers to concentrations of 0.5, 1 and 3 μg/mL while legend is\nfrom 0.25 to 1 μg/mL.\nThank you for noting this discrepancy. We have corrected the figure caption to\naccurately reflect the concentrations shown in the legend\n- Radiance scale is missing from mice bioluminescence data on Figures 5b-e, 7d and\nS4a-d.\nWe have now added the radiance scale bars to all relevant bioluminescence images.\n- Hoechst instead of Hochest on the CLSM images of Figures 4a-c and S3a-c.\nThe spelling has been corrected to \"Hoechst\" on all relevant images\n- Vortex instead of Vertox on Figure 2a.\nThis has been corrected to \"Vortex\" in Figure 2a\nReviewer #2\nThe manuscript proposes advances in the technology of LNP for RNA delivery by\nincluding a core of AuNPs. In particular, the authors propose a structural optimization\nof AuNP core for enhancement of RNA delivery.\nFollowing a physical-chemical characterization, the AuLNPs have been studied in\nterms of in vitro transfection by measuring luciferase activity HeLa and 293 cells and\nmRNA delivery has been investigated by confocal microscopy. The authors showed\nthat AuLNPs have a transfection efficiency significantly superior to that achieved by\nLNP. TEM analysis of cells incubated with Au-LNP showed enlarged vesicles\nattributed to a stronger membrane disruption capability of Au-LNP. More in details,\nthe authors hypothesize that positively charged core of AuNPs attracts negatively\ncharged mRNA, “drawing it closer to the core region, while simultaneously repelling\npositively charged ionizable lipids and pushing them toward the endosomal\nmembrane”. The superiority of the AuLNP has also been demonstrated in vivo by two\ndifferent administration routes. Enhanced immunogenicity of SARS-CoV-2 Spike-\nmRNA Vaccines has been demonstrated; on the other hand, a significant reduction in\ntumor volume and extension in survival rate has been obtained. Although the\nmanuscript reports positive finding, important concerns on rationale and\nmethodologies make very difficult a deep evaluation of the proposed technology. The\nmanuscript is, in my opinion, far to be suitable for publication in Nature\nCommunication.\nGeneral Response:\nWe are deeply grateful for your detailed and thoughtful review of our manuscript,\nwhich provides valuable insights into areas for improvement and recognizes the\npositive findings. Your comments highlight important aspects of rationale,\nmethodology, and mechanistic clarity, which we have addressed through targeted\nrevisions, additional data, and literature support. While we respectfully disagree with\nsome interpretations (e.g., the necessity of surface modifications and the suitability of\nour assays), we have used your feedback to strengthen the manuscript significantly.\nBelow, we provide a point-by-point response grouped by major themes, with proposed\nchanges tracked in the revised manuscript. We believe these enhancements resolve the\nconcerns and position the work as suitable for Nature Communications.\n1.First of all, the rationale for using AuNP as core for LNP is not clear. Actually, the\nauthors motivate the choice of the AuNP with the high electron density together with\nits biocompatibility. However, the electron density cannot be considered an advantage\nfor drug delivery purposes. Moreover, according to the literature, the biocompatibility\nof gold cannot be considered superior to that of other biomaterials, such as lipids or\nother inorganic materials. Moreover, the authors affirm that a further advantage of\nAuNP is their versatile surface modifications. However, in this system the AuNP are\nthe core of the core/shell particles, thus not requiring surface modifications.\nResponse:\nWe thank the reviewer for this insightful feedback and the opportunity to clarify\nour rationale. AuNPs were selected as a proof-of-concept scaffold to demonstrate that\nengineering the internal architecture of LNPs with a pH-responsive core can improve\nendosomal escape. Our goal was not to suggest that gold is an ideal clinical material,\nbut that it provides a chemically versatile platform for mechanistic exploration. In the\nConclusion, we emphasize that the same architectural principle can be readily adapted\nto FDA-approved or biodegradable cores for translational development.\nWith this clarification, we address the reviewer’s specific concerns:\nFirst, the high electron density of gold is not a therapeutic advantage but a\nmethodological one, enabling high-contrast TEM imaging of core–shell morphology\n(Figure 2d) and vesicle disruption during endosomal escape (Figure 4g, h).\nSecond, regarding biocompatibility, we agree that gold is not superior to lipids or\nother biomaterials. We only intended to note that AuNPs are sufficiently biocompatible\nfor in vitro and in vivo proof-of-concept studies, consistent with our own toxicity and\nhistology data (Figure 3c, Figures S5–S7).\nThird, surface modifications are indispensable. We PEGylated AuNPs and then\ncoated them with MC3 by phase transfer, which was essential for compatibility with\nthe lipid shell. Successful modification was validated by complementary methods\nincluding phase transfer, TEM shell thickness, LSPR shift, FTIR/NMR, and zeta\npotential analysis (Figure 2, Supplementary Figure S1, Figure R1). Importantly,\nvarying the AuNP surface coating led to significant differences in delivery efficiency\n(Figure 2a), directly demonstrating that core surface chemistry is an active determinant\nof Au-LNP performance.\nIn summary, AuNPs were chosen for chemical versatility and methodological\nadvantages in proof-of-concept studies, not for inherent therapeutic superiority. Surface\nmodifications were essential for their function and rigorously validated. We believe\nthese clarifications address the reviewer’s concerns.\nFigure R1 a.TEM of Au-PEG. b. UV–Vis absorption spectra of Au-PEG and Au-MC3.\nRevision of Abstract (Line 30-42)\n“Messenger RNA (mRNA) therapeutics rely on lipid nanoparticles (LNPs) for delivery, yet\ninefficient endosomal escape remains a major bottleneck, with only a small fraction of internalized\ncargo reaching the cytoplasm. Conventional LNPs encapsulate mRNA in amorphous lipid cores,\nwhere partial charge neutralization and lack of structural order limit protonation-driven membrane\ndisruption. Here, we present an architectural strategy that engineers LNP internal structure using\nionizable lipid–coated gold nanoparticles (IC-AuNPs) as rigid, pH-responsive cores. The Au cores\ntemplate the formation of radially ordered core–shell architectures that stabilize particles at\nphysiological pH while amplifying charge segregation and curvature stress under acidic endosomal\nconditions. As a result, Au-LNPs achieved a twofold increase in endosomal escape and ~100-fold\ngreater cytoplasmic mRNA diffusion compared to conventional LNPs. Functionally, Au-LNPs\nenhanced mRNA expression in vitro, increased in vivo protein production up to fivefold, boosted\nantibody responses to SARS-CoV-2 vaccines, and improved therapeutic efficacy in a triple-negative\nbreast cancer model.”\nRevision in Introduction (Line 75-81)\n“In this work, we present a distinct architectural approach that shifts the focus from solely chemical\nmodifications to the internal structure of LNPs. Specifically, we leverage ionizable lipid–coated\ngold nanoparticles (IC-AuNPs) as rigid, pH-responsive cores that template the formation of radially\nordered core–shell architectures. This design stabilizes nanoparticles under physiological conditions\n(pH 7.4), preserving their integrity during circulation, while amplifying membrane-disruptive forces\nin the acidic endosomal environment (pH ~5.5–6.0) through enhanced charge segregation and\ncurvature stress (Figure 1).”\nRevision in Results and Discussion (Line 94-113)\n“AuNPs were selected as a proof-of-concept core material primarily due to their chemical versatility,\nincluding high surface reactivity for versatile modifications and precise size control (Figure 2a). To\nexplore whether internal architecture could enhance mRNA delivery, we synthesized IC-AuNPs as\nstructural cores for LNP assembly. AuNPs were first PEGylated and then coated with ionizable\nlipids such as MC3 or SM-102 via phase transfer mediated by hydrophobic forces, yielding\nhydrophobic IC-AuNPs that readily integrated into organic solvents (Figure 2a, Methods).\nSuccessful coating was confirmed by Fourier transform infrared (FTIR) spectroscopy and nuclear\nmagnetic resonance (NMR), which revealed diminished PEG-specific peaks alongside the\nemergence of MC3 signatures (Supplementary Figures S1a–b).\nFor LNP assembly, both conventional LNPs and Au-LNPs were prepared from the same pre-\nmixed lipid master solution (Methods), ensuring identical lipid composition across groups. The\ncritical difference lay in the assembly sequence. In conventional LNPs, mRNA was directly mixed\nwith the lipid solution, resulting in amorphous lipid–mRNA condensates. In contrast, for Au-LNPs,\nmRNA was first condensed onto IC-AuNPs to form a stable mRNA–Au core complex. This\ncondensation step was confirmed by gel electrophoresis, which showed complete retardation of\nmRNA migration and no detectable free mRNA in solution (Supplementary Figure S1c), indicating\nefficient binding to the IC-AuNPs. The resulting mRNA–Au complexes were then encapsulated by\nthe lipid master mix to generate ordered core–shell nanoparticles. This two-step assembly process\npositioned mRNA and ionizable lipids radially around the Au core, establishing a distinct internal\narchitecture compared with conventional LNPs.”\nRevision in Conclusion (Line 452-458)\n“Notably, while the biodistribution and clearance data indicate that Au cores in our formulation do\nnot cause long-term retention under the conditions tested, suggesting that gold itself is not a safety\nconcern in this context. Nevertheless, for eventual clinical translation, the same architectural\nprinciple can be readily extended to FDA-approved or biodegradable core materials that offer well-\nestablished safety profiles. Thus, while Au-LNPs validate the concept, future work will focus on\nadapting this internal core–shell strategy to clinically preferred materials to accelerate translational\npotential.”\nTogether, our findings thus establish a new conceptual framework for designing LNPs, shifting\nthe focus from solely chemical modifications to architectural optimization. By doing so, we provide\na versatile and scalable route to overcoming the longstanding bottleneck of endosomal escape,\nthereby advancing the clinical potential of mRNA therapeutics and vaccines.”\nRevision in Supplementary Figure S1\nFigure S1. Characterization of AuNP modification and Au-LNP assembly.\na. FTIR spectra of IC-AuNPs blue, Au-PEG red, and bare AuNPs black.\nb. 1H NMR spectra of SH-PEG blue, Au-PEG purple, DLin-MC3-DMA red, and Au-\nMC3 black.\nc. Agarose gel electrophoresis images of IC-AuNPs and mRNA at different mass ratios.\nd. TEM analysis of Au-LNPs.\ne. Cryo-EM image of Au-LNPs.\nf. TEM image of conventional LNPs, with cryo-EM image shown in the inset at the top\nright.\ng. Photographs of phase-transfer results of Au-PEG with three ionizable lipids SM-102\nA, DLin-MC3-DMA B, and DOTAP C.\nh. TEM images of Au-LNPs of ALC-0135 and Au-LNPs of DOTAP.\ni. Molecular dynamics simulation snapshots showing a cubic box with edge length\n11.047 nm containing 9 chains of 22-nt polyA RNA and 20 chains of SM-102 lipids,\nsimulated under traditional LNPs and Au-LNPs conditions.\nj. Standard curve for LC–MS quantification of SM-102 and measured SM-102 content\nin Au-LNPs and conventional LNPs from three independent experiments.\nk. Zeta potential profiles of free MC3 molecules versus Au-MC3 nanoparticles under\nidentical pH conditions in 50 mM citrate buffer.\nl. UV–Vis absorption spectra of AuNPs of different diameters 13, 18, 25, and 50 nm.\nm. Firefly luciferase FLuc expression in HeLa cells treated for 24 h with 0.5 μg/mL\nmRNA delivered by Au-LNPs containing gold cores of different diameters 13, 18, 25,\n50 nm; conventional LNPs were used as a positive control.\nn. DLS size distributions of Au-LNPs formulated with 18, 25, and 50 nm Au cores\nReferences\n1. Huehn, J., et al. Selected Standard Protocols for the Synthesis, Phase Transfer,\nand Characterization of Inorganic Colloidal Nanoparticles. Chemistry of\nMaterials 29, 399-461 (2017).\n2. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature\nReviews Molecular Cell Biology 2, 107-117 (2001).\n2．The authors affirm that AuNP are “ideal” marker for localization of LNP by TEM;\nhowever, while AuNP are useful to study NP localization and structure, the choice of\nthe core for drug delivery purpose in not presented. Moreover, also when AuNP are\nused for visualization into the cell, the visualization of AuNP does not correspond to\nthe visualization of the LNP that, once into the cells, should lose their integrity.\nResponse:\nWe thank the reviewer for this helpful comment, which allows us to clarify the\ndual role of AuNPs in our study: (i) as a proof-of-concept core to demonstrate that an\ninternal scaffold can reorganize lipid distribution and enhance endosomal escape, and\n(ii) as an electron-dense marker that facilitates mechanistic tracking by TEM.\nRegarding their use as a delivery core, AuNPs were chosen for their chemical\nversatility, which enables precise surface modification and size control. This allowed\nus to systematically demonstrate how a pH-responsive core can reorganize lipid\narchitecture and improve escape efficiency (Figure 1). As shown in Figure 4d, this\ndesign increased cytoplasmic diffusion of mRNA by nearly 100-fold compared with\nconventional LNPs. We emphasize that gold was used purely as a proof-of-concept\nscaffold; in the Conclusion we note that the strategy is extensible to FDA-approved or\nbiodegradable cores for translational use.\nWith respect to TEM visualization, we agree that LNPs inevitably undergo partial\ndisassembly after cellular uptake, which is necessary for mRNA release. In our system,\nAuNPs were used as a stable electron-dense marker to monitor whether the engineered\ncore–shell particles remained intact during uptake and initiated vesicle disruption upon\nacidification. At physiological pH (7.4), Au-LNPs remain intact during uptake and\nearly trafficking (Figure 2g, Supplementary Figure S2a), consistent with prior reports1-\n3. Disassembly occurs only upon endosomal acidification, at which point the Au cores\nremain visible and mark the original vesicle location. Our TEM images (Figure 4e–h)\nshow Au cores associated with vesicles displaying compromised membranes,\nconfirming that Au-LNPs preserve integrity during uptake and subsequently\ndisassemble in a pH-dependent manner to promote escape.\nTo further improve clarity, we have revised the “Design and Physicochemical\nCharacterization of Gold–Lipid Nanoparticles” subsection, as well as the\nIntroduction and Conclusion.\nReferences\n1. Han, X., et al. Fast and facile synthesis of amidine-incorporated degradable\nlipids for versatile mRNA delivery in vivo. Nature Chemistry 16, 1687-1697\n(2024).\n2. Yap, S.L., et al. Bypassing Endocytic Barriers: Visualizing Membrane Fusion\nand Endosomal Escape of Cubic Phase Lipid Nanoparticles (Cubosomes).\nSmall (Weinheim an der Bergstrasse, Germany), e2502231-e2502231 (2025).\n3. Luozhong, S., et al. Poly(carboxybetaine) lipids enhance mRNA therapeutics\nefficacy and reduce their immunogenicity. Nature Materials (2025).\n3.The authors affirm that “To achieve efficient endosomal escape and mRNA capture, we require Au core that responds well to changes in pH.” However, while for ionizable lipids the mechanism of endosomal escape is well-know, any mechanism of endosomal escape is clearly described here for the AuNPs\nResponse:\nWe thank the reviewer for highlighting this important point. We clarify that the pH\nresponsiveness in our system does not originate from bare AuNPs, but from the\nengineered core–shell structure in which AuNPs are coated with ionizable lipids\n(e.g., MC3; Figure 2a). Upon endosomal acidification (pH ~5.5), MC3 becomes protonated,\nand the presence of the Au core amplifies this response by reorganizing charge\ndistribution within the particle.\nThis architecture produces a dual effect: the positively charged Au–MC3 core\nattracts the negatively charged mRNA toward the interior while repelling the positively\ncharged ionizable lipids outward. This segregation maximizes the exposure of\nprotonated ionizable lipids to the endosomal membrane, promoting the formation of\ndisruptive hexagonal (H_II) phases, a well-established mechanism for ionizable lipid-\nmediated endosomal escape1,2. Consistent with this model, we observed a twofold\nincrease in escape efficiency (Supplementary Figure S3d) and a 100-fold increase in\ncytoplasmic mRNA diffusion area (Figure 4d) compared to conventional LNPs.\nSupporting this mechanism, zeta potential titrations showed that Au-MC3\nnanoparticles underwent a sharper charge reversal than free MC3 (Supplementary\nFigure S1k), indicating that the Au core enforces a more ordered lipid alignment and\nenhances pH sensitivity.\nBased on these findings, the Au core has three functional roles in our system:\n(i) pH-response amplifier, by aligning MC3 lipids at the core interface to generate a\nstronger localized charge burst during acidification (Supplementary Figure S1k);\n(ii) blocker of endosomal maturation, as shown by reduced LAMP1 but unchanged\nRAB5 expression (Figure 5), suggesting inhibition of late endosome formation;\n(iii) TEM marker, leveraging electron density to directly visualize Au cores associated\nwith disrupted endosomal membranes (Figures 4e–h).\nRevisions Made to the Manuscript:\nTo avoid ambiguity, we revised the Results to clarify that the pH responsiveness\narises from MC3 functionalization of the Au core, not from gold itself. We also added\nnew zeta potential data in (Supplementary Figure S1k) showing that Au-MC3\nundergoes the same pH-dependent charge transition as MC3 alone, but with greater\nsharpness, confirming the amplifying effect of the Au core.\nTo enhance clarity, we have revised the entire “Design and Physicochemical\nCharacterization of Gold- Lipid Nanoparticles” and “Enhanced pH-Responsive\nMembrane Disruption by Au-LNPs” subsections, and include description for the\nresults in Supplementary Figure S1k (Line 150-153).\nIn summary, the Au core does not act as a pH-sensitive material by itself. Rather,\nby enforcing spatial organization of ionizable lipids, it amplifies their known\nprotonation-driven endosomal escape mechanism, blocks maturation into degradative\nlysosomes, and simultaneously provides a high-contrast imaging marker.\nReferences:\n1. Semple, S.C., et al. Rational design of cationic lipids for siRNA delivery.\nNature Biotechnology 28, 172-U118 (2010).\n2. Raguram, A., Banskota, S. & Liu, D.R. Therapeutic in vivo delivery of gene\nediting agents. Cell 185, 2806-2827 (2022).\n4.The preparation protocol of the core-shell LNP arises many concerns. The authors\nprepared Au-PEG-NP by reaction of PEG-SH with the preformed AuNPs. Then, they\nperform a ligand exchange demonstrated by change of the solubility of AuNPs from\nthe aqueous to organic solvent. It is not clear what is the mechanism for PEGylation.\nHave the authors performed a chemical conjugation? If the case, mechanism of\n“ligand exchange” is not clear once mixed with the lipids. Finally, in the case the\nPEGylation is required for the core-shell NP in which lipids for the outer layer,\nPEGylated lipids can be used thus avoiding a previous conjugation of PEG to the\nAuNPs.\nResponse:\nWe thank the reviewer for these important questions regarding our preparation\nprotocol (Supplementary Figures S1a-b and Figure R1), which allow us to clarify the\nmechanisms of PEGylation and ionizable lipid coating, and to address the suggestion\nregarding PEG-lipid alternatives. We also acknowledge that our earlier use of the term\n“ligand exchange” was misleading; we have revised the manuscript to describe the\nprocess more accurately as a stepwise surface modification involving covalent\nPEGylation followed by MC3 coating.\nMechanism of PEGylation and Confirmation of Chemical Conjugation.\nYes, we performed a chemical conjugation. Preformed citrate-stabilized AuNPs\nwere reacted with thiol-terminated PEG (PEG-SH) to generate Au-PEG-NPs. This\nproceeds via the well-characterized formation of strong covalent Au–S bonds, in which\nthe thiol groups of PEG-SH displace citrate ligands on the Au surface. This provides\nsteric stabilization and water solubility, preventing aggregation. The success of\nPEGylation was confirmed by (i) retention of aqueous solubility, (ii) FTIR and NMR\nspectra showing PEG-specific signals (C–H stretches at ~2875 cm⁻¹, –CH₂–O– protons\nat 3.78 ppm; Supplementary Figures S1a-b), and (iii) consistency with established\nliterature on Au–S conjugation for PEGylated AuNPs used in biomedical applications1,2.\nMechanism of MC3 Coating via Phase Transfer.\nAfter PEGylation, MC3 was introduced through a biphasic (aqueous–chloroform)\nphase transfer process. The hydrophobicity of MC3 drives its adsorption and\norganization around Au-PEG, facilitated by the high interfacial energy. TEM imaging\nrevealed a 2.5 nm lipid corona surrounding the Au core (Figure 2c), while FTIR and\nNMR spectra confirmed the presence of MC3 signatures (Supplementary Figures S1a-\nb). To probe the binding forces, we treated the particles with Triton X-100 (Figure R2).\nThe treatment reduced but did not eliminate the lipid layer, suggesting that hydrophobic\ninteractions are dominant but reinforced by more persistent interfacial binding. This\nindicates that the coating is not simple physisorption but involves a more stable\ninteraction.\nFigure R2 TEM images of Au-LNPs and Au-MC3 treated by 1% Triton X-100 solution\nResponse to Suggestion: Using PEGylated Lipids Instead of Pre-PEGylation.\nWe appreciate the reviewer’s suggestion and tested this approach by directly\nmodifying AuNPs with DSPE-PEG2000-SH (Au-PEG-lipid) to serve as cores. When\ncompared with conventional LNPs, however, Au-PEG-lipid@LNPs did not enhance\nmRNA delivery efficiency (Figure R3). We attribute this to the lack of pH-responsive\ncharge variation in PEG-lipids, which prevents the charge redistribution mechanism\ncritical for enhanced endosomal escape (Figure 1). In contrast, MC3-modified AuNPs\n(Au-MC3) exhibit sharp pH-dependent zeta potential transitions, enabling the\narchitectural advantages we report. These results confirm that PEG-lipid substitution\ncannot replicate the pH-responsiveness of Au-MC3 and further validate our design\nchoice.\nFigure R3. Evaluation of mRNA delivery efficiency of LNPs and Au-PEG-lipid@LNPs\nin HeLa cells. Fluorescence images and GFP intensity statistical analysis of cells after\nGFP mRNA transfection, and luciferase intensity statistical analysis of cells after\nluciferase mRNA transfection.\nRevisions Made to the Manuscript:\nReplaced the misleading term “ligand exchange” with “MC3 coating”\nTo enhance clarity, we have revised the entire “Design and Physicochemical\nCharacterization of Gold- Lipid Nanoparticles” subsection.\nUpdate Supplementary Figure S1:\nFigure S1. Characterization of AuNP modification and Au-LNP assembly.\na. FTIR spectra of IC-AuNPs blue, Au-PEG red, and bare AuNPs black.\nb. 1H NMR spectra of SH-PEG blue, Au-PEG purple, DLin-MC3-DMA red, and Au-\nMC3 black.\nc. Agarose gel electrophoresis images of IC-AuNPs and mRNA at different mass ratios.\nd. TEM analysis of Au-LNPs.\ne. Cryo-EM image of Au-LNPs.\nf. TEM image of conventional LNPs, with cryo-EM image shown in the inset at the top\nright.\ng. Photographs of phase-transfer results of Au-PEG with three ionizable lipids SM-102\nA, DLin-MC3-DMA B, and DOTAP C.\nh. TEM images of Au-LNPs of ALC-0135 and Au-LNPs of DOTAP.\ni. Molecular dynamics simulation snapshots showing a cubic box with edge length\n11.047 nm containing 9 chains of 22-nt polyA RNA and 20 chains of SM-102 lipids,\nsimulated under traditional LNPs and Au-LNPs conditions.\nj. Standard curve for LC–MS quantification of SM-102 and measured SM-102 content\nin Au-LNPs and conventional LNPs from three independent experiments.\nk. Zeta potential profiles of free MC3 molecules versus Au-MC3 nanoparticles under\nidentical pH conditions in 50 mM citrate buffer.\nl. UV–Vis absorption spectra of AuNPs of different diameters 13, 18, 25, and 50 nm.\nm. Firefly luciferase FLuc expression in HeLa cells treated for 24 h with 0.5 μg/mL\nmRNA delivered by Au-LNPs containing gold cores of different diameters 13, 18, 25,\n50 nm; conventional LNPs were used as a positive control.\nn. DLS size distributions of Au-LNPs formulated with 18, 25, and 50 nm Au cores.\nReferences:\n1. Omping, J., et al. Facile Synthesis of PEGylated Gold Nanoparticles for\nEnhanced Colorimetric Detection of Histamine. ACS Omega 9, 14269-14278\n(2024).\n2. Nitica, S., et al. PEGylated Gold Nanoparticles with Interesting Plasmonic\nProperties Synthesized Using an Original, Rapid, and Easy-to-Implement\nProcedure. Journal of Nanomaterials 2018(2018).\n5.The authors prepared the IL-AuNPs by mixing the AU-PEG NPs with cationic\n(DOTAP) or ionizable (SM102) lipids. The Au-LNP are then prepared by mixing the\nIL-Au NP with the RNA, that should provide lipoplexes. Thus, it is not clear how this\npreparation protocol should provide core-shell LNP encapsulating RNA; on the other\nhand, the mechanism seems to provide lipoplexes for DOTAP, while in the case of\nSM102 any pH change (typically used in the PLN preparation) is used to modify the\ncharge of the ionizable lipid thus inducing the efficient RNA encapsulation. In line\nwith this, it is not clear what kind of interaction, hydrophobic or electrostatic,\ntakes place among the AuNP and the lipids. Finally, MC3 is not mentioned in the\npreparation method.\nResponse:\nWe thank the reviewer for this detailed and constructive comment. We recognize\nthat our original description may have created the impression of simple lipoplex\nformation. In fact, our protocol uses a stepwise assembly strategy that yields bona fide\ncore–shell Au-LNPs, distinct from lipoplexes or polymer–lipid nanoparticles (PLNs).\nFirst, citrate-stabilized AuNPs were covalently PEGylated via Au–S bonds and\nthen coated with MC3 by phase transfer. MC3 was our primary ionizable lipid, now\nexplicitly described in the revised Methods. The coating process is driven mainly by\nhydrophobic interactions at the aqueous–organic interface and results in a uniform ~2.5\nnm corona, confirmed by TEM, FTIR, NMR, and UV–vis analysis (Figure 2c,\nSupplementary Figure S1a–b, Figures R1–R2).\nSecond, these Au-MC3 cores were incorporated into a standard lipid master mix\n(SM-102/DSPC/cholesterol/DMG-PEG2000) by ethanol injection or microfluidics.\nThis drives spontaneous self-assembly into complete Au-LNPs, where the Au-MC3\nacts as a pH-responsive internal scaffold. The final particles are therefore core–shell\nstructures, not simple mRNA–lipid electrostatic complexes.\nThird, we confirmed mRNA association with the Au-MC3 cores by gel\nelectrophoresis, which showed no free mRNA at the optimized ratio (Supplementary\nFigure S1c), and by ITC, which revealed a strong exothermic signal (ΔQ = –46.74 μJ;\nSupplementary Figure S2b). These results indicate robust electrostatic and hydrophobic\ninteractions that stabilize the mRNA–Au-MC3 complex.\nTo avoid confusion, we have revised the Methods section to explicitly describe\nPEGylation, MC3 coating, and final lipid self-assembly, and to emphasize that our\nplatform is mechanistically distinct from lipoplexes or PLNs.\nRevisions Made to the Manuscript:\nRevision of Results (Line 103-106) and (Line 145-147):\n“For LNP assembly, both conventional LNPs and Au-LNPs were prepared from the same pre-mixed\nlipid master solution (Methods), ensuring identical lipid composition across groups. The critical\ndifference lay in the assembly sequence. In conventional LNPs, mRNA was directly mixed with the\nlipid solution, resulting in amorphous lipid–mRNA condensates.”\n“Liquid Chromatography-Mass Spectrometry (LC-MS) analysis confirmed that ionizable lipid\ncontent was indistinguishable between LNPs and Au-LNPs formulations (Supplementary Figure.\nS1i, Methods)”\nRevision of Methods (Line 485-490) and (Line 495-503)\n“Ionizable Lipid Coating (IC-AuNPs)\nAu-PEG (50 μL, 1 mg/mL, aqueous) was added to MC3 in chloroform (200 μL, 25 mg/mL). After\nvortexing (3,000 rpm, 10 min), the AuNPs transferred to the organic phase, confirming\nhydrophobic adsorption. The chloroform phase was washed with ethanol, and particles were\nresuspended in 50 mM acetic acid buffer (pH 4.0). Identical procedures were used for SM-102,\nALC-0135, or DOTAP formulation coating.”\n“LNP and Au-LNP Formulation\nLNPs and Au-LNPs were prepared from the same lipid master mix (SM-\n102:DSPC:cholesterol:DMG-PEG2000 = 50:10:38.5:1.5 mol%). Lipids were dissolved in ethanol\n(22.1 mg/mL).\nLNPs: aqueous phase = mRNA in 50 mM acetic acid (pH 4.0).\nAu-LNPs: aqueous phase = mRNA condensed onto Au-MC3 cores in 50 mM acetic acid (pH 4.0).\nFormulations were generated using a CD-01 microfluidic chip (INano) at total flow rate 12\nmL/min (aqueous:organic ratio 3:1) at room temperature. Suspensions were dialyzed overnight\nagainst PBS (10 kDa MWCO), sterile-filtered (0.22 μm), and stored at 4 °C.”\nUpdate Supplementary Figure S2: Added ITC thermograms (Supplementary Figure\nS2b) and binding parameters demonstrating strong Au-MC3–mRNA interactions.\nFigure S2. Functional and structural evaluation of LNPs and Au-LNPs.\na. Hemolysis percentage of LNPs and Au-LNPs under different pH conditions.\nb. Exothermic interaction between Au-MC3 and mRNA measured by isothermal\ntitration calorimetry (ITC) (ΔQ=-46.74μJ).\nc. Dynamic light scattering analysis of mRNA and Au-MC3–mRNA complexes.\nd. Dose–response analysis of Au-LNPs using seven concentrations of Fluc-mRNA to\ndetermine the optimal dosage.\ne. Flow cytometric analysis of GFP expression in HeLa cells treated with varying\nconcentrations of LNPs and Au-LNPs.\nf. Flow cytometric analysis of GFP expression in 293 cells treated with varying\nconcentrations of LNPs and Au-LNPs.\ng. Chemiluminescence intensity of HeLa cells treated with LNPs and Au-LNPs.\nh. Chemiluminescence intensity of 293 cells treated with LNPs and Au-LNPs.\ni. Bioluminescence intensity of 4T1, EMT6, and JC cells treated with LNPs and Au-\nLNPs.\nj. Bioluminescence intensity of HeLa cells treated with Pfizer–BioNTech LNPs\nformulated with ALC-0135 and Au-LNPs.\nk. Bioluminescence intensity of HeLa cells treated with Moderna LNPs formulated\nwith SM-102 and Au-LNPs.\nl. Bioluminescence intensity of HeLa cells treated with SORT–Lung formulations of\nLNPs and Au-LNPs.\n6. In Figure 2, a core-shell structure is showed; however, it is not reported if the\ndifferent lipids used, especially cationic and ionizable, provide a different assembling\nwith the AU-NPs and, consequently different core/shell structure.\nResponse:\nWe thank the reviewer for raising this important question regarding whether the\nobserved core–shell structure is dependent on the type of lipid used. To address this,\nwe performed additional experiments in which Au-LNPs were formulated using two\nalternative lipids in addition to SM-102: (i) ALC-0315, a clinically validated ionizable\nlipid used in mRNA vaccines, and (ii) DOTAP, a widely studied cationic lipid. As\nshown in the new TEM images (Supplementary Figure S1h), Au-LNPs prepared with\neither ALC-0315 or DOTAP exhibit the same characteristic core–shell morphology\nobserved with SM-102.\nThese results demonstrate that our formulation method is both versatile and robust:\nthe formation of a distinct core–shell structure is reproducible and not limited to a\nparticular lipid species. Instead, the architecture arises from the overall assembly\nprocess of our Au-core functionalization and subsequent LNP formation.\nThis finding is also consistent with recent reports showing that the fundamental\nmorphology of LNPs is relatively conserved across different ionizable lipid chemistries,\nwith functional differences manifesting more in endosomal escape efficiency and\nbiodistribution than in gross structural assembly1,2.\nRevisions Made to the Manuscript:\nText added (Line 126-128):\n“Similar core–shell morphology was also observed when ALC-0315, or DOTAP were\nused in place of SM-102 (Supplementary Figure S1h), demonstrating that the\narchitecture is reproducible across different lipid chemistries and not an artifact of a\nsingle lipid.”\nUpdated Supplementary Figure S1\nFigure S1. Characterization of AuNP modification and Au-LNP assembly.\na. FTIR spectra of IC-AuNPs blue, Au-PEG red, and bare AuNPs black.\nb. 1H NMR spectra of SH-PEG blue, Au-PEG purple, DLin-MC3-DMA red, and Au-\nMC3 black.\nc. Agarose gel electrophoresis images of IC-AuNPs and mRNA at different mass ratios.\nd. TEM analysis of Au-LNPs.\ne. Cryo-EM image of Au-LNPs.\nf. TEM image of conventional LNPs, with cryo-EM image shown in the inset at the top\nright.\ng. Photographs of phase-transfer results of Au-PEG with three ionizable lipids SM-102\nA, DLin-MC3-DMA B, and DOTAP C.\nh. TEM images of Au-LNPs of ALC-0135 and Au-LNPs of DOTAP.\ni. Molecular dynamics simulation snapshots showing a cubic box with edge length\n11.047 nm containing 9 chains of 22-nt polyA RNA and 20 chains of SM-102 lipids,\nsimulated under traditional LNPs and Au-LNPs conditions.\nj. Standard curve for LC–MS quantification of SM-102 and measured SM-102 content\nin Au-LNPs and conventional LNPs from three independent experiments.\nk. Zeta potential profiles of free MC3 molecules versus Au-MC3 nanoparticles under\nidentical pH conditions in 50 mM citrate buffer.\nl. UV–Vis absorption spectra of AuNPs of different diameters 13, 18, 25, and 50 nm.\nm. Firefly luciferase FLuc expression in HeLa cells treated for 24 h with 0.5 μg/mL\nmRNA delivered by Au-LNPs containing gold cores of different diameters 13, 18, 25,\n50 nm; conventional LNPs were used as a positive control.\nn. DLS size distributions of Au-LNPs formulated with 18, 25, and 50 nm Au cores\nReferences\n1. Ojansivu, M., et al. Formulation and Characterization of Novel Ionizable and\nCationic Lipid Nanoparticles for the Delivery of Splice-Switching\nOligonucleotides. Advanced Materials 37(2025).\n2. Song, Z., et al. ALC-0315 Lipid-Based mRNA LNP Induces Stronger Cellular\nImmune Responses Postvaccination. Molecular Pharmaceutics 22, 859-870\n(2025).\n7. The authors speculate on the lipid assembly to justify results of luciferase\nexpression. In particular, at lines 146-150 they affirm that AuNP with higher size\n“interferes with the optimal assembly of the lipid components”. However, they did not\ndemonstrate what kind of assembly arrive by mixing AuNP and the lipids, as well as\nwhen also mixing the RNA.\nResponse:\nWe thank the reviewer for this insightful comment. We agree that our earlier\ndescription was too speculative and did not sufficiently explain the type of assembly\nformed during Au-LNP preparation. Our intent was to emphasize that the assembly\npathway differs between conventional LNPs and Au-LNPs, and that Au core size\nstrongly influences the outcome.\nIn the revised text, we now explicitly describe the process: in Au-LNPs, mRNA\nfirst condenses onto IC-AuNPs to form an mRNA–Au complex, as confirmed by gel\nelectrophoresis showing complete retardation of free mRNA. This complex is\nsubsequently encapsulated by the lipid master solution, yielding a core–shell structure\ndirectly visualized by TEM and cryo-EM (Figure 2d, Supplementary Figure S1e, h).\nDLS analysis further shows that 13 nm Au cores produce uniform particles with low\nPDI, whereas larger cores yield broader distributions and aggregates, consistent with\ndisrupted lipid packing. Complementary MD simulations also indicate that Au-LNPs\nform more thermodynamically stable self-assembled structures than conventional LNPs,\nsupporting the presence of a well-defined core–shell architecture.\nRevision of Results (Line103-113), (Line 159-164) and (Line 172-176):\n“For LNP assembly, both conventional LNPs and Au-LNPs were prepared from the same pre-mixed\nlipid master solution (Methods), ensuring identical lipid composition across groups. The critical\ndifference lay in the assembly sequence. In conventional LNPs, mRNA was directly mixed with the\nlipid solution, resulting in amorphous lipid–mRNA condensates. In contrast, for Au-LNPs, for\nAu-LNPs, mRNA was first condensed onto IC-AuNPs to form a stable mRNA–Au core complex.\nThis condensation step was confirmed by gel electrophoresis, which showed complete retardation of\nmRNA migration and no detectable free mRNA in solution (Supplementary Figure S1c), indicating\nefficient binding to the IC-AuNPs. The resulting mRNA–Au complexes were then encapsulated by\nthe lipid master mix to generate ordered core–shell nanoparticles. This two-step assembly process\npositioned mRNA and ionizable lipids radially around the Au core, establishing a distinct internal\narchitecture compared with conventional LNPs.”\n“We next investigated how the size of the AuNP core influences LNP assembly and mRNA delivery.\nIC-AuNPs with diameters of 13, 18, 25, and 50 nm were synthesized and coated with MC3, then\nincorporated into LNPs using the same lipid master solution as above. TEM and DLS analysis\nrevealed that 13 nm Au cores produced monodisperse Au-LNPs with narrow size distributions,\nwhereas larger cores disrupted lipid organization and yielded broader distributions with oversized\naggregates (Figure. 2e, Supplementary Figure. S1k–m, Methods).”\n“Consistent with these observations, DLS confirmed that only 13 nm Au-MC3 cores produced\nstable nanoparticles with uniform size distributions, whereas larger cores resulted in heterogeneous\nparticles with higher polydispersity (Supplementary Figure. S1m). Together, these results identify\n~13 nm as an optimal Au core size for templating ordered Au-LNPs, directly linking nanoscale\narchitecture to functional mRNA delivery efficiency.”\nUpdate Supplementary Figure S1 and Supplementary Figure S2\nFigure S1. Characterization of AuNP modification and Au-LNP assembly.\na. FTIR spectra of IC-AuNPs blue, Au-PEG red, and bare AuNPs black.\nb. 1H NMR spectra of SH-PEG blue, Au-PEG purple, DLin-MC3-DMA red, and Au-\nMC3 black.\nc. Agarose gel electrophoresis images of IC-AuNPs and mRNA at different mass\nratios.\nd. TEM analysis of Au-LNPs.\ne. Cryo-EM image of Au-LNPs.\nf. TEM image of conventional LNPs, with cryo-EM image shown in the inset at the\ntop right.\ng. Photographs of phase-transfer results of Au-PEG with three ionizable lipids SM-\nh. TEM images of Au-LNPs of ALC-0135 and Au-LNPs of DOTAP.\ni. Molecular dynamics simulation snapshots showing a cubic box with edge length\n11.047 nm containing 9 chains of 22-nt polyA RNA and 20 chains of SM-102 lipids,\nsimulated under traditional LNPs and Au-LNPs conditions.\nj. Standard curve for LC–MS quantification of SM-102 and measured SM-102\ncontent in Au-LNPs and conventional LNPs from three independent experiments.\nk. Zeta potential profiles of free MC3 molecules versus Au-MC3 nanoparticles under\nidentical pH conditions in 50 mM citrate buffer.\nl. UV–Vis absorption spectra of AuNPs of different diameters 13, 18, 25, and 50 nm.\nm. Firefly luciferase FLuc expression in HeLa cells treated for 24 h with 0.5 μg/mL\nmRNA delivered by Au-LNPs containing gold cores of different diameters 13, 18,\n25, 50 nm; conventional LNPs were used as a positive control.\nn. DLS size distributions of Au-LNPs formulated with 18, 25, and 50 nm Au cores.\nFigure S2. Functional and structural evaluation of LNPs and Au-LNPs.\na. Hemolysis percentage of LNPs and Au-LNPs under different pH conditions.\nb. Exothermic interaction between Au-MC3 and mRNA measured by isothermal\ntitration calorimetry (ITC) (ΔQ=-46.74μJ).\nc. Dynamic light scattering analysis of mRNA and Au-MC3–mRNA complexes.\nd. Dose–response analysis of Au-LNPs using seven concentrations of Fluc-mRNA to\ndetermine the optimal dosage.\ne. Flow cytometric analysis of GFP expression in HeLa cells treated with varying\nconcentrations of LNPs and Au-LNPs.\nf. Flow cytometric analysis of GFP expression in 293 cells treated with varying\nconcentrations of LNPs and Au-LNPs.\ng. Chemiluminescence intensity of HeLa cells treated with LNPs and Au-LNPs.\nh. Chemiluminescence intensity of 293 cells treated with LNPs and Au-LNPs.\ni. Bioluminescence intensity of 4T1, EMT6, and JC cells treated with LNPs and Au-\nLNPs.\nj. Bioluminescence intensity of HeLa cells treated with Pfizer–BioNTech LNPs\nformulated with ALC-0135 and Au-LNPs.\nk. Bioluminescence intensity of HeLa cells treated with Moderna LNPs formulated\nwith SM-102 and Au-LNPs.\nl. Bioluminescence intensity of HeLa cells treated with SORT–Lung formulations of\nLNPs and Au-LNPs.\n8. The methods of the self-quenching sulforhodamine B (SRB) to study endosome al\nescape is questionable because suitable to study the liposomes stability, but not the\nstability or ensosomal escape of Au-LNP.\nResponse:\nWe thank the reviewer for raising this important point on the use of the SRB assay.\nWe acknowledge that the SRB liposome model is a simplified in vitro system that does\nnot directly measure the in vivo stability or endosomal escape of Au-LNPs. Rather, it\nserves as a widely used proxy to evaluate the pH-dependent membrane fusion potential\nof lipid-based nanoparticles by quantifying release from artificial liposomal membranes,\nmimicking endosomal conditions1-3.\nTo strengthen our evidence and address the reviewer’s concern, we performed a\npH-dependent hemolysis assay using human red blood cells (RBCs) as a biological\nrelevant membrane model (new Supplementary Figure S2a; Methods updated). The\nresults show that Au-LNPs induced significantly greater RBC membrane disruption at\nacidic pH (65 % hemolysis at pH 5.5 vs. ~57% for conventional LNPs; n=2) compared\nwith conventional LNPs4-6, consistent with the SRB assay's pH 5.5 peak (Figure 2g).\nThis finding is consistent with the SRB assay, which also showed enhanced disruption\nat endosomal pH (~5.5), and together they confirm that Au-LNPs exhibit superior pH-\ntriggered membrane-lytic activity.\nWe have revised the manuscript to clarify the interpretive scope of the SRB assay,\nemphasize its role as a membrane fusion proxy, and now include the complementary\nRBC hemolysis assay as independent validation of the enhanced membrane-disruptive\ncapability of Au-LNPs (new Supplementary Figure S2a).\nWe have revised the manuscript as follows:\nTo enhance clarity, we have revised the entire “Enhanced pH-Responsive Membrane\nDisruption by Au-LNPs” subsection.\nUpdate Supplementary Figure S2a:\nFigure S2. Functional and structural evaluation of LNPs and Au-LNPs.\na. Hemolysis percentage of LNPs and Au-LNPs under different pH conditions.\nb. Exothermic interaction between Au-MC3 and mRNA measured by isothermal\ntitration calorimetry (ITC) (ΔQ=-46.74μJ).\nc. Dynamic light scattering analysis of mRNA and Au-MC3–mRNA complexes.\nd. Dose–response analysis of Au-LNPs using seven concentrations of Fluc-mRNA to\ndetermine the optimal dosage.\ne. Flow cytometric analysis of GFP expression in HeLa cells treated with varying\nconcentrations of LNPs and Au-LNPs.\nf. Flow cytometric analysis of GFP expression in 293 cells treated with varying\nconcentrations of LNPs and Au-LNPs.\ng. Chemiluminescence intensity of HeLa cells treated with LNPs and Au-LNPs.\nh. Chemiluminescence intensity of 293 cells treated with LNPs and Au-LNPs.\ni. Bioluminescence intensity of 4T1, EMT6, and JC cells treated with LNPs and Au-\nLNPs.\nj. Bioluminescence intensity of HeLa cells treated with Pfizer–BioNTech LNPs\nformulated with ALC-0135 and Au-LNPs.\nk. Bioluminescence intensity of HeLa cells treated with Moderna LNPs formulated\nwith SM-102 and Au-LNPs.\nl. Bioluminescence intensity of HeLa cells treated with SORT–Lung formulations of\nLNPs and Au-LNPs.\nReferences:\n1. Aliakbarinodehi, N., et al. Time-Resolved Inspection of Ionizable Lipid-\nFacilitated Lipid Nanoparticle Disintegration and Cargo Release at an Early\nEndosomal Membrane Mimic. Acs Nano 18, 22989-23000 (2024).\n2. Xu, X. & Xia, T. Recent Advances in Site-Specific Lipid Nanoparticles for\nmRNA Delivery. ACS nanoscience Au 3, 192-203 (2023).\n3. Chatterjee, S., Kon, E., Sharma, P. & Peer, D. Endosomal escape: A\nbottleneck for LNP- mediated therapeutics. Proceedings of the National\nAcademy of Sciences of the United States of America 121(2024).\n4. Colonna, W.J., Marti, M.E., Nyman, J.A., Green, C. & Glatz, C.E. Hemolysis\nas a Rapid Screening Technique for Assessing the Toxicity of Native Surfactin\nand a Genetically Engineered Derivative. Environmental Progress &\nSustainable Energy 36, 505-510 (2017).\n5. Dobrovoiskaia, M.A., et al. Method for analysis of nanoparticle hemolytic\nproperties in vitro. Nano Letters 8, 2180-2187 (2008).\n6. Luozhong, S., et al. Poly(carboxybetaine) lipids enhance mRNA therapeutics\nefficacy and reduce their immunogenicity. Nature Materials (2025).\n9.The speculation for the highest transfection efficiency of MC3-AuNP is not\nconvincing because the authors affirm “favorable zeta potential shift of Au-MC3\nunder acidic conditions”; actually, it is well known that MC3 switchs to positive zeta\npotential in acidic condition of the endosomes while the contribution of AuNP is not\nclear. Moreover, at line 218, the authors affirm that the Au-LNP reduces the\nendosomal acidification favoring RNA release, while the acid pH is known to be\nresponsible of RNA release when using LNP. This explanation is too superficial and\nnot suitable to justify the observed results.\nResponse:\nWe thank the reviewer for this perceptive comment, which highlights the need to\nmore clearly distinguish the intrinsic pH-dependent behavior of MC3 from the specific\ncontribution of the core, and to better explain how reduced endosomal acidification\nfavors release without contradicting the role of acid pH in lipid protonation.\nIt is indeed well established that MC3 becomes positively charged at endosomal\npH (pKa ~6.4), enabling protonation-driven membrane disruption. Our data confirm\nthis behavior but also show that Au-MC3 exhibits a sharper and more pronounced zeta\npotential shift under acidic conditions compared to free MC3 (+12 mV at pH 4.0 for\nAu-MC3 vs. +9 mV for MC3 alone; new Supplementary Figure S1k). We attribute this\nto the core acting as a scaffold that enforces more ordered surface alignment of MC3,\nincreasing effective charge density and minimizing neutralization by bound mRNA1-3.\nThis charge segregation amplifies the well-known protonation mechanism of MC3,\nenhancing lipid repulsion and promoting the formation of hexagonal (H_II) phases at\nthe endosomal membrane4-7. The consequence is higher transfection efficiency, as\nreflected by the threefold increase in luciferase expression observed for Au-MC3\ncompared to conventional LNPs (Figure 3a), consistent with recent reports of hybrid\nLNPs where a core material enhances ionizable lipid activity8.\nAt line 218, our statement that “Au-LNPs reduced endosomal acidification” refers\nnot to a loss of the protonation step, but to the prevention of excessive progression\ntoward late lysosomal pH (~4.5). Confocal colocalization with LysoTracker (Figure\n4a–c, Supplementary Figure S3e) showed that Au-LNPs decreased the proportion of\nmRNA trapped in highly acidified compartments. This suggests that Au-LNPs promote\nmembrane disruption and release during the early-to-intermediate acidic stage (pH\n~5.5–6.0), where protonation of MC3 is still effective, while avoiding trafficking into\nmore degradative late lysosomes. Thus, Au-LNPs balance the benefits of acid-triggered\nlipid activation with protection against cargo degradation, leading to greater\ncytoplasmic diffusion (100-fold increase, Figure 4d). This mechanism is consistent with\nrecent LNP studies showing that tuning endosomal acidification can improve delivery\nefficiency by favoring early release before lysosomal degradation. we performed\nadditional experiments examining canonical endosomal markers (new Figure 5.\nWestern blot, qPCR, and immunofluorescence analyses revealed that the expression of\nthe early endosome marker RAB5 was unchanged between Au-LNPs and LNPs groups,\nwhereas the late endosome/lysosome marker LAMP1 was significantly downregulated\nin Au-LNP–treated cells.\nRevisions Made to the Manuscript:\nRevised of Results (Line 148-153):\n“To validate the functional contribution of the Au core, we characterized the Zeta potential of Au-\nMC3 compared with free MC3 lipids across a range of pH values (Methods). Both exhibited\ncharacteristic pH-dependent charge shifts, but Au-MC3 showed a significantly larger change in\nsurface potential (Supplementary Figure. S1j), indicating that the gold core amplifies the\nprotonation-driven responsiveness of ionizable lipids.”\nRevised of Results (Line 283-298):\n“To further investigate whether Au-LNPs modulate endosomal maturation, we examined the\nexpression and localization of canonical endosomal markers9,10. Confocal immunofluorescence\nstaining revealed that LNP-treated cells exhibited strong colocalization of mRNA with the late\nendosomal/lysosomal marker LAMP1, whereas Au-LNP–treated cells showed markedly reduced\nLAMP1 signal, with mRNA distributed more diffusely in the cytoplasm (Figure. 5a). In contrast,\nno significant differences were observed in the early endosomal marker RAB5 between LNPs- and\nAu-LNPs–treated groups (Figure. 5b). Quantification confirmed that LAMP1 intensity was\nsignificantly reduced in Au-LNP–treated cells, while RAB5 intensity remained unchanged (Figure.\n5c).\nThese observations were corroborated by Western blot and qPCR analyses. LAMP1 protein\nand mRNA levels were consistently decreased following Au-LNP treatment at both 4 and 6 h,\nwhereas RAB5 expression showed no significant differences compared with conventional LNPs\n(Fig. 5d–e). Together, these results indicate that Au-LNPs selectively interfere with late endosomal\nmaturation without altering early endosome formation. By attenuating progression to LAMP1\ndegradative compartments, Au-LNPs create a temporal window that favors endosomal escape of\nmRNA at the early-to-late transition stage, consistent with the enhanced cytosolic release observed\nin our colocalization and TEM studies.”\nUpdate Supplementary Figure S1: Added new zeta potential curves (Supplementary\nFigure S1k) comparing MC3 vs. Au-MC3 across finer pH intervals, confirming the\nsharper pH response of Au-MC3.\nFigure S1. Characterization of AuNP modification and Au-LNP assembly.\na. FTIR spectra of IC-AuNPs blue, Au-PEG red, and bare AuNPs black.\nb. 1H NMR spectra of SH-PEG blue, Au-PEG purple, DLin-MC3-DMA red, and Au-\nMC3 black.\nc. Agarose gel electrophoresis images of IC-AuNPs and mRNA at different mass ratios.\nd. TEM analysis of Au-LNPs.\ne. Cryo-EM image of Au-LNPs.\nf. TEM image of conventional LNPs, with cryo-EM image shown in the inset at the top\nright.\ng. Photographs of phase-transfer results of Au-PEG with three ionizable lipids SM-102\nA, DLin-MC3-DMA B, and DOTAP C.\nh. TEM images of Au-LNPs of ALC-0135 and Au-LNPs of DOTAP.\ni. Molecular dynamics simulation snapshots showing a cubic box with edge length\n11.047 nm containing 9 chains of 22-nt polyA RNA and 20 chains of SM-102 lipids,\nsimulated under traditional LNPs and Au-LNPs conditions.\nj. Standard curve for LC–MS quantification of SM-102 and measured SM-102 content\nin Au-LNPs and conventional LNPs from three independent experiments.\nk. Zeta potential profiles of free MC3 molecules versus Au-MC3 nanoparticles under\nidentical pH conditions in 50 mM citrate buffer.\nl. UV–Vis absorption spectra of AuNPs of different diameters 13, 18, 25, and 50 nm.\nm. Firefly luciferase FLuc expression in HeLa cells treated for 24 h with 0.5 μg/mL\nmRNA delivered by Au-LNPs containing gold cores of different diameters 13, 18, 25,\n50 nm; conventional LNPs were used as a positive control.\nn. DLS size distributions of Au-LNPs formulated with 18, 25, and 50 nm Au cores\nUpdate Figure 5\nFigure 5. Mechanistic study of Au-LNP–mediated endosomal escape via\ninhibition of endosomal maturation.\na. b. Confocal fluorescence images of 293T cells treated with 0.5 or 1 µg/mL LNPs\nor Au-LNPs. Nuclei (blue), mRNA (red), and late endosome/lysosome marker\nLAMP1 (green, a) or early endosome marker RAB5 (green, b) were immunostained.\nScale bars, 10 µm.\nc. Quantification of LAMP1 and RAB5 mean fluorescence intensity from images in a\nand b.\nd. Western blot analysis and densitometric quantification of LAMP1 and RAB5\nprotein expression after treatment with LNPs or Au-LNPs for 4 and 6 h.\ne. qPCR analysis of LAMP1 and RAB5 mRNA levels in 293T cells after treatment\nwith LNPs or Au-LNPs for 4 and 6 h.\nStatistical significance was assessed using one-way ANOVA (for >2 groups) or two-\ntailed Student’s t-test (for 2 groups). **P < 0.01, ***P < 0.001, ****P < 0.0001.\nReferences:\n1. Aliakbarinodehi, N., et al. Time-Resolved Inspection of Ionizable Lipid-\nFacilitated Lipid Nanoparticle Disintegration and Cargo Release at an Early\nEndosomal Membrane Mimic. Acs Nano 18, 22989-23000 (2024).\n2. Johansson, J.M., et al. Cellular and biophysical barriers to lipid nanoparticle\nmediated delivery of RNA to the cytosol. Nature Communications 16(2025).\n3. Mrksich, K., Padilla, M.S. & Mitchell, M.J. Breaking the final barrier:\nEvolution of cationic and ionizable lipid structure in lipid nanoparticles to\nescape the endosome. Advanced Drug Delivery Reviews 214(2024).\n4. Cheung, T.H., Fuchs, A. & Shoichet, M.S. Acid-Responsive Polymer\nAdditives Increase RNA Transfection from Lipid Nanoparticles. Advanced\nFunctional Materials 35(2025).\n5. Ponomareva, N., et al. Swelling, Rupture and Endosomal Escape of Biological\nNanoparticles Per Se and Those Fused with Liposomes in Acidic\nEnvironment. Pharmaceutics 16(2024).\n6. Semple, S.C., et al. Rational design of cationic lipids for siRNA delivery.\nNature Biotechnology 28, 172-U118 (2010).\n7. Liu, H., et al. Beyond the Endosomal Bottleneck: Understanding the\nEfficiency of mRNA/LNP Delivery. Advanced Functional Materials\n34(2024).\n8. Cheng, M.H.Y., et al. Liposomal lipid nanoparticles for extrahepatic delivery\nof mRNA. Nature Communications 16, 4135 (2025).\n9. Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature\nReviews Molecular Cell Biology 2, 107-117 (2001).\n10. Saftig, P. & Klumperman, J. Lysosome biogenesis and lysosomal membrane\nproteins: trafficking meets function. Nature Reviews Molecular Cell Biology\n10, 623-635 (2009).\nReviewer #3 (Remarks to the Author):\nThe authors have provided data and explanations to sufficiently answer my questions and comments for the initial review.\nI recommend accepting the paper for publication."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In the revised version of the NCOMMS-25-12523A manuscript on \"Engineered Internal Architecture of Core-Shell Lipid Nanoparticles Promotes Efficient mRNA Endosomal Release\", the authors addressed all the aspects I raised and quantified their data on endosomal escape, adding expression and localization data of two canonical endosomal markers to quantify the endosomal maturation. They also added cryoEM data which confirms the morphological differences between the conventional LNPs and Au-LNPs. I have reservations about the statistical sampling of both TEM and cryoEM images (100 and 10 respectively), which I find extremely limited.\nSome aspects I would like to raise regarding this revision:\n- interpreting the SRB release data in figure 2g, the authors insisted that the Au-LNPs have a pronounced biphasic response with a peak at pH=5.5 while LNPs show a more monotonic decline. The trends are very similar with absolute values 10-20% hihgher (and not 20-30% higher as the authors claim) up to pH=6.5 above which the disruption is identical.\n- in Figure S1, the caption mentions IC-AuNPs while figure S1a,b and c legends all show Au-MC3 which I assume is MC3-AuNP. Could the authors be consistent?\n- I still find the methods section not detailed enough to repeat the experiments.", "reviewer_2": "Although the authors made significant efforts to improve the manuscript, some concerns remain. Actually, the authors clarified that the inclusion of AuNP into the LNP represents an advantage from a methodological point of view, providing an useful tool to study endosomal escape. The same authors specify in the rebuttal letter to the Reviewer 2 that gold have no superior biocompatibility than lipids. This confirms the concerns of the Reviewer on the lack of a robust rationale for including AuNP in the LNP for future therapeutical application for RNA delivery. The accumulation of AuNP into the body should be an issue to address, hampering the use of Au-LNP for systemic administration. In addition, also if the authors report that AuNP core are used as proof-of-concept, the concept of using pH sensitive core sourranded by lipid layer is not novel and already reported with other biomaterials, e.g. calcium/phosphoate (doi: 10.1158/0008-5472.can-21-2269; doi: 10.1016/j.jconrel.2024.11.039). Thus, any advantage of using AuNP is clearly showed compared to previously used core biomaterials.\nIn the emended version of the manuscript, the authors significantly improved the description of the Material and Methods making more clear the preparation protocol of the Au-LNP. Thus, it is now more clear that the MC3 lipid is firstly adsorbed on the Au-Np and then followed by interaction with the lipid mix containing another ionizable lipid SM 102 or the cationic lipid DOTAP. While the effect on the endosomal escape has been ascribed to the AuNP, the contribution of combining the two ionizable or ionizable/cationic lipids is not taken into account.\nFinally, some answers to the Reviewer concerns are not convincing and too speculative. Actually, TEM shown in this manuscript cannot provide a clear visualization of the AuNP associated to the lipid shell, but only of the AuNP. In line with this, these experiments cannot be used to describe the integrity of core/shell NP and the pH-dependent endosomal escape. Moreover, the authors try to strength the significance of SRB assay by associating an hemolytic assay in the emended version of the manuscript. However, the endosomal escape and hemolysis are two different and complex biological phenome and the latter cannot used to support data of the former.", "reviewer_3": "The authors have provided data and explanations to sufficiently answer my questions and comments for the initial review.\nI recommend accepting the paper for publication."}, "author_response": "Response to Reviewers for Manuscript NCOMMS-25-12523A\nWe wish to express our sincere gratitude to the editor and the reviewers for their time and the constructive comments concerning our work. We have carefully reviewed the reviewers’ comments and have revised the manuscript accordingly. All changes made in the revised manuscript have been highlighted in yellow for your convenience. Below is a point-by-point response to the reviewers’ comments\nReviewer #1 (Remarks to the Author):\nIn the revised version of the NCOMMS-25-12523A manuscript on \"Engineered Internal Architecture of Core-Shell Lipid Nanoparticles Promotes Efficient mRNA Endosomal Release\", the authors addressed all the aspects I raised and quantified their data on endosomal escape, adding expression and localization data of two canonical endosomal markers to quantify the endosomal maturation. They also added cryoEM data which confirms the morphological differences between the conventional LNPs and Au-LNPs.\nGeneral Response:\nWe sincerely appreciate the reviewer’s positive assessment of our revised manuscript, particularly the acknowledgement of our efforts in quantifying endosomal escape and characterizing endosomal maturation markers.\nI have reservations about the statistical sampling of both TEM and cryoEM images (100 and 10 respectively), which I find extremely limited.\nResponse:\nWe recognize that the initial sample sizes were limited for drawing robust statistical conclusions. To strictly address this concern and ensure high data reliability, we have significantly expanded our statistical analysis. We increased the sample size to TEM Analysis: 42.37 ± 0.25 nm (n=200) and the size to Cryo-EM Analysis: 69.32 ± 0.34 nm (n=200). Importantly, these values show no significant deviation from our preliminary data.\nRevised Text (Line 116-120):\nWhen assembled into Au-LNPs, these MC3-coated cores yielded nanoparticles with an average dry-state diameter of 42.37 ± 0.25 nm (Figure 2d, Supplementary Figure S1d). In contrast, dynamic light scattering (DLS) indicated a hydrated hydrodynamic size of ~70.2 nm (Figure 2e, Methods), while preliminary cryo-EM measurements confirmed a hydrated size of 69.32 ± 0.34 nm with intact spherical morphology (Supplementary Figure S1e, Methods). \nFigure Update:\nFigure S1d has been updated to reflect these new statistical data.\nFigure S1. Characterization of AuNP modification and Au-LNP assembly.\na. FTIR spectra of Au-MC3 blue, Au-PEG red, and bare AuNPs black.\nb. 1H NMR spectra of SH-PEG blue, Au-PEG purple, DLin-MC3-DMA red, and Au-MC3 black.\nc. Agarose gel electrophoresis images of Au-MC3 and mRNA at different mass ratios.\nd. TEM analysis of Au-LNPs.\ne. Cryo-EM image of Au-LNPs.\nf. TEM image of conventional LNPs, with cryo-EM image shown in the inset at the top right.\ng. Photographs of phase-transfer results of Au-PEG with three ionizable lipids SM-102 A, DLin-MC3-DMA B, and DOTAP C.\nh. TEM images of Au-LNPs of ALC-0135 and Au-LNPs of DOTAP.\ni. Molecular dynamics simulation snapshots showing a cubic box with edge length 11.047 nm containing 9 chains of 22-nt polyA RNA and 20 chains of SM-102 lipids, simulated under traditional LNPs and Au-LNPs conditions.\nj. Standard curve for LC–MS quantification of SM-102 and measured SM-102 content in Au-LNPs and conventional LNPs from three independent experiments.\nk. Hek 293 cells were transfected with varying concentrations of LNPs, MC3-LNPs and Au-LNPs encapsulating GFP- mRNA (0.5μg/mL), then harvested 12 hours later. The proportion of GFP-positive cells was analyzed and quantified via flow cytometry.\nl. Zeta potential profiles of free MC3 molecules versus Au-MC3 nanoparticles under identical pH conditions in 50 mM citrate buffer. (error bars: SD)\nm. UV–Vis absorption spectra of AuNPs of different diameters 13, 18, 25, and 50 nm.\nn. Firefly luciferase FLuc expression in HeLa cells treated for 24 h with 0.5 μg/mL mRNA delivered by Au-LNPs containing gold cores of different diameters 13, 18, 25, 50 nm; conventional LNPs were used as a positive control. (error bars: SD)\no. DLS size distributions of Au-LNPs formulated with 18, 25, and 50 nm Au cores.\nSome aspects I would like to raise regarding this revision:\n1- interpreting the SRB release data in figure 2g, the authors insisted that the Au-LNPs have a pronounced biphasic response with a peak at pH=5.5 while LNPs show a more monotonic decline. The trends are very similar with absolute values 10-20% higher (and not 20-30% higher as the authors claim) up to pH=6.5 above which the disruption is identical.\nResponse:\nWe sincerely thank the reviewer for the careful evaluation of the revised SRB-release data.\nTo clarify our earlier reasoning, we would like to briefly outline the definitions used in our internal analysis. A peak denotes a local maximum, where the signal increases and then decreases, whereas a plateau refers to a region with an approximately zero slope across adjacent measurements. Using these definitions, our internal evaluation indicated a local maximum for Au-LNPs at pH 5.5, whereas conventional LNPs displayed a nearly flat slope between pH 5.0 and 5.5, with no statistically significant difference between these two points. Internally, we also noted that the membrane-disruption rate of Au-LNPs at pH 5.5 was more than double that of LNPs, which contributed to our impression that the trends were not entirely identical.\nHowever, to fully reflect the reviewer’s recommendation, ensure clarity, and avoid emphasizing distinctions that are not central to the paper, we have removed the word “peak” entirely and now describe the SRB-release profile in a conservative and unambiguous manner that remains fully consistent with the data.\n2- in Figure S1, the caption mentions IC-AuNPs while figure S1a, b and c legends all show Au-MC3 which I assume is MC3-AuNP. Could the authors be consistent?\nResponse:\nWe are very grateful to the reviewer for kindly pointing out the inconsistency between our manuscript's title and the figure content. We have now completed the revision of the relevant figure captions and meticulously checked the entire manuscript to ensure consistency between all figures and their descriptions. Thank you for your valuable suggestion, which has significantly helped us improve the quality and clarity of our submission.\n3- I still find the methods section not detailed enough to repeat the experiments.\nResponse:\nWe sincerely thank the reviewer for the constructive suggestions regarding the Methods section. Following your guidance, we have enriched this section by adding more detailed descriptions. We sincerely hope that the updated Methods section now provides sufficient detail to ensure the reproducibility of the experiments.\nReviewer #2 (Remarks to the Author):\nAlthough the authors made significant efforts to improve the manuscript, some concerns remain. Actually, the authors clarified that the inclusion of AuNP into the LNP represents an advantage from a methodological point of view, providing an useful tool to study endosomal escape. The same authors specify in the rebuttal letter to the Reviewer 2 that gold have no superior biocompatibility than lipids. This confirms the concerns of the Reviewer on the lack of a robust rationale for including AuNP in the LNP for future therapeutical application for RNA delivery. The accumulation of AuNP into the body should be an issue to address, hampering the use of Au-LNP for systemic administration.\nResponse:\nWe thank the reviewer for acknowledging our revision efforts and for recognizing the value of Au-LNPs for mechanistic endosomal escape studies. We understand the concern regarding the safety of AuNPs for therapeutic RNA delivery. However, this work is designed as a proof-of-concept study focused on establishing a design principle, rather than developing a clinical formulation. As noted in the Discussion, the size-tunable nature of AuNPs provides a uniquely precise and controllable model system that allows us to dissect how internal architecture affects LNP behavior (Supplementary Figure S1m–o). This rationale is central to our study.\nTo address safety considerations within the scope of this work, we quantified gold content in major organs and blood using ICP-MS (Supplementary Figure S7). Au-LNPs displayed a typical nanoparticle biodistribution profile with transient liver accumulation, followed by rapid clearance. By 48 h post-injection, gold levels had returned to baseline and were indistinguishable from PBS controls.\nAlthough the number of AuNP clinical trials remains modest, existing human data (Table R1) consistently report that systemic AuNP administration has not produced severe adverse events or notable toxicity, demonstrating good short-term tolerance1. These results, together with extensive preclinical evidence, support the suitability of AuNPs as a model carrier for mechanistic studies like ours.\nWe also wish to clarify that an in-depth clinical evaluation is beyond the intended scope of this manuscript. Nonetheless, we have provided experimental data, literature evidence, and clinical results sufficient to show that AuNPs do not exhibit detectable short-term toxicity under the conditions relevant to our mechanistic investigation.\nClinical Trial ID (NCT) AuNP Type Application/Condition Safety Finding\nNCT00356980 TNF-bound colloidal gold Advanced Solid Tumors Exhibited some side effects, but no highly toxic effects associated with AuNPs were observed.\nNCT01270139 Silica-gold iron-bearing Atherosclerosis Evaluation of feasibility and safety confirmed; Long-term follow-up showed improved long-term safety.\nNCT02755870 CNM-Au8 (Gold Nanocrystals) Healthy Volunteers No serious side effects were reported; formulation was tolerated well.\nNCT02837094 C19-A3 GNP, Proinsulin peptide Type 1 Diabetes (Immunotherapy) No serious side effects were reported.\nNCT03020017 NU0129 (SNAs - Spherical Nucleic Acids) Recurrent Glioblastoma Safety assessment demonstrated no treatment-related Grade 4 or 5 toxicity.\nTable R1. Summary of gold nanoparticle clinical trials.\nRevised Line 435-442:\nMetabolism in vivo was assessed using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). At 48 hours post-administration, gold levels in all major organs had substantially decreased and returned to baseline, showing no significant difference from the PBS control group (Supplementary Figure S7). These data indicate that, under the conditions tested, the incorporation of gold nanoparticles does not introduce detectable acute toxicity, supporting the validity of their incorporation as a model component within the Au-LNPs system. We note, however, that comprehensive long-term evaluation will be necessary to fully assess the potential risks of gold nanoparticles in clinical applications.\nFigure S7. Quantitative analysis of Au concentration in major organs of mice at different time points post single injection of Au-LNPs by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).\nReferences:\n1. Ozcicek, I. A critical review of in vitro and in vivo biomedical applications of gold nanoparticles: From toxicology to cancer therapy. Biomedicine & Pharmacotherapy 190(2025).\nIn addition, also if the authors report that AuNP core are used as proof-of-concept, the concept of using pH sensitive core sourranded by lipid layer is not novel and already reported with other biomaterials, e.g. calcium/phosphoate (doi: 10.1158/0008-5472.can-21-2269; doi: 10.1016/j.jconrel.2024.11.039). Thus, any advantage of using AuNP is clearly showed compared to previously used core biomaterials.\nResponse:\nWe thank the reviewer for highlighting prior pH-responsive nanoparticle systems, including CaP-based formulations. While CaP nanoparticles are indeed pH-sensitive, their response arises from general pH-driven chemical changes and does not generate a controlled or tunable surface-charge transition. By contrast, a key innovation of our work is the introduction of an engineered ionizable core whose zeta potential transitions continuously and predictably within the endosomal pH range (pH 6.5–5.5). This design provides two major advantages:\n1. Programmable ionization behavior – the magnitude and pH threshold of the charge transition can be precisely tuned through core size and interfacial chemistry.\n2. Quantitative structure–function control – our platform uniquely enables systematic modulation of internal architecture (core diameter, interfacial composition, and lipid-shell curvature) and allows us to directly correlate these parameters with measured changes in endosomal escape efficiency and mRNA expression (Figures S1m–n and 3a). This level of mechanistic control and structure–function mapping is not achievable with CaP-based systems.\nTaken together, these features highlight the distinct design logic and mechanistic advantages of our ionizable Au-core LNP platform relative to previously reported pH-responsive systems.\nIn the emended version of the manuscript, the authors significantly improved the description of the Material and Methods making more clear the preparation protocol of the Au-LNP. Thus, it is now more clear that the MC3 lipid is firstly adsorbed on the Au-Np and then followed by interaction with the lipid mix containing another ionizable lipid SM 102 or the cationic lipid DOTAP. While the effect on the endosomal escape has been ascribed to the AuNP, the contribution of combining the two ionizable or ionizable/cationic lipids is not taken into account.\nResponse:\nWe thank the reviewer for raising this important question. We agree that combining two ionizable lipids or an ionizable/cationic lipid pair could, in principle, influence endosomal escape, and we performed additional controls to directly evaluate this possibility. LC-MS analysis shows that the SM102 content in conventional LNPs and Au-LNPs is nearly identical (Supplementary Figure S1j), indicating that the outer lipid composition is not the source of enhanced performance.\nTo further assess whether the mixed lipid combination itself could improve endosomal escape, we prepared control LNPs containing free MC3 at the same amount present on the Au-MC3 surface, with MC3 levels confirmed by quantitative NMR1-3. These MC3-LNPs showed no increase in GFP expression compared with standard LNPs (Supplementary Figure S1k), demonstrating that the two ionizable lipids mixture alone does not reproduce the effect observed with Au-LNPs.\nTaken together, these results show that the enhanced endosomal escape does not arise from the presence of multiple ionizable or cationic lipids but from the Au core itself. As illustrated in Figure 1, the Au core creates a structured internal architecture that concentrates MC3 on its surface and spatially separates it from the encapsulated mRNA—an arrangement that cannot be achieved in uniformly mixed LNPs. This Au-templated redistribution of ionizable lipid, rather than the lipid identity or combination, is the key mechanistic driver underlying the improved performance.\nAdd Line 148-153:\nTo determine whether the effect could be attributed simply to combining two ionizable lipids, we generated MC3–SM102 LNPs in which the MC3 content was matched to that of Au-MC3 using quantitative NMR. This mixed-lipid formulation showed no improvement over conventional LNPs, indicating that enhanced performance requires the Au-templated architecture rather than the presence of multiple ionizable lipids (Supplementary Figure S1k)1-3.\nFigure Update:\nWe have updated the Supplementary Figure S1k.\nFigure S1. Characterization of AuNP modification and Au-LNP assembly.\na. FTIR spectra of Au-MC3 blue, Au-PEG red, and bare AuNPs black.\nb. 1H NMR spectra of SH-PEG blue, Au-PEG purple, DLin-MC3-DMA red, and Au-MC3 black.\nc. Agarose gel electrophoresis images of Au-MC3 and mRNA at different mass ratios.\nd. TEM analysis of Au-LNPs.\ne. Cryo-EM image of Au-LNPs.\nf. TEM image of conventional LNPs, with cryo-EM image shown in the inset at the top right.\ng. Photographs of phase-transfer results of Au-PEG with three ionizable lipids SM-102 A, DLin-MC3-DMA B, and DOTAP C.\nh. TEM images of Au-LNPs of ALC-0135 and Au-LNPs of DOTAP.\ni. Molecular dynamics simulation snapshots showing a cubic box with edge length 11.047 nm containing 9 chains of 22-nt polyA RNA and 20 chains of SM-102 lipids, simulated under traditional LNPs and Au-LNPs conditions.\nj. Standard curve for LC–MS quantification of SM-102 and measured SM-102 content in Au-LNPs and conventional LNPs from three independent experiments.\nk. Hek 293 cells were transfected with varying concentrations of LNPs, MC3-LNPs and Au-LNPs encapsulating GFP- mRNA (0.5μg/mL), then harvested 12 hours later. The proportion of GFP-positive cells was analyzed and quantified via flow cytometry.\nl. Zeta potential profiles of free MC3 molecules versus Au-MC3 nanoparticles under identical pH conditions in 50 mM citrate buffer. (error bars: SD)\nm. UV–Vis absorption spectra of AuNPs of different diameters 13, 18, 25, and 50 nm.\nn. Firefly luciferase FLuc expression in HeLa cells treated for 24 h with 0.5 μg/mL mRNA delivered by Au-LNPs containing gold cores of different diameters 13, 18, 25, 50 nm; conventional LNPs were used as a positive control. (error bars: SD)\no. DLS size distributions of Au-LNPs formulated with 18, 25, and 50 nm Au cores\nReferences:\n1. Webster, G.K. & Kumar, S. Expanding the Analytical Toolbox: Pharmaceutical Application of Quantitative NMR. Analytical Chemistry 86, 11474-11480 (2014).\n2. Giraudeau, P. Quantitative NMR spectroscopy of complex mixtures. Chemical Communications 59, 6627-6642 (2023).\n3. Mulder, F.A.A., Tenori, L. & Luchinat, C. Fast and Quantitative NMR Metabolite Analysis Afforded by a Paramagnetic Co-Solute. Angewandte Chemie-International Edition 58, 15283-15286 (2019).\nFinally, some answers to the Reviewer concerns are not convincing and too speculative. Actually, TEM shown in this manuscript cannot provide a clear visualization of the AuNP associated to the lipid shell, but only of the AuNP. In line with this, these experiments cannot be used to describe the integrity of core/shell NP and the pH-dependent endosomal escape.\nResponse:\nWe sincerely thank the reviewer for raising this important point. We fully agree that TEM alone cannot unambiguously resolve the lipid shell surrounding AuNPs and therefore cannot be used as a standalone method to describe the integrity of the core–shell architecture. For this reason, we did not rely solely on TEM for structural interpretation.\nTo more rigorously characterize the core–shell structure, we performed Small-Angle X-ray Scattering (SAXS), which provides ensemble-averaged information and is widely used for probing nanoscale layering and interfacial organization1-3. SAXS analysis of Au-PEG and Au-MC3 (Figure R1a) showed a clear and reproducible size increase from 16.1 ± 2.7 nm (Au-PEG) to 17.2 ± 3.4 nm (Au-MC3), consistent with the formation of a molecularly thin MC3 layer at the Au surface. This size shift aligns with the redshift observed in UV-vis spectra attributed to the LSPR effect and with independent evidence from FTIR/NMR and zeta potential measurements (Figure 2, Supplementary Figure S1, Figure R1)4-6. Together, these orthogonal data sets—SAXS, spectroscopy, and surface charge—provide convergent and statistically robust support for the presence of a lipid shell surrounding the Au core.\nRegarding endosomal escape, we confirm that we do not draw mechanistic conclusions from TEM. Our conclusions are instead based on functional assays. (1) CLSM quantification to measure the cytosolic diffusion area and escape efficiency of Cy5-mRNA (Figures 4a–d, Supplementary Figure S3a–d); (2) LysoTracker staining to assess endosomal acidification Supplementary Figure S3e); (3) Immunofluorescence staining of canonical markers (LAMP1/RAB5) to visualize their spatial distribution and colocalization with mRNA (Figure 5a-c); (4) Western blot and qPCR analyses to confirm the downregulation of late endosomal markers (Figure 5d-e); and (5) the Galectin-8 recruitment assay to provide direct evidence of endosomal membrane rupture (Supplementary Figure S3g). that directly quantify mRNA delivery performance under endosomal conditions. Thus, while TEM offers qualitative visualization of the Au core, the mechanistic insights in the manuscript rely on ensemble-averaged physicochemical characterization and functional readouts rather than on TEM morphology.\nCollectively, these data substantiate the integrity of the Au-templated core–shell structure and its functional impact on pH-mediated endosomal escape, without over-interpreting the TEM images.\nFigure R1 a. SAXS diagram of Au-PEG and Au-MC3 and corresponding size. b. TEM of Au-PEG. c. UV–Vis absorption spectra of Au-PEG and Au-MC3.\nReferences:\n1. Balmer, J.A., et al. Time-Resolved Small-Angle X-ray Scattering Studies of Polymer−Silica Nanocomposite Particles: Initial Formation and Subsequent Silica Redistribution. Journal of the American Chemical Society 133, 826-837 (2010).\n2. Fleury, B., et al. Gold Nanoparticle Internal Structure and Symmetry Probed by Unified Small-Angle X-ray Scattering and X-ray Diffraction Coupled with Molecular Dynamics Analysis. Nano Letters 15, 6088-6094 (2015).\n3. Li, T., Senesi, A.J. & Lee, B. Small Angle X-ray Scattering for Nanoparticle Research. Chemical Reviews 116, 11128-11180 (2016).\n4. Kim, D.-K., et al. Label-Free DNA Biosensor Based on Localized Surface Plasmon Resonance Coupled with Interferometry. Analytical Chemistry 79, 1855-1864 (2007).\n5. Nath, N. & Chilkoti, A. Label-Free Biosensing by Surface Plasmon Resonance of Nanoparticles on Glass: Optimization of Nanoparticle Size. Analytical Chemistry 76, 5370-5378 (2004).\n6. Soliman, M.G., Pelaz, B., Parak, W.J. & del Pino, P. Phase Transfer and Polymer Coating Methods toward Improving the Stability of Metallic Nanoparticles for Biological Applications. Chemistry of Materials 27, 990-997 (2015).\nMoreover, the authors try to strength the significance of SRB assay by associating an hemolytic assay in the emended version of the manuscript. However, the endosomal escape and hemolysis are two different and complex biological phenome and the latter cannot used to support data of the former.\nResponse:\nWe thank the reviewer for this comment and agree that hemolysis cannot be used as a direct indicator of endosomal escape. In our study, hemolysis was applied only as a supporting assay to assess pH-dependent membrane-disruptive activity, while the SRB-loaded liposome leakage assay serves as the primary measurement of pH-triggered fusogenicity.\nTo eliminate ambiguity and provide direct cell-level evidence of endosomal disruption, we additionally performed a Galectin-8 (Gal8) recruitment assay, a widely accepted assay for quantifying endosomal membrane rupture1-3. Gal8-GFP puncta were significantly increased in cells treated with Au-LNPs compared with conventional LNPs (Supplementary Figure S3g), demonstrating enhanced endosomal escape. These results are fully consistent with our Cy5-mRNA cytosolic delivery and endosomal marker analyses (Figures 4–5).\nThus, while hemolysis is used only as an orthogonal fusogenicity assay, the SRB leakage assay and the Gal8 recruitment assay together provide robust and direct mechanistic evidence that Au-LNPs possess superior endosomal escape capability.\nAdded Text (Line 301-304):\nUsing the Galectin-8 (Gal8) recruitment assay which measures endosomal integrity by detecting the binding of cytosolic Gal8 to host glycans exposed upon rupture, we observed significantly increased Gal8-GFP puncta in Au-LNP-treated cells (Supplementary Figure S3g), confirming enhanced endosomal escape1-3.\nAdded Supplementary Information Text (Line 41-48)\nGalectin-8 Recruitment Assay\nHek 293 cells were transiently transfected with a plasmid encoding Galectin-8-GFP using Lipofectamine™ 3000 (Thermo Fisher Scientific), following the manufacturer’s instructions. Twenty-four hours post-transfection, cells were incubated with Au-LNPs or conventional LNPs for 4h (mRNA: 0.5 μg/mL). Cells were subsequently fixed with 4% paraformaldehyde and stained with Hochest33342. The formation of Gal8-GFP puncta, indicative of endosomal rupture, was visualized via confocal microscopy and quantified using Fiji.\nFigure Update:\nWe have included the results from the Gal8-GFP recruitment assay in Supplementary Figure S3g.\nFigure S3. Confocal and ultrastructural analysis of endosomal escape.\na. CLSM images of HeLa cells treated with 0.25 μg/mL Cy5-mRNA for 4 h.\nb. CLSM images of HeLa cells treated with 0.5 μg/mL Cy5-mRNA for 4 h.\nc. CLSM images of HeLa cells treated with 1 μg/mL Cy5-mRNA for 4 h.\nd. Quantification of endosomal escape rate in HeLa cells treated with 0.25, 0.5, and 1 μg/mL Cy5-mRNA for 4 h or 6 h. (error bars: SD)\ne. Quantification of overlap between lysosome signal and mRNA signal. (error bars: SD)\nf. TEM image of untreated HeLa cell.\ng. CLSM images of Gal8-GFP expression HEK293 cells treated by PBS, LNPs, and Au-LNPs (mRNA: 0.5 μg/mL, 4h) and endosomal disruption level quantified by the intensity of Gal8-GFP per cell.\nReferences:\n1. Kilchrist, K.V., et al. Gal8 Visualization of Endosome Disruption Predicts Carrier-Mediated Biologic Drug Intracellular Bioavailability. ACS Nano (2019).\n2. Rui, Y., et al. Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing. Science Advances 5, eaay3255 (2019).\n3. Golba, B., et al. Combinatorial Synthesis and Evaluation of Trialkyl Galloyl Amidoamine Ionizable Lipids for mRNA Formulation. Journal of the American Chemical Society 147, 26934-26943 (2025).\nReviewer #3\nWe thank the reviewer for reviewing our manuscript.\nManuscript ID：NCOMMS-25-12523B\nTitle: Engineered Internal Architecture of Core-Shell Lipid Nanoparticles Promotes Efficient mRNA Endosomal Release\nWe would like to express our sincere gratitude to the editors and reviewers for their time and effort in reviewing our manuscript. We are delighted to learn that the reviewers are satisfied with our revisions. Their constructive comments and suggestions have significantly improved the quality and rigor of this work.\nReviewer #1\nWe sincerely thank the reviewer for their positive evaluation and for recommending our manuscript for publication.\nReviewer #2\nWe thank the reviewer for their comprehensive assessment and for endorsing the publication of our work."}]

Decision letter