Phase-Separated Peptide Coacervates as Delivery Vehicles for mRNA Vaccines

Phase-Separated Peptide Coacervates as Delivery Vehicles for mRNA Vaccines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Phase-Separated Peptide Coacervates as Delivery Vehicles for mRNA Vaccines Jiang Xia, Zhiyi Xu, Chaiyaporn Kuwentrai, Yaxin Hu, Renhao Li, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7559034/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Nucleic acid therapeutics, such as mRNA vaccines, require effective delivery vehicles; yet widely used systems like lipid nanoparticles or cationic polymers are often associated with adverse responses. Developing low-molecular-weight compounds as delivery vehicles for nucleic acids is promising but remains challenging. Here, we identify a disulfide-bond-linked, six-residue peptide, WWKssKWW (WWK), which forms coacervates through liquid-liquid phase separation (LLPS) in aqueous solutions. The WWK coacervates encapsulate diverse nucleic acids—including single-stranded DNA, siRNA, mRNA, and plasmids—and deliver them into cells with efficiencies equivalent to or exceeding conventional agents (lipofectamine, InstantFECT, PEI, and lipid nanoparticles). Subcutaneous and intramuscular injection of mRNA-loaded WWK coacervates in mice results in robust protein expression and a favorable safety profile. Importantly, when human PBMCs were transfected with various mRNA formulations, WWK/mRNA induced significantly lower levels of the inflammatory cytokine IL-1β compared to liposomes and lipid nanoparticles, similar to naked mRNA, suggesting reduced toxicity risk. WWK coacervates also enabled efficient intramuscular delivery of SARS-CoV-2 spike mRNA, producing spike-specific cellular responses comparable to the commercial Comirnaty vaccine. Furthermore, in a B16-OVA cancer model, WWK/OVA mRNA coacervates exhibited potent anti-tumor effects and significantly enhanced tumor-free survival. Overall, our findings demonstrate that peptide coacervates are promising vehicles for mRNA delivery, offering improved safety and efficacy for gene therapy and vaccine development. Biological sciences/Biotechnology/Nanobiotechnology/Nanostructures Physical sciences/Chemistry/Supramolecular chemistry Liquid-liquid phase separation peptide coacervates mRNA therapeutics transfection cancer immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 SIGNIFICANCE STATEMENT A redox-responsive peptide made up of six residues forms coacervates, serving as an innovative transfection agent for nucleic acid therapeutics, with efficacy comparable to or exceeding that of lipofectamine, InstantFECT cationic liposome, and polyethylenimine. To our knowledge, with a molecular weight of 1152 Daltons, this six-residue peptide is the smallest transfection agent. This study also presents the first in vivo investigation of coacervate-mediated mRNA vaccine delivery. Introduction In recent decades, nucleic acids have emerged as highly appealing treatment modalities. DNA, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and messenger RNAs (mRNAs) have been widely utilized in the therapeutic landscape for treating various diseases. [1] Nucleic acid medications have the unique ability to modulate gene expression, offering precise treatment for a diverse array of conditions at the genetic level—a capability often beyond the reach of traditional small-molecule and protein drugs. [2] In 1998, the FDA approved the first antisense oligonucleotide drug for treating cytomegalovirus retinitis in AIDS patients. [3] Despite its discontinuation due to reduced demand, this ground-breaking medication spurred the advancement of different therapeutic nucleic acids. [4-6] In 2018, patisiran became the inaugural siRNA therapy available on the market, targeting hereditary transthyretin amyloidosis (hATTR) and employing lipid nanoparticles (LNPs) for hepatic delivery. [7] By 2024, over 23 nucleic acid drugs had received global approval, with 55% designed for rare diseases and the remaining aimed at chronic conditions. [8] Within the array of nucleic acid treatments, mRNA therapy stands out as a particularly promising avenue. [9, 10] mRNA, a form of RNA, serves as a messenger transporting genetic instructions from DNA and orchestrating protein synthesis via translation. Upon entry into the patient's cells, mRNA stimulates protein production, guiding the cells to generate particular proteins and internally modulating the human system through these functional proteins. [11] In recent years, mRNA-based therapies have garnered widespread attention due to their clinical benefits, including strong treatment outcomes, self-adjuvant properties, and relatively rapid clearance from the body. [12-14] mRNA therapies saw a surge in development during the COVID-19 pandemic, fueled by the impressive efficacy of mRNA vaccines. BNT162b2 from Pfizer-BioNTech and mRNA-1273 from Moderna received emergency approvals in 2020, showing over 94% efficacy against SARS-CoV-2. [15] Despite these successes, delivering mRNA into target cells continues to be a significant challenge. [16] Naked mRNAs are unstable due to RNase-based degradation, requiring carriers to encapsulate and transport the mRNAs to their intended sites. Currently, the most widely used delivery methods for mRNA include viral vectors, lipid nanoparticles (LNPs), polymeric micelles, and metal-based nanoparticles. [17-22] Although viral vectors are highly effective for gene delivery, they carry risks of immunogenicity and insertional mutagenesis. [23, 24] LNPs provide excellent self-assembly capability and efficient delivery but may experience limited stability and potential issues in toxicity. [25] Polymeric micelles offer flexible modification capabilities and enhanced stability; however, they may encounter challenges related to high toxicity and low delivery efficiency. [26] Metal-based nanoparticles present unique properties, including high surface area and stability; however, they can induce adverse biological responses, necessitating careful design to minimize cytotoxicity and involving complex synthesis steps. [27] The limited options for transfection agents restrict the broad use of mRNAs as therapeutics in disease treatment. [28, 29] Therefore, developing novel reagents to effectively deliver mRNA drug payloads is essential for enhancing mRNA-based therapies. [30-33] On another note, phase-separated droplets, also known as coacervates, have emerged as a promising drug delivery system, offering excellent intracellular delivery capabilities, good biodegradability, low cellular toxicity, and stable drug release. [34-38] Conventional coacervates are formed through liquid-liquid phase separation (LLPS) of biomacromolecules, which is driven primarily by multivalent macromolecular interactions and the intrinsically disordered regions. [39, 40] LLPS has been found to be a fundamental process in cellular biology that leads to the formation of membrane-less organelles such as nucleoli, stress granules, and P-bodies with various biomolecules, including DNA, RNA, and proteins. [41] Molecules containing multiple weakly interacting motifs (for example, hydrophobic groups) and a hydrophilic linker, meeting the “stick-and-spacer model”, are prone to undergo LLPS and form microdroplets or coacervates [42]. Starting from coacervating polymers [43,44] to biomolecules and phase-separating peptides, including the mixture of the histone and DNA [45,46], L17E trimer [47], HBpep-SR [35, 48-52], and others [53-55], coacervates have recently been shown to encapsulate and deliver proteins into liposomes and cells. [56-58] Miserez and co-workers developed a peptide coacervate system (23 amino acids in length) responsive to glutathione (GSH) for the delivery of plasmids, mRNA, and proteins in vitro with decent efficiency. [35, 48-52] However, the efficacy of peptide-based delivery systems in vivo , together with the biological activity and immunological responses, is currently unknown. Moreover, this study employs a peptide with 23 amino acids to form coacervates; peptides of this length may trigger antibody responses, posing unknown risks [59, 60]. In another example, Dou and co-workers designed a DNA-peptide complex coacervate for intracellular plasmid delivery. [61] We, on the other hand, discovered that low-molecular-weight compounds can also form coacervates via LLPS, and coacervates formed by small molecules can spontaneously deliver antibodies into cells. In addition, the coacervates can release their cargo through various chemical reactions within the cell. For example, we designed a photo-responsive, phase-separating fluorescent molecule (PPFM) with a molecular weight of 666.6 Daltons based on pyrene that can undergo LLPS in the aqueous solution, carry proteins into cells, and release the cargo to the cytosol upon photo illumination [37]. We also designed a triphenylphosphine-based compound that can deliver proteins into cells and respond to an azide compound for cytosolic release [38]. These coacervate delivery systems offer innovative and universal solutions for delivering proteins to cultured cells. Some of the coacervates formed by small molecules have also shown the capability of encapsulating and delivering nucleic acids into cells, enabling gene transfection. However, the efficacy of coacervate-mediated gene transfection in vivo , especially the comparison of coacervates to transfection agents, has not been addressed. In this study, we design a peptide consisting of only 6 amino acids, including hydrophobic residues and a positively charged lysine residue, and dimerized through a disulfide bond, with a molecular weight of 1152 Daltons. A meticulously designed balance of hydrophobicity, positive charge, and a flexible linker drives the coacervate formation via LLPS in aqueous solution. The peptide coacervates can encapsulate nucleic acids of different types and lengths into mammalian cells with high efficiency. The intracellular glutathione reduces the disulfide bond, dissociates the coacervates, and releases the complexed nucleic acid cargo into the cytosol, achieving highly efficient gene transfection. We then demonstrate that peptide coacervates can serve as vehicles for mRNA vaccines in vivo , with transfection efficiencies equivalent to or higher than those of known transfection reagents, such as lipofectamine, InstantFECT (a cationic liposome), and polyethylenimine (PEI). Results and Discussion 1. Synthesis and selection of phase-separating peptide coacervates Molecules containing spacer-and-sticker sequences are known to have the propensity to form coacervates via LLPS. Here, we designed disulfide-linked dimers of tripeptides with hydrophilic, positively charged spacers (KssK and RssR) and hydrophobic stickers (FF, FW, WF, WW) ( Figure 1a ). Tryptophan and tyrosine were selected as the stickers due to their hydrophobicity and the π-π stacking property. The incorporation of two lysine, histidine, or arginine residues, linked by a disulfide bond, served as the spacer, conferring positive charges and hydrophilicity to the peptide. The positive charge facilitates the attraction of nucleic acids with strong negative charges, and the disulfide bond enables cargo release within the cytoplasm. We synthesized a small library of six-residue peptide dimers and screened for their capability to form coacervates ( Table S1 ). These peptides were first dissolved in DMSO at 100 mg/mL as stock solutions and then diluted to aqueous solutions. In PBS buffer, peptide WWKssKWW ( Figure 1a ) formed aggregates at room temperature, and at higher temperatures, the aggregates gradually turned into liquid droplets. Other peptides formed either aggregates or non-uniform droplets, despite the high similarity in the sequence ( Figure S1 ). Therefore, we chose the peptide WWKssKWW (hereafter referred to as WWK ) for the following studies. The WWK coacervates were formed at higher temperature and concentration ( Figure S2a ). The size of droplets became smaller with the increase of the 1,6-HD concentration, suggesting that hydrophobic interactions play a key role in LLPS ( Figure S2b ). After 1 μL of the stock solution was added to 100 μL of Dulbecco’s Modified Eagle Medium (DMEM) at 37℃, the medium gradually became turbid, and uniform droplets could be observed under the microscope ( Figure 1b ). The coacervates showed sizes typically ranging from 300 to 1300 nm based on dynamic light scattering (DLS) ( Figure 1c ). The WWK coacervates displayed dynamic fusion within minutes, showing their liquid properties ( Figure 1d ). After labelling the coacervates with Nile Red, we photobleached a region of the droplet and observed the recovery of the signal in the photobleached region to about 70% in 200 seconds ( Figure 1e ). Turbidity of the coacervate solution decreased in the first 30 minutes and then gradually plateaued over the following hours ( Figures S2c and S2d ), indicating that the coacervates were sufficiently stable for the following cell-based experiments. Ionic strength did not significantly affect the coacervate formation ( Figure S2e ). In the presence of 10 mM reduced glutathione (GSH), the turbid solution became clear, and coacervates disappeared, showing that disulfide reduction disintegrated the coacervates at 37℃ ( Figures 1b ). 5 mM GSH was adequate for complete disintegration of 1 mg/mL WWK coacervates in the medium based on the HPLC traces ( Figure S3 ), showing the feasibility of dissociating coacervates in the cytosol. 2. Encapsulation of nucleic acids for intracellular delivery Next, we investigated whether WWK coacervates could encapsulate nucleic acids, using single-stranded DNA labeled with a fluorescent dye, ROX (ROX-ssDNA) as the cargo. Fluorescent microscopy analysis showed successful encapsulation of ROX-ssDNA in WWK coacervates ( Figure S4a ). Next, 15 μg of plasmids with a size of 7.6 Kb were encapsulated in 1 mg/mL WWK coacervates in 25 μL Opti-MEM medium at 37℃, indicating a high loading capacity of approximately 60% and the encapsulation efficiency close to 100% ( Figure S4b ). These data show that WWK coacervates can encapsulate nucleic acids, primarily driven by the charge-charge and hydrophobic interactions. Before cellular experiments, we tested the cytotoxicity of the WWK coacervates based on the lactate dehydrogenase (LDH) assay. WWK coacervates showed minimal cytotoxicity to HEK293 cells at concentrations below 0.9 mg/mL ( Figure S5 ). Next, we incubated ROX-ssDNA-loaded WWK coacervates at 0.9 mg/mL with HEK293 cells for 24 hours and observed that many droplets carrying the fluorescent ROX signal were taken up by the cells. The ROX-ssDNA was also found in the cytoplasm, suggesting its release into the cytoplasm ( Figure S6a ). 3D confocal microscopy images showed a two-step uptake procedure: within 6 hours, WWK coacervates primarily adhered to the cell membrane, and after 12 hours, many coacervates were endocytosed into the cells ( Figure S6b ). The delivery efficiency of WWK coacervates was comparable to that of Lipofectamine® 3000 and higher than PEI, based on the flow cytometry results ( Figure S6c ). These data show that WWK coacervates can encapsulate and deliver ssDNA into cells with minimal cytotoxicity and decently high efficiency. Next, we examined the delivery of siRNA molecules with the WWK coacervates, focusing on the intracellular function of the nucleic acids ( Figure 2a ). Small interfering RNA (siRNA) is a promising therapeutic modality with the ability to silence disease-related genes by targeting specific messenger RNAs (mRNAs) and facilitating their degradation with the assistance of the enzyme Dicer. [62, 63] In 2018, the first siRNA drug, patisiran, was approved for the treatment of hereditary transthyretin-mediated amyloidosis. [7] Here, we delivered a GAPDH-specific siRNA (siGAPDH) as a model. Western blotting analysis demonstrated that the expression level of GAPDH declined to approximately 70% of its original level within 24 hours following the addition of siGAPDH-loaded WWK coacervates. The knockdown efficiency of GAPDH was sustained for up to 48 hours. In contrast, the commercial transfection agent, lipofectamine ® 3000, only reduced 25% of the GAPDH expression level ( Figure 2b ). Then, we utilized WWK coacervates to deliver plasmids for protein overexpression in cells. Plasmids have been used as biopharmaceuticals for the treatment of hereditary disorders, multifactorial diseases, and the prevention of infectious diseases. [64] The first gene therapy product approved for clinical use, Gendicine, is a plasmid that delivers the tumor suppressor p53 gene to treat head and neck squamous cell carcinoma. [65] Two plasmids of different sizes were used as the cargo: a plasmid expressing mCherry protein, pCMV-mCherry (~4 kb), and the second expressing mCherry-TRIM21 recombinant protein (a model protein), pCI-neo.mCherry-TRIM21 (~7.6 kb). Fluorescent microscopy images show that in all three groups, mCherry was successfully expressed ( Figure S7 ). Western blotting analysis revealed that the expression level of mCherry in the WWK coacervate (0.3 mg/mL) group was 1.5 times higher than that in the PEI group and about 1.2 times higher than the lipofectamine® 3000 (Lipo3000) group in 48 hours ( Figure 2c ). Similarly, the WWK coacervate groups gave higher expression levels of mCherry-TRIM21 than the other two groups ( Figure 2d ). In this experiment, we observed a surprisingly low expression level in the Lipo3000 group for unknown reasons ( Figure 2d ). Similar results were obtained in another cell line, MDA-MB-231 cells ( Figure S8a ). We also explored the delivery of mRNA by WWK coacervates. An EGFP mRNA was delivered into HEK293 cells and MDA-MB-231 cells with WWK coacervates, and the expression level of EGFP in the transfected cells was found to be similar to that of the Lipo3000 group in HEK293 cells ( Figure 2e ), while notably higher than the Lipo3000 group in MDA-MB-231 cells ( Figure S8b ), suggesting a possible cell type variation. Altogether, these data show that WWK coacervates can deliver a variety of nucleic acids into cells, with efficiency equivalent to or higher than Lipo3000 and PEI. 3. Cellular uptake mechanism of WWK coacervates Next, we explored the mechanism by which WWK coacervates enter cells, using fluorescent trackers and antibodies specifically targeting F-actin, early endosomes, late endosomes, lysosomes, mitochondria, and the endoplasmic reticulum (ER). WWK coacervates loaded with fluorescent ssDNA were used in this case. Based on the confocal images of the colocalization experiments, polymerization of F-actin was found around the coacervates on the surface of the cell, which seems to facilitate the coacervates’ entry into the cells. During the early stage of cellular uptake, the fluorescent signal of WWK coacervates partially overlapped with the Rab5A signal, a marker of the early endosome. After 6 hours, based on the signal of Rab7A, a late endosome marker, and a LysoTracker, we found no noticeable overlap between the coacervates and late endosome or lysosome. This result suggests that although some of the coacervates may be endocytosed via the endosomal uptake, they may have escaped from the endosomes ( Figure 3a ). In addition, WWK coacervates did not colocalize with mitochondria and ER ( Figure S12 ). Inhibitors targeting various endocytosis pathways, including dynasore (an inhibitor of dynamin-mediated endocytosis), amiloride (an inhibitor of pinocytosis), chloroquine (an inhibitor of clathrin-mediated endocytosis), cytochalasin B (an inhibitor of pinocytosis and phagocytosis), and 2-deoxy-D-glucose (an inhibitor of energy-dependent endocytosis), were also utilized to evaluate the uptake mechanisms. [66] Flow cytometry results indicated that the serum-starved group exhibited a reduction of over 50% in coacervate uptake in HEK293 cells, compared to the group with serum, suggesting that WWK coacervates mainly enter cells via energy-dependent pathways. The dynasore-treated group showed a pronounced decline in coacervate uptake, showing that WWK coacervates partially penetrated the cell membrane via the dynamin-mediated endocytosis pathway. In contrast, the amiloride- and cytochalasin B-treated groups displayed an uptake level comparable to that of the positive control, indicating that macropinocytosis is not the major cellular uptake pathway in HEK293 cells ( Figure 3b ). However, the uptake route was different in MDA-MB-231 cells. Cytochalasin B significantly inhibited the uptake of WWK coacervates into MDA-MB-231 cells, indicating that pinocytosis was the major uptake pathway. Instead, the dynamin-mediated endocytosis inhibitor dynasore did not show any influence on the coacervate uptake ( Figure 3c ). Therefore, the uptake mechanism of WWK coacervates differs in various cells. The major pathway for WWK coacervates to enter HEK293 cells is dynamin-mediated endocytosis, but for MDA-MB-231 cells pinocytosis dominates. 4. Comparison of mRNA delivery in vitro Next, we compared WWK coacervates with commercially available transfection agents, including PEI, InstantFECT, Lipofectamine MessengerMax (LipoMessengerMax), and protamine, in mRNA delivery to HEK293 cells ( Figure 4a ). In addition, we applied various concentrations of WWK coacervates for the transfection of the firefly luciferase mRNA and analyzed the luminescence 24 hours after the transfection ( Figure 4b ). The empty vector induced negligible luminescence and thus was used as a negative control. When 1 µg of luciferase mRNA was used as the cargo, WWK coacervate transfection induced strong luminescence intensities, which peaked at the peptide concentration of 0.9 mg/mL ( Figure 4c and Figure S10a ). Cell viability assay also showed that WWK coacervates up to 0.9 mg/mL were well-tolerated by cells ( Figure S5 ). Consequently, this peptide concentration was chosen as the optimal condition for further experiments. Cytotoxicity may be an important reason for the reduced level of luminescence at WWK concentrations above 0.9 mg/mL ( Figures 4b and 4c ). Surprisingly, compared to the commercial transfection agents, coacervate-mediated mRNA transfection at 0.9 mg/mL of WWK produced significantly stronger luciferase signal than LipoMessengerMax, InstantFECT cationic liposome, PEI, and protamine ( Figures 4e and 4f ). Naked mRNA alone also induced negligible luminescence ( Figure 4f and Figure S10b ). In dendritic cells (DC2.4 cells), WWK coacervate-mediated mRNA transfection yielded marked luciferase expression, whereas the luminescence signal in the LipoMessengerMax group was undetectable ( Figure S11 ). Taken together, these data show that WWK coacervates can efficiently deliver mRNA into cells, with an efficiency on par with or higher than commercial transfection agents. Next, we measured the inflammatory profile of different mRNA/transfection agent formulations by measuring the level of the inflammatory cytokine IL-1β in the supernatants of human Peripheral Blood Mononuclear Cell (PBMC) cultures after mRNA transfection ( Figure 4g ). Liposomes and lipid nanoparticles induced high levels of IL-1β, whereas WWK /mRNA formulation induced a low level of IL-1β release, similar to the naked mRNA control. [17] Although inflammation partly contributes to the immunogenicity of mRNA vaccines, excessive responses result in toxicities that are not favorable for gene therapy. This result suggests that WWK coacervates may have a more favorable safety profile in vivo . 5. Transfection of luciferase mRNA using WWK coacervates in animals Next, we investigated the efficacy of WWK coacervate-mediated mRNA transfection in animals using a WWK concentration of 0.9 mg/mL and 10 mg luciferase mRNA per animal. We performed an in vivo imaging using the IVIS (In Vivo Imaging System) 24 and 48 hours after subcutaneous or intramuscular injections of various luciferase mRNA formulations ( Figure 5a ). Consistent with the in vitro transfection results, the WWK/ mRNA formulation achieved robust luciferase expression after subcutaneous and intramuscular injections ( Figure 5b ). At 24 hours, subcutaneously injected WWK /mRNA formulation induced luminescence in mice with significantly higher levels than those of the PEI/mRNA formulation and vehicle controls, comparable to that of the LipoMessengerMax /mRNA formulation, albeit lower than InstantFECT/mRNA formulation ( Figures 5c and Figure S12 ). At 48 hours, the WWK /mRNA formulation administered through the subcutaneous route produced a luminescence level comparable to that of the InstantFECT/mRNA formulation, and significantly higher than the LipoMessengerMax/mRNA formulation, PEI/mRNA formulation, and vehicle controls ( Figure 5d ). For the intramuscular administration route, the WWK /mRNA formulation generated a luminescence level comparable to the vehicle controls, InstantFECT/mRNA, PEI/mRNA, and LipoMessengerMax/mRNA formulations at 24 hours ( Figure 5e ). At 48 hours, the three groups, WWK coacervates, InstantFECT, and LipoMessengerMax, were comparable, higher than the vehicle control and the PEI group ( Figure 5f ). Importantly, the mice that received WWK coacervate injections did not exhibit any signs of weight loss, organ abnormalities, discomfort, or side effects ( Figures S13 and S14 ). In contrast, subcutaneous and intramuscular injections of the PEI/mRNA formulation led to severe discomfort, and some mice died by 24 hours. A histological analysis of mouse organs at the 24-hour time point revealed that the InstantFECT/mRNA and LipoMessengerMax/mRNA formulations resulted in congestive hepatopathy in the liver, whereas the WWK /mRNA group remained the same as the healthy animals ( Figure 5g ). Although most cases of congestive hepatopathy due to lipid carriers are asymptomatic, some cases may cause abdominal discomfort. [67-69] No other organs exhibited abnormalities ( Figure 5h ). These findings show that WWK coacervates are safe vehicles to deliver mRNA vaccines with high efficiency. 6. Cell-mediated immune responses induced by different ovalbumin (OVA) mRNA and SARS-CoV-2 spike mRNA formulations Next, we evaluated the cell-mediated immune responses of the WWK coacervate-delivered mRNA vaccines, with InstantFECT and LipoMessengerMax as controls. The antigen-specific cellular response to biological vaccines could indicate the level of immune protection. To study the immune responses of WWK coacervate-delivered OVA mRNA vaccines and adjuvants, we performed ELISPOT analysis for interferon-g (IFN-g) using the isolated splenocytes of mice subcutaneously immunized with different OVA mRNA vaccine formulations, including WWK /OVA mRNA, LipoMessengerMax/OVA mRNA, WWK /OVA mRNA plus TLR7, WWK /TLR7, TLR7 alone, and vehicle controls ( Figure 6a ). Interestingly, the WWK /OVA mRNA formulation could stimulate a significantly greater OVA-specific cellular response than the LipoMessengerMax/OVA mRNA formulation and vehicle controls, which was comparable to that of the InstantFECT/OVA mRNA formulation ( Figures 6b and 6c ). These findings suggest that WWK coacervates effectively deliver the OVA mRNA vaccine into antigen-presenting cells. Additionally, WWK coacervates permitted TLR7 adjuvant delivery, and the combined subcutaneous treatment with OVA mRNA augmented OVA-specific cellular responses in the immunized mice compared to the WWK /OVA mRNA alone ( Figures 6d and 6e ). WWK coacervates also facilitated the efficient intramuscular delivery of a SARS-CoV-2 spike mRNA and produced spike-specific cellular responses comparable to those of the commercial Comirnaty mRNA vaccine ( Figures 6f-6h ). The cell-mediated immune response in the WWK /spike mRNA group at this dosage was adequate to induce anti-spike antibodies, albeit the antibody levels were lower than those of the Comirnaty mRNA vaccine group ( Figure S15 ), suggesting that different mRNA carriers may adopt different vaccine uptake mechanisms. Overall, these results demonstrate that WWK coacervate is a viable vehicle for mRNA vaccines and the adjuvant TLR7 in vivo . 7. Antibody responses induced by coacervate-delivered SARS-CoV-2 spike mRNA The generation of antibody responses, through antigen-specific antibody production, can indicate immune protection against infectious diseases, such as SARS-CoV-2. At a dosage of 25 µg/mouse, spike mRNA was delivered by WWK coacervates, LipoMessgenerMAX, InstantFECT, and Comirnaty via intramuscular injection at days 0 and 21 ( Figure 7a ). We performed serum ELISA after one and two months of immunization. The WWK /spike mRNA immunizations generated a significant level of spike-specific IgG antibodies, comparable to those of the InstantFECT/spike mRNA group ( Figures 7b and 7c ). Nonetheless, the Comirnaty COVID-19 mRNA vaccine produced the highest level of antibodies in the animal ( Figures 7b and 7c ). This result differs from the cellular data, which showed that the Comirnaty formulation had comparable levels of cellular immunity ( Figures 6g and 6h ). The heightened humoral response produced by the Comirnaty vaccine in vivo highlights the power of commercial vaccine development, including optimization of compound size, temperature, pH, aggregation, and moiety modifications during the preparation stage [70]. The IgG1: IgG2a ratios amongst the antibodies generated by the different spike mRNA vaccines were comparable, which suggests equivalent levels of Th2-based and Th1-based immunity, respectively ( Figure 7d ). Neutralization antibodies against viral infections remain the gold standard indicator of antiviral immune protection generated by vaccines. All formulations generated high levels of neutralization antibodies against the wild type (WT), Delta, and Alpha SARS-CoV-2 strains, judged by the higher serum dilution factor to achieve 50% of the inhibition ( Figure 7e-g ). The WWK /mRNA vaccine generated a similar level of neutralizing antibodies as InstantFECT and LipoMessengerMAX, but lower than the Comirnaty vaccine. Altogether, the WWK /mRNA vaccine induced potent antigen-specific humoral immunity through intramuscular immunization, highlighting their promise in the context of infectious disease prevention. 8. WWK/OVA mRNA immunization against B16-OVA cancer Lastly, we examine the therapeutic potential of WWK coacervates for delivering mRNA vaccine payloads for cancer treatment. We established a B16-OVA subcutaneous tumor model (2 ´ 10⁵ cells/per mouse) to compare the efficacy of various OVA mRNA formulations (namely, OVA mRNA delivered by WWK coacervates, InstantFECT, LipoMessengerMAX, and control groups without mRNA) ( Figure 8a ). The WWK /OVA mRNA group demonstrated a significant tumor suppression (average tumor size = 42.5 mm 3 ), compared to the LipoMessengerMAX/mRNA group (average tumor size = 292 mm 3 ), the LipoMessengerMAX only group (average tumor size = 1256 mm 3 ), and the WWK peptide only group (average tumor size = 1024 mm 3 ) by the tumor endpoint ( Figures 8b-8i ). The tumor sizes and survival rates were comparable between the WWK /OVA mRNA group and the InstantFECT/mRNA group ( Figures 8i and 8j ). The WWK /OVA mRNA treatment significantly prolonged the tumor-free survival rate in mice (2/5 mice survived) compared to all vehicle controls (all mice died) by day 40 ( Figure 8j ). Tumor-infiltrating cytotoxic CD8+ T cells may exhibit potent tumor-killing capabilities, leading to the observed cancer eradication. Therefore, we conducted flow cytometry analysis on the harvested tumors from the various groups at the tumor endpoint. A significantly elevated level of CD8+ T cells (but not CD4+ T cells) was detected in the WWK /OVA mRNA group compared to the LipoMessengerMAX/OVA mRNA group and the vehicle control groups ( Figure 8k and 8l ). These data suggest that cytotoxic T cells are the key players responsible for the cancer-suppressive tumor environment induced by the WWK /OVA mRNA treatment. Conclusion The field of therapeutic biomacromolecules, like nucleic acids, faces challenges in crossing cell membranes because of their large size and high electronegativity, requiring the use of delivery vehicles. Existing options for delivery vehicles are limited in the market and have issues such as potential toxicity and suboptimal delivery efficacy. Here, we introduce a coacervate system designed for the efficient and safe delivery of nucleic acids. WWK coacervates exhibit high encapsulation efficiency and loading capacity for nucleic acids, minimal cytotoxicity, a responsive release mechanism triggered by redox signals, and effective cytosolic delivery. Remarkably, the WWK coacervate delivery system shows superior delivery of siRNA, mRNA, DNA, and plasmids compared to commercially available transfection agents. In animal models, the WWK coacervates effectively convey OVA mRNA, SARS-CoV-2 spike mRNA, and TLR7 adjuvants in vivo , triggering cell-mediated immune responses and antibody responses. Employed as a protective vaccine against SARS-CoV-2, WWK /spike mRNA therapy elicits heightened levels of antigen-specific antibodies, neutralizing WT, Delta, and Alpha strains of SARS-CoV-2. In the realm of cancer immunotherapy, WWK /OVA mRNA treatment efficiently restrains B16-OVA tumor progression and significantly extends the lifespan of tumor-bearing mice. This innovative research sheds light on the development of peptide coacervate-based mRNA vaccines, providing valuable insights for potential clinical translation. Declarations Competing interest statement : Z. X., C. K., J. H., and J. X. have filed a US patent application based on this research, with a title: phase-separating peptide coacervates for nucleic acid transfection and mRNA vaccine delivery, and an application No: 63/788315. ACKNOWLEDGMENT This work was partially funded by grants from the University Grants Committee of Hong Kong (GRF grants 14306222, 14301922, and 14307523), C5026-24G, R5013-19, and CUHK (ICSG, CRIMS, 1+1+1 CUHK/CUHKSZ grant, and Direct Grant 4053563). References Belgrad, J.; Fakih, H. H.; Khvorova, A. Nucleic Acid Therapeutics: Successes, Milestones, and Upcoming Innovation. Nuc. Acid Ther. 2024 , 34 , 52–72. https://doi.org/10.1089/nat.2023.0068. Moccia, M.; Pascucci, B.; Saviano, M.; Maria Teresa Cerasa; Terzidis, M. A.; Chryssostomos Chatgilialoglu; Masi, A. Advances in Nucleic Acid Research: Exploring the Potential of Oligonucleotides for Therapeutic Applications and Biological Studies. Int. J. Mol. Sci. 2023 , 25 , 146–146. https://doi.org/10.3390/ijms25010146. Lundin, K. E.; Gissberg, O.; Smith, C. I. E. Oligonucleotide Therapies: The Past and the Present. Hum. Gene Ther. 2015 , 26 , 475–485. https://doi.org/10.1089/hum.2015.070. Zhang, L.; Tang, X.; Xi, Z.; Chattopadhyaya, J. Nucleic Acids in Medicinal Chemistry and Chemical Biology ; Wiley, 2022. https://doi.org/10.1002/9781119692799. Kulkarni, J. A.; Witzigmann, D.; Thomson, S. B.; Chen, S.; Leavitt, B. R.; Cullis, P. R.; van der Meel, R. The Current Landscape of Nucleic Acid Therapeutics. Nat. Nanotechnol.  2021 , 16 , 630–643. https://doi.org/10.1038/s41565-021-00898-0. Egli, M.; Manoharan, M. Chemistry, Structure and Function of Approved Oligonucleotide Therapeutics. Nucleic Acids Res 2023 , 51 , 2529–2573. https://doi.org/10.1093/nar/gkad067. Adams, D. Patisiran, an Investigational RNAi Therapeutic for Patients with Hereditary Transthyretin-Mediated (HATTR) Amyloidosis : Results from the Phase 3 APOLLO Study. Rev. Med. Interne. 2018 , 39 , A185. https://doi.org/10.1016/j.revmed.2018.10.163. Liu, C.; Shi, Q.; Huang, X.; Koo, S.; Kong, N.; Tao, W. MRNA-Based Cancer Therapeutics. Nat. Rev. Cancer 2023 , 23 , 1–18. https://doi.org/10.1038/s41568-023-00586-2. Kowalski, P. S.; Rudra, A.; Miao, L.; Anderson, D. G. Delivering the Messenger: Advances in Technologies for Therapeutic MRNA Delivery. Mol. Ther. 2019 , 27 , 710–728. https://doi.org/10.1016/j.ymthe.2019.02.012. Duan, Q.; Hu, T.; Zhu, Q.; Jin, X.; Chi, F.; Chen, X. How Far Are the New Wave of MRNA Drugs from Us? MRNA Product Current Perspective and Future Development. Front. immunol. 2022 , 13 , 974433. https://doi.org/10.3389/fimmu.2022.974433. Dai, H.; Abdullah, R.; Wu, X.; Li, F.; Ma, Y.; Lu, A.; Zhang, G. Pancreatic Cancer: Nucleic Acid Drug Discovery and Targeted Therapy. Front. Cell Dev. Biol. 2022 , 10 , 855474. https://doi.org/10.3389/fcell.2022.855474. Lorentzen, C. L.; Haanen, J. B.; Met, Ö.; Svane, I. M. Clinical Advances and Ongoing Trials of MRNA Vaccines for Cancer Treatment. Lancet Oncol. 2022 , 23 , e450–e458. https://doi.org/10.1016/S1470-2045(22)00372-2. Wang, X. L.; Terrence Tsz-Tai Yuen; Yu, D.; Hu, J.; Li, R. L.; Zhang, Z.; Lin, X.; Gong, H.-R.; Chan, C. N.; Yoon, C.; Huiping Shuai; Deborah Tip-Yin Ho; Ivan Fan-Ngai Hung; Zhang, B.; Chu, H.; Huang, J.-D. Vaccine-Induced Protection against SARS-CoV-2 Requires IFN-γ-Driven Cellular Immune Response. Nat. Commun. 2023 , 14 , 3440. https://doi.org/10.1038/s41467-023-39096-y. Xu, S.; Yang, K.; Li, R.; Zhang, L. MRNA Vaccine Era—Mechanisms, Drug Platform and Clinical Prospection. Int. J. Mol. Sci. 2020 , 21 , 6582. https://doi.org/10.3390/ijms21186582. Sun, X.; Setrerrahmane, S.; Li, C.; Hu, J.; Xu, H. Nucleic Acid Drugs: Recent Progress and Future Perspectives. Signal Transduct Tar. 2024 , 9 , 316. https://doi.org/10.1038/s41392-024-02035-4. Dong, L.; Deng, X.; Li, Y.; Zhu, X.; Shu, M.; Chen, J.; Luo, H.; An, K.; Cheng, M.; Zhang, P.; Tan, W. Stimuli-Responsive MRNA Vaccines to Induce Robust CD8 + T Cell Response via ROS-Mediated Innate Immunity Boosting. J. Am. Chem. Soc. 2024 , 146 , 19218–19228. https://doi.org/10.1021/jacs.4c04331. Kon, E.; Elia, U.; Peer, D. Principles for Designing an Optimal MRNA Lipid Nanoparticle Vaccine. Curr. Opin. Biotechnol. 2022 , 73 , 329–336. https://doi.org/10.1016/j.copbio.2021.09.016. Arevalo, C. P.; Bolton, M. J.; Le Sage, V.; Ye, N.; Furey, C.; Muramatsu, H.; Alameh, M.-G.; Pardi, N.; Drapeau, E. M.; Parkhouse, K.; Garretson, T.; Morris, J. S.; Moncla, L. H.; Tam, Y. K.; Fan, S. H. Y.; Lakdawala, S. S.; Weissman, D.; Hensley, S. E. A Multivalent Nucleoside-Modified MRNA Vaccine against All Known Influenza Virus Subtypes. Science 2022 , 378 , 899–904. https://doi.org/10.1126/science.abm0271. Crommelin, D. J. A.; Anchordoquy, T. J.; Volkin, D. B.; Jiskoot, W.; Mastrobattista, E. Addressing the Cold Reality of MRNA Vaccine Stability. J. Pharm. Sci. 2020 , 110 . https://doi.org/10.1016/j.xphs.2020.12.006. Bird, L. MRNA Vaccine for Treating Pancreatic Cancer. Nat. Rev. Immunol. 2023 , 23 , 413–413. https://doi.org/10.1038/s41577-023-00899-1. Xu, C.; Cao, C.; Tan, T.; Deng, S.; Wu, B.; Yang, Q.; Wu, M.; Wu, H. MRNA Vaccine-A New Cancer Treatment Strategy. Curr. Cancer Drug Tar. 2023 , 23 , 669–681. https://doi.org/10.2174/1568009623666230222124424. Vishweshwaraiah, Y. L.; Dokholyan, N. V. MRNA Vaccines for Cancer Immunotherapy. Front. Immunol. 2022 , 13 , 1029069. https://doi.org/10.3389/fimmu.2022.1029069. Sengupta, P.; Banerjee, N.; Dutta, A.; Chaudhuri , M.; Chatterjee , S. Nucleic Acid Therapeutics in Cancer Biology. In Nucleic Acid Biology and Its Application in Human Diseases ; Springer: Singapore, 2023; pp. 321–348. Yamada, Y. Nucleic Acid Drugs—Current Status, Issues, and Expectations for Exosomes. Cancers 2021 , 13 , 5002. https://doi.org/10.3390/cancers13195002. Scheideler, M.; Vidakovic, I.; Prassl, R. Lipid Nanocarriers for MicroRNA Delivery. Chem. Phys. Lipids 2020 , 226 , 104837. https://doi.org/10.1016/j.chemphyslip.2019.104837. Qin, Y.; Ou, L.; Zha, L.; Zeng, Y.; Li, L. Delivery of Nucleic Acids Using Nanomaterials. Mol. Biomed. 2023 , 4 . https://doi.org/10.1186/s43556-023-00160-0. Sharma, A. R.; Lee, Y.-H.; Bat-Ulzii, A.; Bhattacharya, M.; Chakraborty, C.; Lee, S.-S. Recent Advances of Metal-Based Nanoparticles in Nucleic Acid Delivery for Therapeutic Applications. J. Nanobiotechnol. 2022 , 20 . https://doi.org/10.1186/s12951-022-01650-z. Das, S.; Vera, M.; Gandin, V.; Singer, R. H.; Tutucci, E. Intracellular MRNA Transport and Localized Translation. Nat. Rev. Mol. Cell Biol. 2021 , 22 , 483–504. https://doi.org/10.1038/s41580-021-00356-8. Wu, B.; Eliscovich, C.; Yoon, Y. J.; Singer, R. H. Translation Dynamics of Single MRNAs in Live Cells and Neurons. Science 2016 , 352 , 1430–1435. https://doi.org/10.1126/science.aaf1084. Pardi, N.; Hogan, M. J.; Porter, F. W.; Weissman, D. MRNA Vaccines — a New Era in Vaccinology. Nat. Rev. Drug Discov. 2018 , 17 , 261–279. Goel, R. R.; Painter, M. M.; Apostolidis, S. A.; Mathew, D.; Meng, W.; Rosenfeld, A. M.; Lundgreen, K. A.; Reynaldi, A.; Khoury, D. S.; Pattekar, A.; Gouma, S.; Kuri-Cervantes, L.; Hicks, P.; Dysinger, S.; Hicks, A.; Sharma, H.; Herring, S.; Korte, S.; Baxter, A. E.; Oldridge, D. A. MRNA Vaccines Induce Durable Immune Memory to SARS-CoV-2 and Variants of Concern. Science 2021 , 374 , abm0829. https://doi.org/10.1126/science.abm0829. Verbeke, R.; Lentacker, I.; De Smedt, S.; Dewitte, H. The Dawn of MRNA Vaccines: The COVID-19 Case. J. Controlled Release 2021 , 333 , 511–520. https://doi.org/10.1016/j.jconrel.2021.03.043. Biggs, A. T.; Littlejohn, L. F. Describing MRNA Vaccine Technology for a Military Audience. Mil. Med. 2022 , 188 , 547–554. https://doi.org/10.1093/milmed/usac129. Cerrato, C. P.; Leppert, A.; Sun, Y.; Lane, D. P.; Arsenian-Henriksson, M.; Miserez, A.; Landreh, M. Monitoring Disassembly and Cargo Release of Phase-Separated Peptide Coacervates with Native Mass Spectrometry. Ana. Chem. 2023 , 95 , 10869–10872. https://doi.org/10.1021/acs.analchem.3c02384. Sun, Y.; Lau, S. Y.; Lim, Z. W.; Chang, S. C.; Ghadessy, F.; Partridge, A.; Miserez, A. Phase-Separating Peptides for Direct Cytosolic Delivery and Redox-Activated Release of Macromolecular Therapeutics. Nat. Chem. 2022 , 14 , 274–283. https://doi.org/10.1038/s41557-021-00854-4. Ramat, A.; Simonelig, M. Activating Translation with Phase Separation. Science 2022 , 377 , 712–713. https://doi.org/10.1126/science.add6323. Bao, Y.; Chen, H.; Xu, Z.; Gao, J.; Jiang, L.; Xia, J. Photo‐Responsive Phase‐Separating Fluorescent Molecules for Intracellular Protein Delivery. Angew. Chem. Int. Ed. 2023 , 62 (42), e202307045. https://doi.org/10.1002/anie.202307045. Bao, Y.; Xu, Z.; Cheng, K.; Li, X.; Chen, F.; Yuan, D.; Zhang, F.; Che, A. R.-Y.; Zeng, X.; Zhao, Y.-D.; Xia, J. Staudinger Reaction-Responsive Coacervates for Cytosolic Antibody Delivery and TRIM21-Mediated Protein Degradation. J. Am. Chem. Soc. 2025 , 147 , 3830–3839. https://doi.org/10.1021/jacs.4c17054. Banani, S. F.; Lee, H. O.; Hyman, A. A.; Rosen, M. K. Biomolecular Condensates: Organizers of Cellular Biochemistry. Nat. Rev. Mol. Cell Bio. 2017 , 18 , 285–298. https://doi.org/10.1038/nrm.2017.7. Zhang, B.; Liu, B.; Wu, Z.; Oyama, K.; Ikari, M.; Yagi, H.; Naoya Tochio; Takanori Kigawa; Tsutomu Mikawa; Miyake, T. A Hybrid Nanotube Stamp System in Intracellular Protein Delivery for Cancer Treatment and NMR Analytical Techniques. Anal. Chem. 2024 , 96 , 8349–8355. https://doi.org/10.1021/acs.analchem.3c05331. Dinic, J.; Marciel, A. B.; Tirrell, M. V. Polyampholyte Physics: Liquid–Liquid Phase Separation and Biological Condensates. Curr. Opin. Colloid Interface Sci. 2021 , 54 , 101457. https://doi.org/10.1016/j.cocis.2021.101457. Ladbury, J. E.; Lin, C.-C.; Suen, K. M. Phase Separation Enhances Probability of Receptor Signalling and Drug Targeting. Trends Biochem. Sci. 2023 , 48 , 428–436. https://doi.org/10.1016/j.tibs.2023.01.005. Jo, H.; Gajendiran, M.; Kim, K. Development of Polymer Coacersome Structure with Enhanced Colloidal Stability for Therapeutic Protein Delivery. Macro. Biosci. 2019 , 19 , e1900207. https://doi.org/10.1002/mabi.201900207. Xiao, W.; Jakimowicz, M. D.; Ioannis Zampetakis; Neely, S.; Scarpa, F.; Davis, S. A.; Williams, D. S.; Perriman, A. W. Biopolymeric Coacervate Microvectors for the Delivery of Functional Proteins to Cells. Adv. Biosyst. 2020 , 4 , e2000101. https://doi.org/10.1002/adbi.202000101. Wen, P.; Huang, H.; Zhang, R.; Zheng, H.; Liang, T.; Zhuang, C.; Wu, Q.; Wang, J.; Liu, F.; Zhang, K.; Wu, W.; He, K.; Liu, F.; Li, H.; Gu, Z. Coacervate Vesicles Assembled by Liquid–Liquid Phase Separation Improve Delivery of Biopharmaceuticals. Nat. Chem. 2025 , 17 , 279–288. https://doi.org/10.1038/s41557-024-01705-8. Mashima, T.; van Stevendaal, M. H. M. E.; Cornelissens, F. R. A.; Mason, A. F.; Rosier, B. J. H. M.; Altenburg, W. J.; Oohora, K.; Hirayama, S.; Hayashi, T.; van Hest, J. C. M.; Brunsveld, L. DNA‐Mediated Protein Shuttling between Coacervate‐Based Artificial Cells. Angew. Chem. Int. Ed. 2022 , 61 , e202115041. https://doi.org/10.1002/anie.202115041. Yim, W.; Jin, Z.; Chang, Y.-C.; Brambila, C.; Creyer, M. N.; Ling, C.; He, T.; Li, Y.; Retout, M.; Penny, W. F.; Zhou, J.; Jokerst, J. V. Polyphenol-Stabilized Coacervates for Enzyme-Triggered Drug Delivery. Nat. Commun. 2024 , 15 , 7295. https://doi.org/10.1038/s41467-024-51218-8. Iwata, T.; Hirose, H.; Sakamoto, K.; Hirai, Y.; Vincent, J.; Misao Akishiba; Imanishi, M.; Shiroh Futaki. Liquid Droplet Formation and Facile Cytosolic Translocation of IgG in the Presence of Attenuated Cationic Amphiphilic Lytic Peptides. Angew. Chem. Int. Ed. 2021 , 60 , 198 04–19812. https://doi.org/10.1002/anie.202105527. un, Y.; Xu, X.; Chen, L.; Chew, W. L.; Ping, Y.; Miserez, A. Redox-Responsive Phase-Separating Peptide as a Universal Delivery Vehicle for CRISPR/Cas9 Genome Editing Machinery. ACS Nano 2023 , 17 , 16597–16606. https://doi.org/10.1021/acsnano.3c02669. Sun, Y.; Wu, X.; Li, J.; Milad Radiom; Raffaele Mezzenga; Verma, C. S.; Yu, J.; Miserez, A. Phase-Separating Peptide Coacervates with Programmable Material Properties for Universal Intracellular Delivery of Macromolecules. Nat. Commun. 2024 , 15 , 10094. https://doi.org/10.1038/s41467-024-54463-z. Cerrato, C. P.; Krkoška, M.; Sun, Y.; Liaño-Pons, J.; Neo, Q. Y.; Vosselman, T.; Alzrigat, M.; Vojtěšek, B.; Lane, D. P.; Arsenian Henriksson, M.; Miserez, A.; Landreh, M. Engineered Peptide Coacervates Enable Efficient Intracellular Delivery of the MYC Inhibitor OmoMYC. Mol. Pharms. 2025 , 22 , 3479–3490. https://doi.org/10.1021/acs.molpharmaceut.5c00468. Sun, Y.; Wu, X.; Shen, K.; Guo, K.; Lim, D. S.; Chew, W. L.; Yu, J.; Miserez, A. Polyethylene-Glycol-Conjugated Peptide Coacervates with Tunable Size for Intracellular MRNA Delivery. ACS Nano 2025 , 10.1021/acsnano.4c16908 , Advance online publication. https://doi.org/10.1021/acsnano.4c16908. Shebanova, A.; Perrin, Q. M.; Zhu, K.; Sushanth Gudlur; Chen, Z.; Sun, Y.; Huang, C.; Lim, Z. W.; Mondarte, E. A.; Sun, R.; Lim, S.; Yu, J.; Miao, Y.; Parikh, A. N.; Ludwig, A.; Miserez, A. Cellular Uptake of Phase‐Separating Peptide Coacervates. Adv. Sci. 2024 , 11 , e2402652. https://doi.org/10.1002/advs.202402652. Xie, X.; Li, T.; Ma, L.; Wu, J.; Qi, Y.; Yang, B.; Li, Z.; Yang, Z.; Zhang, K.; Chu, Z.; Ngai, T.; Xia, J.; Wang, Y.; Zhao, P.; Bian, L. A Designer Minimalistic Model Parallels the Phase-Separation-Mediated Assembly and Biophysical Cues of Extracellular Matrix. Nat. Chem. 2025 , 10.1038/s41557-025-01837-5 , Advance online publication. https://doi.org/10.1038/s41557-025-01837-5. Nasr, S. S.; Lee, S.; Durairaj Thiyagarajan; Boese, A.; Loretz, B.; Lehr, C.-M. Co-Delivery of MRNA and PDNA Using Thermally Stabilized Coacervate-Based Core-Shell Nanosystems. Pharmaceutics 2021 , 13 , 1924–1924. https://doi.org/10.3390/pharmaceutics13111924. Lu, T.; Liese, S.; Ludo Schoenmakers; Weber, C. A.; Suzuki, H.; Wilhelm; Spruijt, E. Endocytosis of Coacervates into Liposomes. J. Am. Chem. Soc. 2022 , 144 , 13451–13455. https://doi.org/10.1021/jacs.2c04096. Ianeselli, A.; Damla Tetiker; Stein, J.; Kühnlein, A.; Mast, C. B.; Braun, D.; T-Y Dora Tang. Non-Equilibrium Conditions inside Rock Pores Drive Fission, Maintenance and Selection of Coacervate Protocells. Nat. Chem. 2021 , 14 , 32–39. https://doi.org/10.1038/s41557-021-00830-y. Chen, H.; Bao, Y.; Li, X.; Chen, F.; Sugimura, R.; Zeng, X.; Xia, J. Cell Surface Engineering by Phase‐Separated Coacervates for Antibody Display and Targeted Cancer Cell Therapy. Angew. Chem. Int. Ed. 2024 , 63 , e202410566. https://doi.org/10.1002/anie.202410566. Welling, G. W.; Fries, H. Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein. FEBS Lett. 1985 , 182 , 81-4. https://doi.org/10.1016/0014-5793(85)81158-3. Liew, O. W.; Ling, S. S. M.; Lilyanna, S.; Zhou, Y.; Wang, P.; Chong, J. P. C.; Ng, Y. X.; Lim, A. E. S.; Leong, E. R. Y.; Lin, Q.; Lim, T. K.; Lin, Q.; Ng, E. M. W.; Ng, T. W.; Richards, A. M. Epitope-directed monoclonal antibody production using a mixed antigen cocktail facilitates antibody characterization and validation. Commun Biol . 2021 , 4 , 441. https://doi.org/10.1038/s42003-021-01965-x. Wang, C.; You, J.; Gao, M.; Zhang, P.; Xu, G.; Dou, H. Bio-Inspired Gene Carriers with Low Cytotoxicity Constructed via the Assembly of Dextran Nanogels and Nano-Coacervates. Nanomedicine 2020 , 15 , 1285–1296. https://doi.org/10.2217/nnm-2020-0065. Ketting, R. F. Dicer Functions in RNA Interference and in Synthesis of Small RNA Involved in Developmental Timing in C. Elegans. Gene Dev. 2001 , 15 , 2654–2659. https://doi.org/10.1101/gad.927801. Friedrich, M.; Aigner, A. Therapeutic SiRNA: State-of-The-Art and Future Perspectives. BioDrugs 2022 , 36 , 549–571. https://doi.org/10.1007/s40259-022-00549-3. Prazeres, D. M. F.; Monteiro, G. A. Plasmid Biopharmaceuticals. Microbiol. Spectr. 2014 , 2 , 10.1128/microbiolspec.PLAS0022-2014. https://doi.org/10.1128/microbiolspec.plas-0022-2014. Peng, Z. Current Status of Gendicine in China: Recombinant Human Ad-P53 Agent for Treatment of Cancers. Hum. Gene Ther. 2005 , 16 , 1016–1027. https://doi.org/10.1089/hum.2005.16.1016. Rennick, J. J.; Johnston, A. P. R.; Parton, R. G. Key Principles and Methods for Studying the Endocytosis of Biological and Nanoparticle Therapeutics. Nat. Nanotechnol. 2021 , 16 , 266–276. https://doi.org/10.1038/s41565-021-00858-8. Mendonça, M. C. P.; Radaic, A.; Garcia-Fossa, F.; da Cruz-Höfling, M. A.; Vinolo, M. A. R.; de Jesus, M. B. The in Vivo Toxicological Profile of Cationic Solid Lipid Nanoparticles. Drug Deliv. Transl. Res. 2019 , 10 , 34–42. https://doi.org/10.1007/s13346-019-00657-8. Husain, H.; Latief, U.; Ahmad, R. Pomegranate Action in Curbing the Incidence of Liver Injury Triggered by Diethylnitrosamine by Declining Oxidative Stress via Nrf2 and NFκB Regulation. Sci. Rep. 2018 , 8 , 8606. https://doi.org/10.1038/s41598-018-26611-1. Yoon, H. Y.; Son, S.; Lee, S. J.; You, D. G.; Yhee, J. Y.; Park, J. H.; Swierczewska, M.; Lee, S.; Kwon, I. C.; Kim, S. H.; Kim, K.; Pomper, M. G. Glycol Chitosan Nanoparticles as Specialized Cancer Therapeutic Vehicles: Sequential Delivery of Doxorubicin and Bcl-2 SiRNA. Sci. Rep. 2014 , 4 , 6878. https://doi.org/10.1038/srep06878. Yasmin, F.; Najeeb, H.; Naeem, U.; Moeed, A.; Atif, A. R.; Asghar, M. S.; Nimri, N.; Saleem, M.; Bandyopadhyay, D.; Krittanawong, C.; Fadelallah Eljack, M. M.; Tahir, M. J.; Waqar, F. Adverse Events Following COVID-19 MRNA Vaccines: A Systematic Review of Cardiovascular Complication, Thrombosis, and Thrombocytopenia. Immun. Inflamm. Dis. 2023 , 11 , e807. https://doi.org/10.1002/iid3.807. Additional Declarations Yes there is potential Competing Interest. Z. X., C. K., J. H., and J. X. have filed a US patent application based on this research, with a title: phase-separating peptide coacervates for nucleic acid transfection and mRNA vaccine delivery, and an application No: 63/788315. Supplementary Files SI2025818.pdf Supporting information file TOCfigure.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7559034","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":518680106,"identity":"e584eac2-f1f8-4643-90a6-f7571d1eca56","order_by":0,"name":"Jiang Xia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYBACxmYwJcHAz8DAxtgAFGhg4GEmTotkA7Fa4MDgALFamNuZnz382mYhb3z8jNnDGQw2shsO8B42wO8wNnNjmTMShtvO5JgbbmBIM95wgC85gYBfzKQlKiQYt93gMZN8wHA4ccMBHuMD+LWwf5OWMJCw3zwDrOU/MVqAKj9USCRukAAyNjAcAGsh4DCeMmmGMxLJM86klUnOMEg2nnmYxxiv9w37j2+T/NlWZ9vffnibZE+FnWzf8R5jCbxaGoABzQPngownFJHyIMf9IKBoFIyCUTAKRjgAAAUdRWRnYOojAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-8112-7625","institution":"The Chinese University of Hong Kong","correspondingAuthor":true,"prefix":"","firstName":"Jiang","middleName":"","lastName":"Xia","suffix":""},{"id":518680107,"identity":"09b7b12e-55db-485c-be84-bc3e20d92919","order_by":1,"name":"Zhiyi Xu","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Zhiyi","middleName":"","lastName":"Xu","suffix":""},{"id":518680108,"identity":"bb93021a-3f8a-49e7-b660-31bf83da31e5","order_by":2,"name":"Chaiyaporn Kuwentrai","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Chaiyaporn","middleName":"","lastName":"Kuwentrai","suffix":""},{"id":518680109,"identity":"e287a9b6-adc3-446a-bc02-f71c3342309c","order_by":3,"name":"Yaxin Hu","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Yaxin","middleName":"","lastName":"Hu","suffix":""},{"id":518680110,"identity":"ee5c1dc8-da20-4240-86d7-06622b5b2439","order_by":4,"name":"Renhao Li","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Renhao","middleName":"","lastName":"Li","suffix":""},{"id":518680111,"identity":"fdf07f88-cf44-4694-8010-9369105077ae","order_by":5,"name":"Pengchao Wang","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Pengchao","middleName":"","lastName":"Wang","suffix":""},{"id":518680112,"identity":"79700e98-c123-4ede-a489-dc009bc9d7d2","order_by":6,"name":"Zhong Zheng","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Zhong","middleName":"","lastName":"Zheng","suffix":""},{"id":518680113,"identity":"9f9645fc-6cfc-40e2-8113-acea51c05b0e","order_by":7,"name":"Liqiang Feng","email":"","orcid":"","institution":"Guangzhou Institutes of Biomedicine and Health","correspondingAuthor":false,"prefix":"","firstName":"Liqiang","middleName":"","lastName":"Feng","suffix":""},{"id":518680114,"identity":"ee831790-2c0d-4a56-9203-1927caea2e12","order_by":8,"name":"Jian-Dong Huang","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Jian-Dong","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-09-08 01:40:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7559034/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7559034/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91963156,"identity":"3944accc-da6e-44c3-8f04-3107eaeed871","added_by":"auto","created_at":"2025-09-23 08:00:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1062088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesign and characterization of phase-separating peptides.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e. Chemical structure of peptide WWKssKWW (\u003cstrong\u003eWWK\u003c/strong\u003e). \u003cstrong\u003eb\u003c/strong\u003e. Formation of \u003cstrong\u003eWWK\u003c/strong\u003e coacervates in DMEM and disintegration in the presence of reduced GSH. \u003cstrong\u003ec\u003c/strong\u003e. Size of \u003cstrong\u003eWWK\u003c/strong\u003ecoacervates measured by DLS. \u003cstrong\u003ed\u003c/strong\u003e. Confocal microscopy images showing the fusion of coacervate droplets within 75 seconds. \u003cstrong\u003ee\u003c/strong\u003e. Fluorescence Recovery After Photobleaching (FRAP) analysis of coacervates showing the fluorescence recovery of the photobleached spot in the coacervates. White circles show the photobleached spot.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7559034/v1/b1fecce7a32c709d351a4149.png"},{"id":91963151,"identity":"788c7c4e-b26d-479e-bae2-8dfabc7bfb7b","added_by":"auto","created_at":"2025-09-23 08:00:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1789905,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWWK coacervates efficiently deliver nucleic acids into HEK293 cells. a\u003c/strong\u003e. Schematic illustration showing nucleic acid transfection into cells by \u003cstrong\u003eWWK\u003c/strong\u003e coacervates followed by cytosolic release upon reaction with glutathione. \u003cstrong\u003eb\u003c/strong\u003e. Western blot results showing that siGAPDH delivered by different transfection agents induces knockdown of GAPDH in HEK293 cells. \u003cstrong\u003ec\u003c/strong\u003e. Western blot results showing the overexpression of mCherry in HEK293 cells 24 h or 48 h after transfection of a pCMV-mcherry plasmid (~4 kb) plasmid by different transfection agents. \u003cstrong\u003ed\u003c/strong\u003e. Western blot results showing the overexpression of mCherry-TRIM21 in HEK293 cells 24 h or 48 h after transfection of a pCI-neo.mCherry-TRIM21 plasmid (~7.6 kb) by different transfection agents. Unpaired t-test was used for statistical assessment. n=3; ***, P \u0026lt; 0.001. **, P \u0026lt; 0.01; *, P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7559034/v1/f7f1839b2e49d09007e5673b.png"},{"id":91963667,"identity":"c25405ad-cafe-4aba-85af-5156d875becd","added_by":"auto","created_at":"2025-09-23 08:08:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1338375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWWK coacervates’ cellular uptake pathways.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e. Confocal microscopy images of fluorescently labeled \u003cstrong\u003eWWK\u003c/strong\u003e coacervates and different cellular organelles labeled with organelle-specific dyes in HEK293 cells. The fluorescent signals along a line drawn across the cell were depicted to show the colocalization of the coacervates and organelles. \u003cstrong\u003eb.\u003c/strong\u003e Flow cytometry data showing cellular uptake of fluorescently labeled \u003cstrong\u003eWWK\u003c/strong\u003e coacervates in inhibitor-treated HEK293 cells. \u003cstrong\u003ec\u003c/strong\u003e. Flow cytometry data showing the uptake of fluorescently labeled \u003cstrong\u003eWWK\u003c/strong\u003e coacervates in inhibitor-treated MDA-MB-231 cells.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7559034/v1/331bd13ff8dcbb881c46a846.png"},{"id":91963162,"identity":"89d3784e-42a4-4e23-9886-b6c9a8638e82","added_by":"auto","created_at":"2025-09-23 08:00:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2765724,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of transfection agents in delivering luciferase mRNA to HEK293 cells. a\u003c/strong\u003e. Schematic illustration showing the comparison of \u003cstrong\u003eWWK\u003c/strong\u003e coacervates with commonly used transfection reagents, PEI, InstantFECT, LipoMessengerMax, and protamine to deliver a luciferase mRNA into HEK293 cells and the quantification of luminescence by overexpressed luciferase. \u003cstrong\u003eb\u003c/strong\u003e. Fluorescent well-plate imaging of luminescent signals by overexpressed luciferase in HEK293 cells transfected by luciferase mRNA (1 µg/well) and various concentrations (0.1 mg/ml, 0.3 mg/ml, 0.6 mg/ml, 0.9 mg/ml, 1.2 mg/ml) of \u003cstrong\u003eWWK\u003c/strong\u003e coacervates. \u003cstrong\u003ec\u003c/strong\u003e. Quantification of luciferase luminescence from various groups in \u003cstrong\u003eFigure 4b\u003c/strong\u003e (n=4). \u003cstrong\u003ed\u003c/strong\u003e. Viability of HEK293 cells treated by varying concentrations of \u003cstrong\u003eWWK\u003c/strong\u003e coacervates containing mRNA (1 µg/well). ns, no significance. \u003cstrong\u003ee\u003c/strong\u003e. Fluorescent well-plate imaging of luminescence transfected by luciferase mRNA (1 µg/well) delivered by \u003cstrong\u003eWWK\u003c/strong\u003ecoacervates (0.9 mg/ml), LipoMessengerMax, InstantFECT, PEI, and protamine, with the naked mRNA as the negative control. \u003cstrong\u003ef\u003c/strong\u003e. Quantification of luciferase luminescence of various groups in \u003cstrong\u003eFigure 4e\u003c/strong\u003e (n=4). \u003cstrong\u003eg\u003c/strong\u003e. ELISA of IL-1β from human PBMC treated with various mRNA formulations for 17 h (n=3). Experiments were repeated at least in duplicate. Unpaired t-test was used for statistical assessment. ***, P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7559034/v1/4ae8d8762a4ce90e17f5960b.png"},{"id":91963668,"identity":"6212f8e9-6648-42d5-9be9-46125e36443f","added_by":"auto","created_at":"2025-09-23 08:08:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3929834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcutaneous or intramuscular injection of mRNA delivery systems \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a\u003c/strong\u003e. Schematic illustration showing the formation of \u003cstrong\u003eWWK\u003c/strong\u003e/mRNA formulation followed by subcutaneous (S.C.) or intramuscular (I. M.) injection into mice. \u003cstrong\u003eb\u003c/strong\u003e. In vivo imaging of mice that received S. C. or I. M. administration of various luciferase mRNA formulations (10 µg mRNA/mouse). \u003cstrong\u003ec-f.\u003c/strong\u003e Quantification of the luminescence of IVIS images at 24 hours (\u003cstrong\u003ec\u003c/strong\u003e, S.C.; \u003cstrong\u003ee\u003c/strong\u003e, I. M.), and 48 hours (\u003cstrong\u003ed\u003c/strong\u003e, S. C.; \u003cstrong\u003ef\u003c/strong\u003e, I. M.) (n = 4). Experiments were repeated at least in duplicate. Unpaired t-test was used for statistical assessment. ****, P \u0026lt; 0.0001; ns, no significance. \u003cstrong\u003eg\u003c/strong\u003e. Histological liver images 24 hours after injection of various mRNA formulations, showing congestion in InstantFECT and LipoMessengerMax groups (arrows), but no signs of congestion in the \u003cstrong\u003eWWK\u003c/strong\u003ecoacervate group. Scale bars, 50 µm. \u003cstrong\u003eh\u003c/strong\u003e. Histological assessment of other major organs (heart, lung, spleen, and kidney) 24 hours after injection of mRNA formulations, showing no signs of toxicity. Scale bars, 50µm.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7559034/v1/6852db574448fa6eca9159ba.png"},{"id":91963152,"identity":"a56b3fde-c27a-48c5-9b80-d63ff50eae2f","added_by":"auto","created_at":"2025-09-23 08:00:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1977477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell-mediated immune responses of antigen mRNA and TLR 7 delivered by different transfection agents. a. \u003c/strong\u003eSchematic illustrating showing subcutaneous injection of OVA antigen mRNA (25 µg/mouse) or TLR7 to mice delivered by \u003cstrong\u003eWWK\u003c/strong\u003e coacervates. \u003cstrong\u003eb\u003c/strong\u003e. IFN-g ELISPOT images from mRNA delivered by \u003cstrong\u003eWWK\u003c/strong\u003ecoacervates, LipoMessengerMax, InstantFECT, and vehicle control injected mice.\u003cstrong\u003ec\u003c/strong\u003e. Quantification of ELISPOT images in \u003cstrong\u003eFigure 6b\u003c/strong\u003e (n = 4). \u003cstrong\u003ed\u003c/strong\u003e. IFN-g ELISPOT images from \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA, \u003cstrong\u003eWWK\u003c/strong\u003e/TLR7, \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA+TLR7, and TLR7 only groups.\u003cstrong\u003e e.\u003c/strong\u003e Quantification of ELISPOT wells from groups in \u003cstrong\u003eFigure 6d\u003c/strong\u003e (n=4). \u003cstrong\u003ef\u003c/strong\u003e. Schematic illustration showing intramuscular injections of spike antigen mRNA (15 µg/mouse) delivered by different mRNA formulations. \u003cstrong\u003eg\u003c/strong\u003e. IFN-g ELISPOT images from \u003cstrong\u003eWWK\u003c/strong\u003e/spike mRNA, Comirnaty mRNA vaccine, InstantFECT/spike mRNA, LipoMessengerMax/spike mRNA, and \u003cstrong\u003eWWK\u003c/strong\u003e coacervate only control groups. \u003cstrong\u003eh\u003c/strong\u003e. Quantification of ELISPOT wells from groups in \u003cstrong\u003eFigure 5g\u003c/strong\u003e(n=3). Experiments were repeated at least twice independently. Unpaired t-test was used for statistical assessment. ****, P \u0026lt; 0.0001; ***, P \u0026lt; 0.001; **, P \u0026lt; 0.01; *, P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7559034/v1/becfefa5ba26b7336e99e341.png"},{"id":91963157,"identity":"22597819-0ecd-4a99-9f87-306ef8b7fd4e","added_by":"auto","created_at":"2025-09-23 08:00:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1368796,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibody responses induced by SARS-CoV-2 spike mRNA formulations.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e. Schematic illustration showing intramuscular injections of spike antigen mRNA (25 µg/mouse) by various vaccine formulations. \u003cstrong\u003eb\u003c/strong\u003e. SARS-CoV-2 spike protein-specific antibody responses at 1 month after final vaccination from \u003cstrong\u003eWWK\u003c/strong\u003e/spike mRNA, Comirnaty mRNA vaccine, InstantFECT/spike mRNA, LipoMessengerMAX/spike mRNA, and \u003cstrong\u003eWWK\u003c/strong\u003e coacervate only control groups. \u003cstrong\u003ec\u003c/strong\u003e. SARS-CoV-2 spike protein-specific antibody responses 2 months after final vaccination from \u003cstrong\u003eWWK\u003c/strong\u003e/spike mRNA, Comirnaty mRNA vaccine, InstantFECT/spike mRNA, LipoMessengerMAX/spike mRNA, and \u003cstrong\u003eWWK\u003c/strong\u003e coacervate only control groups. \u003cstrong\u003ed\u003c/strong\u003e. Ratio of IgG1: IgG2A from various treatment groups after 2 months. The Kolmogorov-Smirnov test was used for statistical analysis. \u003cstrong\u003ee\u003c/strong\u003e. Neutralizing antibody titers against the WT strain of SARS-CoV-2 from various experimental groups 2 months after final vaccination.\u003cstrong\u003e f\u003c/strong\u003e. Neutralizing antibody titers against the Delta strain of SARS-CoV-2 from various experimental groups 2 months after final vaccination. \u003cstrong\u003eg\u003c/strong\u003e. Neutralizing antibody titers against the Alpha strain of SARS-CoV-2 from various experimental groups 2 months after final vaccination. **, P \u0026lt; 0.01. ns, no significance.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7559034/v1/6e8e6f072314537708a4f030.png"},{"id":91963158,"identity":"9f3d03f6-d4b0-4c4e-a103-d01d0e16150d","added_by":"auto","created_at":"2025-09-23 08:00:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1432950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcutaneous injections of OVA mRNA formulations to treat B16-OVA cancer.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e. Schematic illustration showing subcutaneous injections of different OVA mRNA formulations to mice carrying B16 tumors. \u003cstrong\u003eb\u003c/strong\u003e. Images of endpoint tumors from groups in \u003cstrong\u003eFigure 8a\u003c/strong\u003e(n = 5/group). \u003cstrong\u003ec-h\u003c/strong\u003e, tumor growth profiles in different groups: \u003cstrong\u003ec\u003c/strong\u003e, LipoMessengerMax only CTRL; \u003cstrong\u003ed\u003c/strong\u003e. LipoMessengerMax/OVA mRNA; \u003cstrong\u003ee\u003c/strong\u003e. \u003cstrong\u003eWWK\u003c/strong\u003ecoacervates CTRL; \u003cstrong\u003ef\u003c/strong\u003e. WWK/OVA mRNA; \u003cstrong\u003eg\u003c/strong\u003e. InstantFECT CTRL; \u003cstrong\u003eh\u003c/strong\u003e. InstantFECT OVA mRNA. \u003cstrong\u003ei\u003c/strong\u003e. Quantification of tumor volumes (n = 5/group). \u003cstrong\u003ej\u003c/strong\u003e. Survival curve of mice in different groups (n = 5/group). Significance was assessed by Mantel-Cox statistical analysis. ***, P \u0026lt; 0.001; **, P \u0026lt; 0.01; *, P \u0026lt; 0.05; ns, no significance. \u003cstrong\u003ek-l\u003c/strong\u003e, Quantification of tumor-infiltrating CD8-positive T cells (\u003cstrong\u003ek\u003c/strong\u003e) and CD4-positive T cells (\u003cstrong\u003el\u003c/strong\u003e) in different treatment groups. Unpaired t-test was used for statistical assessment. ****, P \u0026lt; 0.0001; ns, no significance.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7559034/v1/4d637015f6ee507ed0babe7e.png"},{"id":95798385,"identity":"70db6945-c84a-4d83-9ec3-22f8a95f2eaa","added_by":"auto","created_at":"2025-11-13 08:16:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20463623,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7559034/v1/1440ed87-437a-497e-b8ae-7a45fcd135d9.pdf"},{"id":91963153,"identity":"8d70ebae-b0b0-4b0f-90e2-57144e127965","added_by":"auto","created_at":"2025-09-23 08:00:52","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3863194,"visible":true,"origin":"","legend":"Supporting information file","description":"","filename":"SI2025818.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7559034/v1/79a5571bf27a52651c38ff54.pdf"},{"id":91963150,"identity":"b465ab00-5d25-4048-9645-93882ddb8b02","added_by":"auto","created_at":"2025-09-23 08:00:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":108431,"visible":true,"origin":"","legend":"","description":"","filename":"TOCfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7559034/v1/a188fe8c79ea447e28d5c530.docx"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nZ. X., C. K., J. H., and J. X. have filed a US patent application based on this research, with a title: phase-separating peptide coacervates for nucleic acid transfection and mRNA vaccine delivery, and an application No: 63/788315.","formattedTitle":"Phase-Separated Peptide Coacervates as Delivery Vehicles for mRNA Vaccines","fulltext":[{"header":"SIGNIFICANCE STATEMENT","content":"\u003cp\u003eA redox-responsive peptide made up of six residues forms coacervates, serving as an innovative transfection agent for nucleic acid therapeutics, with efficacy comparable to or exceeding that of lipofectamine, InstantFECT cationic liposome, and polyethylenimine. To our knowledge, with a molecular weight of 1152 Daltons, this six-residue peptide is the smallest transfection agent. This study also presents the first \u003cem\u003ein vivo\u003c/em\u003e investigation of coacervate-mediated mRNA vaccine delivery.\u003c/p\u003e\n"},{"header":"Introduction","content":"\u003cp\u003eIn recent decades, nucleic acids have emerged as highly appealing treatment modalities. DNA, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and messenger RNAs (mRNAs) have been widely utilized in the therapeutic landscape for treating various diseases. [1] Nucleic acid medications have the unique ability to modulate gene expression, offering precise treatment for a diverse array of conditions at the genetic level\u0026mdash;a capability often beyond the reach of traditional small-molecule and protein drugs. [2] In 1998, the FDA approved the first antisense oligonucleotide drug for treating cytomegalovirus retinitis in AIDS patients. [3] Despite its discontinuation due to reduced demand, this ground-breaking medication spurred the advancement of different therapeutic nucleic acids. [4-6] In 2018, patisiran became the inaugural siRNA therapy available on the market, targeting hereditary transthyretin amyloidosis (hATTR) and employing lipid nanoparticles (LNPs) for hepatic delivery. [7] By 2024, over 23 nucleic acid drugs had received global approval, with 55% designed for rare diseases and the remaining aimed at chronic conditions. [8]\u003c/p\u003e\n\u003cp\u003eWithin the array of nucleic acid treatments, mRNA therapy stands out as a particularly promising avenue. [9, 10] mRNA, a form of RNA, serves as a messenger transporting genetic instructions from DNA and orchestrating protein synthesis via translation. Upon entry into the patient\u0026apos;s cells, mRNA stimulates protein production, guiding the cells to generate particular proteins and internally modulating the human system through these functional proteins. [11] In recent years, mRNA-based therapies have garnered widespread attention due to their clinical benefits, including strong treatment outcomes, self-adjuvant properties, and relatively rapid clearance from the body. [12-14] mRNA therapies saw a surge in development during the COVID-19 pandemic, fueled by the impressive efficacy of mRNA vaccines. BNT162b2 from Pfizer-BioNTech and mRNA-1273 from Moderna received emergency approvals in 2020, showing over 94% efficacy against SARS-CoV-2. [15]\u003c/p\u003e\n\u003cp\u003eDespite these successes, delivering mRNA into target cells continues to be a significant challenge. [16] Naked mRNAs are unstable due to RNase-based degradation, requiring carriers to encapsulate and transport the mRNAs to their intended sites. Currently, the most widely used delivery methods for mRNA include viral vectors, lipid nanoparticles (LNPs), polymeric micelles, and metal-based nanoparticles. [17-22] Although viral vectors are highly effective for gene delivery, they carry risks of immunogenicity and insertional mutagenesis. [23, 24] LNPs provide excellent self-assembly capability and efficient delivery but may experience limited stability and potential issues in toxicity. [25] Polymeric micelles offer flexible modification capabilities and enhanced stability; however, they may encounter challenges related to high toxicity and low delivery efficiency. [26] Metal-based nanoparticles present unique properties, including high surface area and stability; however, they can induce adverse biological responses, necessitating careful design to minimize cytotoxicity and involving complex synthesis steps. [27] The limited options for transfection agents restrict the broad use of mRNAs as therapeutics in disease treatment. [28, 29] Therefore, developing novel reagents to effectively deliver mRNA drug payloads is essential for enhancing mRNA-based therapies. [30-33]\u003c/p\u003e\n\u003cp\u003eOn another note, phase-separated droplets, also known as coacervates, have emerged as a promising drug delivery system, offering excellent intracellular delivery capabilities, good biodegradability, low cellular toxicity, and stable drug release. [34-38] Conventional coacervates are formed through liquid-liquid phase separation (LLPS) of biomacromolecules, which is driven primarily by multivalent macromolecular interactions and the intrinsically disordered regions. [39, 40] LLPS has been found to be a fundamental process in cellular biology that leads to the formation of membrane-less organelles such as nucleoli, stress granules, and P-bodies with various biomolecules, including DNA, RNA, and proteins. [41] Molecules containing multiple weakly interacting motifs (for example, hydrophobic groups) and a hydrophilic linker, meeting the \u0026ldquo;stick-and-spacer model\u0026rdquo;, are prone to undergo LLPS and form microdroplets or coacervates [42]. Starting from coacervating polymers [43,44] to biomolecules and phase-separating peptides, including the mixture of the histone and DNA [45,46], L17E trimer [47], HBpep-SR [35, 48-52], and others [53-55], coacervates have recently been shown to encapsulate and deliver proteins into liposomes and cells. [56-58] Miserez and co-workers developed a peptide coacervate system (23 amino acids in length) responsive to glutathione (GSH) for the delivery of plasmids, mRNA, and proteins \u003cem\u003ein vitro\u003c/em\u003e with decent efficiency. [35, 48-52] However, the efficacy of peptide-based delivery systems \u003cem\u003ein vivo\u003c/em\u003e, together with the biological activity and immunological responses, is currently unknown. Moreover, this study employs a peptide with 23 amino acids to form coacervates; peptides of this length may trigger antibody responses, posing unknown risks [59, 60]. In another example, Dou and co-workers designed a DNA-peptide complex coacervate for intracellular plasmid delivery. [61]\u003c/p\u003e\n\u003cp\u003eWe, on the other hand, discovered that low-molecular-weight compounds can also form coacervates via LLPS, and coacervates formed by small molecules can spontaneously deliver antibodies into cells. In addition, the coacervates can release their cargo through various chemical reactions within the cell. For example, we designed a photo-responsive, phase-separating fluorescent molecule (PPFM) with a molecular weight of 666.6 Daltons based on pyrene that can undergo LLPS in the aqueous solution, carry proteins into cells, and release the cargo to the cytosol upon photo illumination [37]. We also designed a triphenylphosphine-based compound that can deliver proteins into cells and respond to an azide compound for cytosolic release [38]. These coacervate delivery systems offer innovative and universal solutions for delivering proteins to cultured cells. Some of the coacervates formed by small molecules have also shown the capability of encapsulating and delivering nucleic acids into cells, enabling gene transfection. However, the efficacy of coacervate-mediated gene transfection \u003cem\u003ein vivo\u003c/em\u003e, especially the comparison of coacervates to transfection agents, has not been addressed.\u003c/p\u003e\n\u003cp\u003eIn this study, we design a peptide consisting of only 6 amino acids, including hydrophobic residues and a positively charged lysine residue, and dimerized through a disulfide bond, with a molecular weight of 1152 Daltons. A meticulously designed balance of hydrophobicity, positive charge, and a flexible linker drives the coacervate formation via LLPS in aqueous solution. The peptide coacervates can encapsulate nucleic acids of different types and lengths into mammalian cells with high efficiency. The intracellular glutathione reduces the disulfide bond, dissociates the coacervates, and releases the complexed nucleic acid cargo into the cytosol, achieving highly efficient gene transfection. We then demonstrate that peptide coacervates can serve as vehicles for mRNA vaccines \u003cem\u003ein vivo\u003c/em\u003e, with transfection efficiencies equivalent to or higher than those of known transfection reagents, such as lipofectamine, InstantFECT (a cationic liposome), and polyethylenimine (PEI).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e1. Synthesis and selection of phase-separating peptide coacervates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecules containing spacer-and-sticker sequences are known to have the propensity to form coacervates via LLPS. Here, we designed disulfide-linked dimers of tripeptides with hydrophilic, positively charged spacers (KssK and RssR) and hydrophobic stickers (FF, FW, WF, WW) (\u003cstrong\u003eFigure 1a\u003c/strong\u003e). Tryptophan and tyrosine were selected as the stickers due to their hydrophobicity and the \u0026pi;-\u0026pi; stacking property. The incorporation of two lysine, histidine, or arginine residues, linked by a disulfide bond, served as the spacer, conferring positive charges and hydrophilicity to the peptide. The positive charge facilitates the attraction of nucleic acids with strong negative charges, and the disulfide bond enables cargo release within the cytoplasm. \u003c/p\u003e\n\u003cp\u003eWe synthesized a small library of six-residue peptide dimers and screened for their capability to form coacervates (\u003cstrong\u003eTable S1\u003c/strong\u003e). These peptides were first dissolved in DMSO at 100 mg/mL as stock solutions and then diluted to aqueous solutions. In PBS buffer, peptide WWKssKWW (\u003cstrong\u003eFigure 1a\u003c/strong\u003e) formed aggregates at room temperature, and at higher temperatures, the aggregates gradually turned into liquid droplets. Other peptides formed either aggregates or non-uniform droplets, despite the high similarity in the sequence (\u003cstrong\u003eFigure S1\u003c/strong\u003e). Therefore, we chose the peptide WWKssKWW (hereafter referred to as \u003cstrong\u003eWWK\u003c/strong\u003e) for the following studies. The \u003cstrong\u003eWWK\u003c/strong\u003e coacervates were formed at higher temperature and concentration (\u003cstrong\u003eFigure S2a\u003c/strong\u003e). The size of droplets became smaller with the increase of the 1,6-HD concentration, suggesting that hydrophobic interactions play a key role in LLPS (\u003cstrong\u003eFigure S2b\u003c/strong\u003e). After 1 \u0026mu;L of the stock solution was added to 100 \u0026mu;L of Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) at 37℃, the medium gradually became turbid, and uniform droplets could be observed under the microscope (\u003cstrong\u003eFigure 1b\u003c/strong\u003e). The coacervates showed sizes typically ranging from 300 to 1300 nm based on dynamic light scattering (DLS) (\u003cstrong\u003eFigure 1c\u003c/strong\u003e). The \u003cstrong\u003eWWK\u003c/strong\u003e coacervates displayed dynamic fusion within minutes, showing their liquid properties (\u003cstrong\u003eFigure 1d\u003c/strong\u003e). After labelling the coacervates with Nile Red, we photobleached a region of the droplet and observed the recovery of the signal in the photobleached region to about 70% in 200 seconds (\u003cstrong\u003eFigure 1e\u003c/strong\u003e). Turbidity of the coacervate solution decreased in the first 30 minutes and then gradually plateaued over the following hours (\u003cstrong\u003eFigures S2c and S2d\u003c/strong\u003e), indicating that the coacervates were sufficiently stable for the following cell-based experiments. Ionic strength did not significantly affect the coacervate formation (\u003cstrong\u003eFigure S2e\u003c/strong\u003e). In the presence of 10 mM reduced glutathione (GSH), the turbid solution became clear, and coacervates disappeared, showing that disulfide reduction disintegrated the coacervates at 37℃ (\u003cstrong\u003eFigures 1b\u003c/strong\u003e). 5 mM GSH was adequate for complete disintegration of 1 mg/mL \u003cstrong\u003eWWK\u003c/strong\u003e coacervates in the medium based on the HPLC traces (\u003cstrong\u003eFigure S3\u003c/strong\u003e), showing the feasibility of dissociating coacervates in the cytosol. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Encapsulation of nucleic acids for intracellular delivery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we investigated whether \u003cstrong\u003eWWK\u003c/strong\u003e coacervates could encapsulate nucleic acids, using single-stranded DNA labeled with a fluorescent dye, ROX (ROX-ssDNA) as the cargo. Fluorescent microscopy analysis showed successful encapsulation of ROX-ssDNA in \u003cstrong\u003eWWK\u003c/strong\u003e coacervates (\u003cstrong\u003eFigure S4a\u003c/strong\u003e). Next, 15 \u0026mu;g of plasmids with a size of 7.6 Kb were encapsulated in 1 mg/mL \u003cstrong\u003eWWK\u003c/strong\u003e coacervates in 25 \u0026mu;L Opti-MEM medium at 37℃, indicating a high loading capacity of approximately 60% and the encapsulation efficiency close to 100% (\u003cstrong\u003eFigure S4b\u003c/strong\u003e). These data show that \u003cstrong\u003eWWK\u003c/strong\u003e coacervates can encapsulate nucleic acids, primarily driven by the charge-charge and hydrophobic interactions. \u003c/p\u003e\n\u003cp\u003eBefore cellular experiments, we tested the cytotoxicity of the \u003cstrong\u003eWWK\u003c/strong\u003e coacervates based on the lactate dehydrogenase (LDH) assay. \u003cstrong\u003eWWK\u003c/strong\u003e coacervates showed minimal cytotoxicity to HEK293 cells at concentrations below 0.9 mg/mL (\u003cstrong\u003eFigure S5\u003c/strong\u003e). Next, we incubated ROX-ssDNA-loaded \u003cstrong\u003eWWK\u003c/strong\u003e coacervates at 0.9 mg/mL with HEK293 cells for 24 hours and observed that many droplets carrying the fluorescent ROX signal were taken up by the cells. The ROX-ssDNA was also found in the cytoplasm, suggesting its release into the cytoplasm (\u003cstrong\u003eFigure S6a\u003c/strong\u003e). 3D confocal microscopy images showed a two-step uptake procedure: within 6 hours, \u003cstrong\u003eWWK\u003c/strong\u003e coacervates primarily adhered to the cell membrane, and after 12 hours, many coacervates were endocytosed into the cells (\u003cstrong\u003eFigure S6b\u003c/strong\u003e). The delivery efficiency of \u003cstrong\u003eWWK\u003c/strong\u003e coacervates was comparable to that of Lipofectamine\u0026reg; 3000 and higher than PEI, based on the flow cytometry results (\u003cstrong\u003eFigure S6c\u003c/strong\u003e). These data show that \u003cstrong\u003eWWK\u003c/strong\u003e coacervates can encapsulate and deliver ssDNA into cells with minimal cytotoxicity and decently high efficiency. \u003c/p\u003e\n\u003cp\u003eNext, we examined the delivery of siRNA molecules with the \u003cstrong\u003eWWK\u003c/strong\u003e coacervates, focusing on the intracellular function of the nucleic acids (\u003cstrong\u003eFigure 2a\u003c/strong\u003e). Small interfering RNA (siRNA) is a promising therapeutic modality with the ability to silence disease-related genes by targeting specific messenger RNAs (mRNAs) and facilitating their degradation with the assistance of the enzyme Dicer. [62, 63] In 2018, the first siRNA drug, patisiran, was approved for the treatment of hereditary transthyretin-mediated amyloidosis. [7] Here, we delivered a GAPDH-specific siRNA (siGAPDH) as a model. Western blotting analysis demonstrated that the expression level of GAPDH declined to approximately 70% of its original level within 24 hours following the addition of siGAPDH-loaded \u003cstrong\u003eWWK\u003c/strong\u003e coacervates. The knockdown efficiency of GAPDH was sustained for up to 48 hours. In contrast, the commercial transfection agent, lipofectamine\u003csup\u003e\u0026reg;\u003c/sup\u003e 3000, only reduced 25% of the GAPDH expression level (\u003cstrong\u003eFigure 2b\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eThen, we utilized \u003cstrong\u003eWWK\u003c/strong\u003e coacervates to deliver plasmids for protein overexpression in cells. Plasmids have been used as biopharmaceuticals for the treatment of hereditary disorders, multifactorial diseases, and the prevention of infectious diseases. [64] The first gene therapy product approved for clinical use, Gendicine, is a plasmid that delivers the tumor suppressor p53 gene to treat head and neck squamous cell carcinoma. [65] Two plasmids of different sizes were used as the cargo: a plasmid expressing mCherry protein, pCMV-mCherry (~4 kb), and the second expressing mCherry-TRIM21 recombinant protein (a model protein), pCI-neo.mCherry-TRIM21 (~7.6 kb). Fluorescent microscopy images show that in all three groups, mCherry was successfully expressed (\u003cstrong\u003eFigure S7\u003c/strong\u003e). Western blotting analysis revealed that the expression level of mCherry in the \u003cstrong\u003eWWK\u003c/strong\u003e coacervate (0.3 mg/mL) group was 1.5 times higher than that in the PEI group and about 1.2 times higher than the lipofectamine\u0026reg; 3000 (Lipo3000) group in 48 hours (\u003cstrong\u003eFigure 2c\u003c/strong\u003e). Similarly, the \u003cstrong\u003eWWK\u003c/strong\u003e coacervate groups gave higher expression levels of mCherry-TRIM21 than the other two groups (\u003cstrong\u003eFigure 2d\u003c/strong\u003e). In this experiment, we observed a surprisingly low expression level in the Lipo3000 group for unknown reasons (\u003cstrong\u003eFigure 2d\u003c/strong\u003e). Similar results were obtained in another cell line, MDA-MB-231 cells (\u003cstrong\u003eFigure S8a\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe also explored the delivery of mRNA by \u003cstrong\u003eWWK\u003c/strong\u003e coacervates. An EGFP mRNA was delivered into HEK293 cells and MDA-MB-231 cells with \u003cstrong\u003eWWK\u003c/strong\u003e coacervates, and the expression level of EGFP in the transfected cells was found to be similar to that of the Lipo3000 group in HEK293 cells (\u003cstrong\u003eFigure 2e\u003c/strong\u003e), while notably higher than the Lipo3000 group in MDA-MB-231 cells (\u003cstrong\u003eFigure \u003c/strong\u003e\u003cstrong\u003eS8b\u003c/strong\u003e), suggesting a possible cell type variation. Altogether, these data show that \u003cstrong\u003eWWK\u003c/strong\u003e coacervates can deliver a variety of nucleic acids into cells, with efficiency equivalent to or higher than Lipo3000 and PEI. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Cellular uptake mechanism of WWK coacervates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we explored the mechanism by which \u003cstrong\u003eWWK\u003c/strong\u003e coacervates enter cells, using fluorescent trackers and antibodies specifically targeting F-actin, early endosomes, late endosomes, lysosomes, mitochondria, and the endoplasmic reticulum (ER). \u003cstrong\u003eWWK\u003c/strong\u003e coacervates loaded with fluorescent ssDNA were used in this case. Based on the confocal images of the colocalization experiments, polymerization of F-actin was found around the coacervates on the surface of the cell, which seems to facilitate the coacervates\u0026rsquo; entry into the cells. During the early stage of cellular uptake, the fluorescent signal of \u003cstrong\u003eWWK\u003c/strong\u003e coacervates partially overlapped with the Rab5A signal, a marker of the early endosome. After 6 hours, based on the signal of Rab7A, a late endosome marker, and a LysoTracker, we found no noticeable overlap between the coacervates and late endosome or lysosome. This result suggests that although some of the coacervates may be endocytosed via the endosomal uptake, they may have escaped from the endosomes (\u003cstrong\u003eFigure 3a\u003c/strong\u003e). In addition, \u003cstrong\u003eWWK\u003c/strong\u003e coacervates did not colocalize with mitochondria and ER (\u003cstrong\u003eFigure S12\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eInhibitors targeting various endocytosis pathways, including dynasore (an inhibitor of dynamin-mediated endocytosis), amiloride (an inhibitor of pinocytosis), chloroquine (an inhibitor of clathrin-mediated endocytosis), cytochalasin B (an inhibitor of pinocytosis and phagocytosis), and 2-deoxy-D-glucose (an inhibitor of energy-dependent endocytosis), were also utilized to evaluate the uptake mechanisms. [66] Flow cytometry results indicated that the serum-starved group exhibited a reduction of over 50% in coacervate uptake in HEK293 cells, compared to the group with serum, suggesting that \u003cstrong\u003eWWK\u003c/strong\u003e coacervates mainly enter cells via energy-dependent pathways. The dynasore-treated group showed a pronounced decline in coacervate uptake, showing that \u003cstrong\u003eWWK\u003c/strong\u003e coacervates partially penetrated the cell membrane via the dynamin-mediated endocytosis pathway. In contrast, the amiloride- and cytochalasin B-treated groups displayed an uptake level comparable to that of the positive control, indicating that macropinocytosis is not the major cellular uptake pathway in HEK293 cells (\u003cstrong\u003eFigure 3b\u003c/strong\u003e). However, the uptake route was different in MDA-MB-231 cells. Cytochalasin B significantly inhibited the uptake of \u003cstrong\u003eWWK\u003c/strong\u003e coacervates into MDA-MB-231 cells, indicating that pinocytosis was the major uptake pathway. Instead, the dynamin-mediated endocytosis inhibitor dynasore did not show any influence on the coacervate uptake (\u003cstrong\u003eFigure 3c\u003c/strong\u003e). Therefore, the uptake mechanism of \u003cstrong\u003eWWK\u003c/strong\u003e coacervates differs in various cells. The major pathway for \u003cstrong\u003eWWK\u003c/strong\u003e coacervates to enter HEK293 cells is dynamin-mediated endocytosis, but for MDA-MB-231 cells pinocytosis dominates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Comparison of mRNA delivery \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we compared \u003cstrong\u003eWWK\u003c/strong\u003e coacervates with commercially available transfection agents, including PEI, InstantFECT, Lipofectamine MessengerMax (LipoMessengerMax), and protamine, in mRNA delivery to HEK293 cells (\u003cstrong\u003eFigure 4a\u003c/strong\u003e). In addition, we applied various concentrations of \u003cstrong\u003eWWK\u003c/strong\u003e coacervates for the transfection of the firefly luciferase mRNA and analyzed the luminescence 24 hours after the transfection (\u003cstrong\u003eFigure 4b\u003c/strong\u003e). The empty vector induced negligible luminescence and thus was used as a negative control. When 1 \u0026micro;g of luciferase mRNA was used as the cargo, \u003cstrong\u003eWWK\u003c/strong\u003e coacervate transfection induced strong luminescence intensities, which peaked at the peptide concentration of 0.9 mg/mL (\u003cstrong\u003eFigure 4c and Figure S10a\u003c/strong\u003e). Cell viability assay also showed that \u003cstrong\u003eWWK\u003c/strong\u003e coacervates up to 0.9 mg/mL were well-tolerated by cells (\u003cstrong\u003eFigure S5\u003c/strong\u003e). Consequently, this peptide concentration was chosen as the optimal condition for further experiments. Cytotoxicity may be an important reason for the reduced level of luminescence at \u003cstrong\u003eWWK\u003c/strong\u003e concentrations above 0.9 mg/mL (\u003cstrong\u003eFigures 4b and 4c\u003c/strong\u003e). Surprisingly, compared to the commercial transfection agents, coacervate-mediated mRNA transfection at 0.9 mg/mL of \u003cstrong\u003eWWK\u003c/strong\u003e produced significantly stronger luciferase signal than LipoMessengerMax, InstantFECT cationic liposome, PEI, and protamine (\u003cstrong\u003eFigures 4e and 4f\u003c/strong\u003e). Naked mRNA alone also induced negligible luminescence (\u003cstrong\u003eFigure 4f and Figure S10b\u003c/strong\u003e). In dendritic cells (DC2.4 cells), \u003cstrong\u003eWWK\u003c/strong\u003e coacervate-mediated mRNA transfection yielded marked luciferase expression, whereas the luminescence signal in the LipoMessengerMax group was undetectable (\u003cstrong\u003eFigure S11\u003c/strong\u003e). Taken together, these data show that \u003cstrong\u003eWWK\u003c/strong\u003e coacervates can efficiently deliver mRNA into cells, with an efficiency on par with or higher than commercial transfection agents.\u003c/p\u003e\n\u003cp\u003eNext, we measured the inflammatory profile of different mRNA/transfection agent formulations by measuring the level of the inflammatory cytokine IL-1\u0026beta; in the supernatants of human Peripheral Blood Mononuclear Cell (PBMC) cultures after mRNA transfection (\u003cstrong\u003eFigure 4g\u003c/strong\u003e). Liposomes and lipid nanoparticles induced high levels of IL-1\u0026beta;, whereas \u003cstrong\u003eWWK\u003c/strong\u003e/mRNA formulation induced a low level of IL-1\u0026beta; release, similar to the naked mRNA control. [17] Although inflammation partly contributes to the immunogenicity of mRNA vaccines, excessive responses result in toxicities that are not favorable for gene therapy. This result suggests that \u003cstrong\u003eWWK\u003c/strong\u003e coacervates may have a more favorable safety profile \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. Transfection of luciferase mRNA using WWK coacervates in animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we investigated the efficacy of \u003cstrong\u003eWWK\u003c/strong\u003e coacervate-mediated mRNA transfection in animals using a \u003cstrong\u003eWWK\u003c/strong\u003e concentration of 0.9 mg/mL and 10 mg luciferase mRNA per animal. We performed an \u003cem\u003ein vivo\u003c/em\u003e imaging using the IVIS (In Vivo Imaging System) 24 and 48 hours after subcutaneous or intramuscular injections of various luciferase mRNA formulations (\u003cstrong\u003eFigure 5a\u003c/strong\u003e). Consistent with the \u003cem\u003ein vitro\u003c/em\u003e transfection results, the \u003cstrong\u003eWWK/\u003c/strong\u003emRNA formulation achieved robust luciferase expression after subcutaneous and intramuscular injections (\u003cstrong\u003eFigure 5b\u003c/strong\u003e). At 24 hours, subcutaneously injected \u003cstrong\u003eWWK\u003c/strong\u003e/mRNA formulation induced luminescence in mice with significantly higher levels than those of the PEI/mRNA formulation and vehicle controls, comparable to that of the LipoMessengerMax /mRNA formulation, albeit lower than InstantFECT/mRNA formulation (\u003cstrong\u003eFigures 5c and Figure S12\u003c/strong\u003e). At 48 hours, the \u003cstrong\u003eWWK\u003c/strong\u003e/mRNA formulation administered through the subcutaneous route produced a luminescence level comparable to that of the InstantFECT/mRNA formulation, and significantly higher than the LipoMessengerMax/mRNA formulation, PEI/mRNA formulation, and vehicle controls (\u003cstrong\u003eFigure 5d\u003c/strong\u003e). For the intramuscular administration route, the \u003cstrong\u003eWWK\u003c/strong\u003e/mRNA formulation generated a luminescence level comparable to the vehicle controls, InstantFECT/mRNA, PEI/mRNA, and LipoMessengerMax/mRNA formulations at 24 hours (\u003cstrong\u003eFigure 5e\u003c/strong\u003e). At 48 hours, the three groups, \u003cstrong\u003eWWK\u003c/strong\u003e coacervates, InstantFECT, and LipoMessengerMax, were comparable, higher than the vehicle control and the PEI group (\u003cstrong\u003eFigure 5f\u003c/strong\u003e). Importantly, the mice that received \u003cstrong\u003eWWK\u003c/strong\u003e coacervate injections did not exhibit any signs of weight loss, organ abnormalities, discomfort, or side effects (\u003cstrong\u003eFigures S13 and S14\u003c/strong\u003e). In contrast, subcutaneous and intramuscular injections of the PEI/mRNA formulation led to severe discomfort, and some mice died by 24 hours. A histological analysis of mouse organs at the 24-hour time point revealed that the InstantFECT/mRNA and LipoMessengerMax/mRNA formulations resulted in congestive hepatopathy in the liver, whereas the \u003cstrong\u003eWWK\u003c/strong\u003e/mRNA group remained the same as the healthy animals (\u003cstrong\u003eFigure 5g\u003c/strong\u003e). Although most cases of congestive hepatopathy due to lipid carriers are asymptomatic, some cases may cause abdominal discomfort. [67-69] No other organs exhibited abnormalities (\u003cstrong\u003eFigure 5h\u003c/strong\u003e). These findings show that \u003cstrong\u003eWWK\u003c/strong\u003e coacervates are safe vehicles to deliver mRNA vaccines with high efficiency. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6. Cell-mediated immune responses induced by different ovalbumin (OVA) mRNA and SARS-CoV-2 spike mRNA formulations \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we evaluated the cell-mediated immune responses of the \u003cstrong\u003eWWK\u003c/strong\u003e coacervate-delivered mRNA vaccines, with InstantFECT and LipoMessengerMax as controls. The antigen-specific cellular response to biological vaccines could indicate the level of immune protection. To study the immune responses of \u003cstrong\u003eWWK \u003c/strong\u003ecoacervate-delivered OVA mRNA vaccines and adjuvants, we performed ELISPOT analysis for interferon-g (IFN-g) using the isolated splenocytes of mice subcutaneously immunized with different OVA mRNA vaccine formulations, including \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA, LipoMessengerMax/OVA mRNA, \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA plus TLR7, \u003cstrong\u003eWWK\u003c/strong\u003e/TLR7, TLR7 alone, and vehicle controls (\u003cstrong\u003eFigure 6a\u003c/strong\u003e). Interestingly, the \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA formulation could stimulate a significantly greater OVA-specific cellular response than the LipoMessengerMax/OVA mRNA formulation and vehicle controls, which was comparable to that of the InstantFECT/OVA mRNA formulation (\u003cstrong\u003eFigures 6b and 6c\u003c/strong\u003e). These findings suggest that \u003cstrong\u003eWWK\u003c/strong\u003e coacervates effectively deliver the OVA mRNA vaccine into antigen-presenting cells. Additionally, \u003cstrong\u003eWWK\u003c/strong\u003e coacervates permitted TLR7 adjuvant delivery, and the combined subcutaneous treatment with OVA mRNA augmented OVA-specific cellular responses in the immunized mice compared to the \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA alone (\u003cstrong\u003eFigures 6d and 6e\u003c/strong\u003e). \u003cstrong\u003eWWK\u003c/strong\u003e coacervates also facilitated the efficient intramuscular delivery of a SARS-CoV-2 spike mRNA and produced spike-specific cellular responses comparable to those of the commercial Comirnaty mRNA vaccine (\u003cstrong\u003eFigures 6f-6h\u003c/strong\u003e). \u003c/p\u003e\n\u003cp\u003eThe cell-mediated immune response in the \u003cstrong\u003eWWK\u003c/strong\u003e/spike mRNA group at this dosage was adequate to induce anti-spike antibodies, albeit the antibody levels were lower than those of the Comirnaty mRNA vaccine group (\u003cstrong\u003eFigure S15\u003c/strong\u003e), suggesting that different mRNA carriers may adopt different vaccine uptake mechanisms. Overall, these results demonstrate that \u003cstrong\u003eWWK\u003c/strong\u003e coacervate is a viable vehicle for mRNA vaccines and the adjuvant TLR7 \u003cem\u003ein vivo\u003c/em\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e7. Antibody responses induced by coacervate-delivered SARS-CoV-2 spike mRNA \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe generation of antibody responses, through antigen-specific antibody production, can indicate immune protection against infectious diseases, such as SARS-CoV-2. At a dosage of 25 \u0026micro;g/mouse, spike mRNA was delivered by \u003cstrong\u003eWWK\u003c/strong\u003e coacervates, LipoMessgenerMAX, InstantFECT, and Comirnaty via intramuscular injection at days 0 and 21 (\u003cstrong\u003eFigure 7a\u003c/strong\u003e). We performed serum ELISA after one and two months of immunization. The \u003cstrong\u003eWWK\u003c/strong\u003e/spike mRNA immunizations generated a significant level of spike-specific IgG antibodies, comparable to those of the InstantFECT/spike mRNA group (\u003cstrong\u003eFigures 7b and 7c\u003c/strong\u003e). Nonetheless, the Comirnaty COVID-19 mRNA vaccine produced the highest level of antibodies in the animal (\u003cstrong\u003eFigures 7b and 7c\u003c/strong\u003e). This result differs from the cellular data, which showed that the Comirnaty formulation had comparable levels of cellular immunity (\u003cstrong\u003eFigures 6g and 6h\u003c/strong\u003e). The heightened humoral response produced by the Comirnaty vaccine \u003cem\u003ein vivo\u003c/em\u003e highlights the power of commercial vaccine development, including optimization of compound size, temperature, pH, aggregation, and moiety modifications during the preparation stage [70]. The IgG1: IgG2a ratios amongst the antibodies generated by the different spike mRNA vaccines were comparable, which suggests equivalent levels of Th2-based and Th1-based immunity, respectively (\u003cstrong\u003eFigure 7d\u003c/strong\u003e). Neutralization antibodies against viral infections remain the gold standard indicator of antiviral immune protection generated by vaccines. All formulations generated high levels of neutralization antibodies against the wild type (WT), Delta, and Alpha SARS-CoV-2 strains, judged by the higher serum dilution factor to achieve 50% of the inhibition (\u003cstrong\u003eFigure 7e-g\u003c/strong\u003e). The \u003cstrong\u003eWWK\u003c/strong\u003e/mRNA vaccine generated a similar level of neutralizing antibodies as InstantFECT and LipoMessengerMAX, but lower than the Comirnaty vaccine. Altogether, the \u003cstrong\u003eWWK\u003c/strong\u003e/mRNA vaccine induced potent antigen-specific humoral immunity through intramuscular immunization, highlighting their promise in the context of infectious disease prevention.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. WWK/OVA mRNA immunization against B16-OVA cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLastly, we examine the therapeutic potential of \u003cstrong\u003eWWK\u003c/strong\u003e coacervates for delivering mRNA vaccine payloads for cancer treatment. We established a B16-OVA subcutaneous tumor model (2 \u0026acute; 10⁵ cells/per mouse) to compare the efficacy of various OVA mRNA formulations (namely, OVA mRNA delivered by \u003cstrong\u003eWWK\u003c/strong\u003e coacervates, InstantFECT, LipoMessengerMAX, and control groups without mRNA) (\u003cstrong\u003eFigure 8a\u003c/strong\u003e). The \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA group demonstrated a significant tumor suppression (average tumor size = 42.5 mm\u003csup\u003e3\u003c/sup\u003e), compared to the LipoMessengerMAX/mRNA group (average tumor size = 292 mm\u003csup\u003e3\u003c/sup\u003e), the LipoMessengerMAX only group (average tumor size = 1256 mm\u003csup\u003e3\u003c/sup\u003e), and the \u003cstrong\u003eWWK\u003c/strong\u003e peptide only group (average tumor size = 1024 mm\u003csup\u003e3\u003c/sup\u003e) by the tumor endpoint (\u003cstrong\u003eFigures 8b-8i\u003c/strong\u003e). The tumor sizes and survival rates were comparable between the \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA group and the InstantFECT/mRNA group (\u003cstrong\u003eFigures 8i and 8j\u003c/strong\u003e). The \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA treatment significantly prolonged the tumor-free survival rate in mice (2/5 mice survived) compared to all vehicle controls (all mice died) by day 40 (\u003cstrong\u003eFigure 8j\u003c/strong\u003e). Tumor-infiltrating cytotoxic CD8+ T cells may exhibit potent tumor-killing capabilities, leading to the observed cancer eradication. Therefore, we conducted flow cytometry analysis on the harvested tumors from the various groups at the tumor endpoint. A significantly elevated level of CD8+ T cells (but not CD4+ T cells) was detected in the \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA group compared to the LipoMessengerMAX/OVA mRNA group and the vehicle control groups (\u003cstrong\u003eFigure 8k and 8l\u003c/strong\u003e). These data suggest that cytotoxic T cells are the key players responsible for the cancer-suppressive tumor environment induced by the \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA treatment. \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe field of therapeutic biomacromolecules, like nucleic acids, faces challenges in crossing cell membranes because of their large size and high electronegativity, requiring the use of delivery vehicles. Existing options for delivery vehicles are limited in the market and have issues such as potential toxicity and suboptimal delivery efficacy. Here, we introduce a coacervate system designed for the efficient and safe delivery of nucleic acids. \u003cstrong\u003eWWK\u003c/strong\u003e coacervates exhibit high encapsulation efficiency and loading capacity for nucleic acids, minimal cytotoxicity, a responsive release mechanism triggered by redox signals, and effective cytosolic delivery. Remarkably, the \u003cstrong\u003eWWK\u003c/strong\u003e coacervate delivery system shows superior delivery of siRNA, mRNA, DNA, and plasmids compared to commercially available transfection agents. In animal models, the \u003cstrong\u003eWWK\u003c/strong\u003e coacervates effectively convey OVA mRNA, SARS-CoV-2 spike mRNA, and TLR7 adjuvants \u003cem\u003ein vivo\u003c/em\u003e, triggering cell-mediated immune responses and antibody responses. Employed as a protective vaccine against SARS-CoV-2, \u003cstrong\u003eWWK\u003c/strong\u003e/spike mRNA therapy elicits heightened levels of antigen-specific antibodies, neutralizing WT, Delta, and Alpha strains of SARS-CoV-2. In the realm of cancer immunotherapy, \u003cstrong\u003eWWK\u003c/strong\u003e/OVA mRNA treatment efficiently restrains B16-OVA tumor progression and significantly extends the lifespan of tumor-bearing mice. This innovative research sheds light on the development of peptide coacervate-based mRNA vaccines, providing valuable insights for potential clinical translation.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interest statement\u003c/strong\u003e: Z. X., C. K., J. H., and J. X. have filed a US patent application based on this research, with a title: phase-separating peptide coacervates for nucleic acid transfection and mRNA vaccine delivery, and an application No: 63/788315.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eACKNOWLEDGMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially funded by grants from the University Grants Committee of Hong Kong (GRF grants 14306222, 14301922, and 14307523), C5026-24G, R5013-19, and CUHK (ICSG, CRIMS, 1+1+1 CUHK/CUHKSZ grant, and Direct Grant 4053563).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBelgrad, J.; Fakih, H. H.; Khvorova, A. Nucleic Acid Therapeutics: Successes, Milestones, and Upcoming Innovation. \u003cem\u003eNuc. Acid Ther.\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e34\u003c/em\u003e, 52\u0026ndash;72. https://doi.org/10.1089/nat.2023.0068.\u003c/li\u003e\n\u003cli\u003eMoccia, M.; Pascucci, B.; Saviano, M.; Maria Teresa Cerasa; Terzidis, M. A.; Chryssostomos Chatgilialoglu; Masi, A. Advances in Nucleic Acid Research: Exploring the Potential of Oligonucleotides for Therapeutic Applications and Biological Studies. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e25\u003c/em\u003e, 146\u0026ndash;146. https://doi.org/10.3390/ijms25010146.\u003c/li\u003e\n\u003cli\u003eLundin, K. E.; Gissberg, O.; Smith, C. I. E. Oligonucleotide Therapies: The Past and the Present. \u003cem\u003eHum. Gene Ther.\u003c/em\u003e \u003cstrong\u003e2015\u003c/strong\u003e, \u003cem\u003e26\u003c/em\u003e, 475\u0026ndash;485. https://doi.org/10.1089/hum.2015.070.\u003c/li\u003e\n\u003cli\u003eZhang, L.; Tang, X.; Xi, Z.; Chattopadhyaya, J. \u003cem\u003eNucleic Acids in Medicinal Chemistry and Chemical Biology\u003c/em\u003e; Wiley, 2022. https://doi.org/10.1002/9781119692799.\u003c/li\u003e\n\u003cli\u003eKulkarni, J. A.; Witzigmann, D.; Thomson, S. B.; Chen, S.; Leavitt, B. R.; Cullis, P. R.; van der Meel, R. The Current Landscape of Nucleic Acid Therapeutics. \u003cem\u003eNat. Nanotechnol.\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e16\u003c/em\u003e, 630\u0026ndash;643. https://doi.org/10.1038/s41565-021-00898-0.\u003c/li\u003e\n\u003cli\u003eEgli, M.; Manoharan, M. Chemistry, Structure and Function of Approved Oligonucleotide Therapeutics. \u003cem\u003eNucleic Acids Res\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e51\u003c/em\u003e, 2529\u0026ndash;2573. https://doi.org/10.1093/nar/gkad067.\u003c/li\u003e\n\u003cli\u003eAdams, D. Patisiran, an Investigational RNAi Therapeutic for Patients with Hereditary Transthyretin-Mediated (HATTR) Amyloidosis : Results from the Phase 3 APOLLO Study. \u003cem\u003eRev. Med. Interne. \u003c/em\u003e\u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e39\u003c/em\u003e, A185. https://doi.org/10.1016/j.revmed.2018.10.163.\u003c/li\u003e\n\u003cli\u003eLiu, C.; Shi, Q.; Huang, X.; Koo, S.; Kong, N.; Tao, W. MRNA-Based Cancer Therapeutics. \u003cem\u003eNat. Rev. Cancer \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e23\u003c/em\u003e, 1\u0026ndash;18. https://doi.org/10.1038/s41568-023-00586-2.\u003c/li\u003e\n\u003cli\u003eKowalski, P. S.; Rudra, A.; Miao, L.; Anderson, D. G. Delivering the Messenger: Advances in Technologies for Therapeutic MRNA Delivery. \u003cem\u003eMol. Ther.\u003c/em\u003e \u003cstrong\u003e2019\u003c/strong\u003e, \u003cem\u003e27\u003c/em\u003e, 710\u0026ndash;728. https://doi.org/10.1016/j.ymthe.2019.02.012.\u003c/li\u003e\n\u003cli\u003eDuan, Q.; Hu, T.; Zhu, Q.; Jin, X.; Chi, F.; Chen, X. How Far Are the New Wave of MRNA Drugs from Us? MRNA Product Current Perspective and Future Development. \u003cem\u003eFront. immunol.\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e13\u003c/em\u003e, 974433. https://doi.org/10.3389/fimmu.2022.974433.\u003c/li\u003e\n\u003cli\u003eDai, H.; Abdullah, R.; Wu, X.; Li, F.; Ma, Y.; Lu, A.; Zhang, G. Pancreatic Cancer: Nucleic Acid Drug Discovery and Targeted Therapy. \u003cem\u003eFront. Cell Dev. Biol.\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e10\u003c/em\u003e, 855474. https://doi.org/10.3389/fcell.2022.855474.\u003c/li\u003e\n\u003cli\u003eLorentzen, C. L.; Haanen, J. B.; Met, \u0026Ouml;.; Svane, I. M. Clinical Advances and Ongoing Trials of MRNA Vaccines for Cancer Treatment. \u003cem\u003eLancet Oncol.\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e23\u003c/em\u003e, e450\u0026ndash;e458. https://doi.org/10.1016/S1470-2045(22)00372-2.\u003c/li\u003e\n\u003cli\u003eWang, X. L.; Terrence Tsz-Tai Yuen; Yu, D.; Hu, J.; Li, R. L.; Zhang, Z.; Lin, X.; Gong, H.-R.; Chan, C. N.; Yoon, C.; Huiping Shuai; Deborah Tip-Yin Ho; Ivan Fan-Ngai Hung; Zhang, B.; Chu, H.; Huang, J.-D. Vaccine-Induced Protection against SARS-CoV-2 Requires IFN-\u0026gamma;-Driven Cellular Immune Response. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e14\u003c/em\u003e, 3440. https://doi.org/10.1038/s41467-023-39096-y.\u003c/li\u003e\n\u003cli\u003eXu, S.; Yang, K.; Li, R.; Zhang, L. MRNA Vaccine Era\u0026mdash;Mechanisms, Drug Platform and Clinical Prospection. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e21\u003c/em\u003e, 6582. https://doi.org/10.3390/ijms21186582.\u003c/li\u003e\n\u003cli\u003eSun, X.; Setrerrahmane, S.; Li, C.; Hu, J.; Xu, H. Nucleic Acid Drugs: Recent Progress and Future Perspectives. \u003cem\u003eSignal Transduct Tar.\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e9\u003c/em\u003e, 316. https://doi.org/10.1038/s41392-024-02035-4.\u003c/li\u003e\n\u003cli\u003eDong, L.; Deng, X.; Li, Y.; Zhu, X.; Shu, M.; Chen, J.; Luo, H.; An, K.; Cheng, M.; Zhang, P.; Tan, W. Stimuli-Responsive MRNA Vaccines to Induce Robust CD8\u003csup\u003e+\u003c/sup\u003e T Cell Response via ROS-Mediated Innate Immunity Boosting. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e146\u003c/em\u003e, 19218\u0026ndash;19228. https://doi.org/10.1021/jacs.4c04331.\u003c/li\u003e\n\u003cli\u003eKon, E.; Elia, U.; Peer, D. Principles for Designing an Optimal MRNA Lipid Nanoparticle Vaccine. \u003cem\u003eCurr. Opin. Biotechnol. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e73\u003c/em\u003e, 329\u0026ndash;336. https://doi.org/10.1016/j.copbio.2021.09.016.\u003c/li\u003e\n\u003cli\u003eArevalo, C. P.; Bolton, M. J.; Le Sage, V.; Ye, N.; Furey, C.; Muramatsu, H.; Alameh, M.-G.; Pardi, N.; Drapeau, E. M.; Parkhouse, K.; Garretson, T.; Morris, J. S.; Moncla, L. H.; Tam, Y. K.; Fan, S. H. Y.; Lakdawala, S. S.; Weissman, D.; Hensley, S. E. A Multivalent Nucleoside-Modified MRNA Vaccine against All Known Influenza Virus Subtypes. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e378\u003c/em\u003e, 899\u0026ndash;904. https://doi.org/10.1126/science.abm0271.\u003c/li\u003e\n\u003cli\u003eCrommelin, D. J. A.; Anchordoquy, T. J.; Volkin, D. B.; Jiskoot, W.; Mastrobattista, E. Addressing the Cold Reality of MRNA Vaccine Stability. \u003cem\u003eJ. Pharm. Sci. \u003c/em\u003e\u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e110\u003c/em\u003e. https://doi.org/10.1016/j.xphs.2020.12.006.\u003c/li\u003e\n\u003cli\u003eBird, L. MRNA Vaccine for Treating Pancreatic Cancer. \u003cem\u003eNat. Rev. Immunol.\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e23\u003c/em\u003e, 413\u0026ndash;413. https://doi.org/10.1038/s41577-023-00899-1.\u003c/li\u003e\n\u003cli\u003eXu, C.; Cao, C.; Tan, T.; Deng, S.; Wu, B.; Yang, Q.; Wu, M.; Wu, H. MRNA Vaccine-A New Cancer Treatment Strategy. \u003cem\u003eCurr. Cancer Drug Tar. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e23\u003c/em\u003e, 669\u0026ndash;681. https://doi.org/10.2174/1568009623666230222124424.\u003c/li\u003e\n\u003cli\u003eVishweshwaraiah, Y. L.; Dokholyan, N. V. MRNA Vaccines for Cancer Immunotherapy. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e13\u003c/em\u003e, 1029069. https://doi.org/10.3389/fimmu.2022.1029069.\u003c/li\u003e\n\u003cli\u003eSengupta, P.; Banerjee, N.; Dutta, A.; Chaudhuri , M.; Chatterjee , S. Nucleic Acid Therapeutics in Cancer Biology. In \u003cem\u003eNucleic Acid Biology and Its Application in Human Diseases\u003c/em\u003e; Springer: Singapore, 2023; pp. 321\u0026ndash;348.\u003c/li\u003e\n\u003cli\u003eYamada, Y. Nucleic Acid Drugs\u0026mdash;Current Status, Issues, and Expectations for Exosomes. \u003cem\u003eCancers\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e13\u003c/em\u003e, 5002. https://doi.org/10.3390/cancers13195002.\u003c/li\u003e\n\u003cli\u003eScheideler, M.; Vidakovic, I.; Prassl, R. Lipid Nanocarriers for MicroRNA Delivery. \u003cem\u003eChem. Phys. Lipids\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e226\u003c/em\u003e, 104837. https://doi.org/10.1016/j.chemphyslip.2019.104837.\u003c/li\u003e\n\u003cli\u003eQin, Y.; Ou, L.; Zha, L.; Zeng, Y.; Li, L. Delivery of Nucleic Acids Using Nanomaterials. \u003cem\u003eMol. Biomed.\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e4\u003c/em\u003e. https://doi.org/10.1186/s43556-023-00160-0.\u003c/li\u003e\n\u003cli\u003eSharma, A. R.; Lee, Y.-H.; Bat-Ulzii, A.; Bhattacharya, M.; Chakraborty, C.; Lee, S.-S. Recent Advances of Metal-Based Nanoparticles in Nucleic Acid Delivery for Therapeutic Applications. \u003cem\u003eJ. Nanobiotechnol.\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e20\u003c/em\u003e. https://doi.org/10.1186/s12951-022-01650-z.\u003c/li\u003e\n\u003cli\u003eDas, S.; Vera, M.; Gandin, V.; Singer, R. H.; Tutucci, E. Intracellular MRNA Transport and Localized Translation. \u003cem\u003eNat. Rev. Mol. Cell Biol.\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e22\u003c/em\u003e, 483\u0026ndash;504. https://doi.org/10.1038/s41580-021-00356-8.\u003c/li\u003e\n\u003cli\u003eWu, B.; Eliscovich, C.; Yoon, Y. J.; Singer, R. H. Translation Dynamics of Single MRNAs in Live Cells and Neurons. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, \u003cem\u003e352\u003c/em\u003e, 1430\u0026ndash;1435. https://doi.org/10.1126/science.aaf1084.\u003c/li\u003e\n\u003cli\u003ePardi, N.; Hogan, M. J.; Porter, F. W.; Weissman, D. MRNA Vaccines \u0026mdash; a New Era in Vaccinology. \u003cem\u003eNat. Rev. Drug Discov.\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e17\u003c/em\u003e, 261\u0026ndash;279.\u003c/li\u003e\n\u003cli\u003eGoel, R. R.; Painter, M. M.; Apostolidis, S. A.; Mathew, D.; Meng, W.; Rosenfeld, A. M.; Lundgreen, K. A.; Reynaldi, A.; Khoury, D. S.; Pattekar, A.; Gouma, S.; Kuri-Cervantes, L.; Hicks, P.; Dysinger, S.; Hicks, A.; Sharma, H.; Herring, S.; Korte, S.; Baxter, A. E.; Oldridge, D. A. MRNA Vaccines Induce Durable Immune Memory to SARS-CoV-2 and Variants of Concern. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e374\u003c/em\u003e, abm0829. https://doi.org/10.1126/science.abm0829.\u003c/li\u003e\n\u003cli\u003eVerbeke, R.; Lentacker, I.; De Smedt, S.; Dewitte, H. The Dawn of MRNA Vaccines: The COVID-19 Case. \u003cem\u003eJ. Controlled Release\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e333\u003c/em\u003e, 511\u0026ndash;520. https://doi.org/10.1016/j.jconrel.2021.03.043.\u003c/li\u003e\n\u003cli\u003eBiggs, A. T.; Littlejohn, L. F. Describing MRNA Vaccine Technology for a Military Audience. \u003cem\u003eMil. Med.\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e188\u003c/em\u003e, 547\u0026ndash;554. https://doi.org/10.1093/milmed/usac129.\u003c/li\u003e\n\u003cli\u003eCerrato, C. P.; Leppert, A.; Sun, Y.; Lane, D. P.; Arsenian-Henriksson, M.; Miserez, A.; Landreh, M. Monitoring Disassembly and Cargo Release of Phase-Separated Peptide Coacervates with Native Mass Spectrometry. \u003cem\u003eAna. Chem. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e95\u003c/em\u003e, 10869\u0026ndash;10872. https://doi.org/10.1021/acs.analchem.3c02384.\u003c/li\u003e\n\u003cli\u003eSun, Y.; Lau, S. Y.; Lim, Z. W.; Chang, S. C.; Ghadessy, F.; Partridge, A.; Miserez, A. Phase-Separating Peptides for Direct Cytosolic Delivery and Redox-Activated Release of Macromolecular Therapeutics. \u003cem\u003eNat. Chem.\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e14\u003c/em\u003e, 274\u0026ndash;283. https://doi.org/10.1038/s41557-021-00854-4.\u003c/li\u003e\n\u003cli\u003eRamat, A.; Simonelig, M. Activating Translation with Phase Separation. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e377\u003c/em\u003e, 712\u0026ndash;713. https://doi.org/10.1126/science.add6323.\u003c/li\u003e\n\u003cli\u003eBao, Y.; Chen, H.; Xu, Z.; Gao, J.; Jiang, L.; Xia, J. Photo‐Responsive Phase‐Separating Fluorescent Molecules for Intracellular Protein Delivery. \u003cem\u003eAngew. Chem. Int. Ed.\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e62\u003c/em\u003e (42), e202307045. https://doi.org/10.1002/anie.202307045.\u003c/li\u003e\n\u003cli\u003eBao, Y.; Xu, Z.; Cheng, K.; Li, X.; Chen, F.; Yuan, D.; Zhang, F.; Che, A. R.-Y.; Zeng, X.; Zhao, Y.-D.; Xia, J. Staudinger Reaction-Responsive Coacervates for Cytosolic Antibody Delivery and TRIM21-Mediated Protein Degradation. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e2025\u003c/strong\u003e, \u003cem\u003e147\u003c/em\u003e, 3830\u0026ndash;3839. https://doi.org/10.1021/jacs.4c17054.\u003c/li\u003e\n\u003cli\u003eBanani, S. F.; Lee, H. O.; Hyman, A. A.; Rosen, M. K. Biomolecular Condensates: Organizers of Cellular Biochemistry. \u003cem\u003eNat. Rev. Mol. Cell Bio.\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, \u003cem\u003e18\u003c/em\u003e, 285\u0026ndash;298. https://doi.org/10.1038/nrm.2017.7.\u003c/li\u003e\n\u003cli\u003eZhang, B.; Liu, B.; Wu, Z.; Oyama, K.; Ikari, M.; Yagi, H.; Naoya Tochio; Takanori Kigawa; Tsutomu Mikawa; Miyake, T. A Hybrid Nanotube Stamp System in Intracellular Protein Delivery for Cancer Treatment and NMR Analytical Techniques. \u003cem\u003eAnal. Chem.\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e96\u003c/em\u003e, 8349\u0026ndash;8355. https://doi.org/10.1021/acs.analchem.3c05331.\u003c/li\u003e\n\u003cli\u003eDinic, J.; Marciel, A. B.; Tirrell, M. V. Polyampholyte Physics: Liquid\u0026ndash;Liquid Phase Separation and Biological Condensates. \u003cem\u003eCurr. Opin. Colloid Interface Sci.\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e54\u003c/em\u003e, 101457. https://doi.org/10.1016/j.cocis.2021.101457.\u003c/li\u003e\n\u003cli\u003eLadbury, J. E.; Lin, C.-C.; Suen, K. M. Phase Separation Enhances Probability of Receptor Signalling and Drug Targeting. \u003cem\u003eTrends Biochem. Sci. \u003c/em\u003e\u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e48\u003c/em\u003e, 428\u0026ndash;436. https://doi.org/10.1016/j.tibs.2023.01.005.\u003c/li\u003e\n\u003cli\u003eJo, H.; Gajendiran, M.; Kim, K. Development of Polymer Coacersome Structure with Enhanced Colloidal Stability for Therapeutic Protein Delivery. \u003cem\u003eMacro. Biosci.\u003c/em\u003e \u003cstrong\u003e2019\u003c/strong\u003e, \u003cem\u003e19\u003c/em\u003e, e1900207. https://doi.org/10.1002/mabi.201900207.\u003c/li\u003e\n\u003cli\u003eXiao, W.; Jakimowicz, M. D.; Ioannis Zampetakis; Neely, S.; Scarpa, F.; Davis, S. A.; Williams, D. S.; Perriman, A. W. Biopolymeric Coacervate Microvectors for the Delivery of Functional Proteins to Cells. \u003cem\u003eAdv. Biosyst.\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e4\u003c/em\u003e, e2000101. https://doi.org/10.1002/adbi.202000101.\u003c/li\u003e\n\u003cli\u003eWen, P.; Huang, H.; Zhang, R.; Zheng, H.; Liang, T.; Zhuang, C.; Wu, Q.; Wang, J.; Liu, F.; Zhang, K.; Wu, W.; He, K.; Liu, F.; Li, H.; Gu, Z. Coacervate Vesicles Assembled by Liquid\u0026ndash;Liquid Phase Separation Improve Delivery of Biopharmaceuticals. \u003cem\u003eNat. Chem.\u003c/em\u003e \u003cstrong\u003e2025\u003c/strong\u003e, \u003cem\u003e17\u003c/em\u003e, 279\u0026ndash;288. https://doi.org/10.1038/s41557-024-01705-8.\u003c/li\u003e\n\u003cli\u003eMashima, T.; van Stevendaal, M. H. M. E.; Cornelissens, F. R. A.; Mason, A. F.; Rosier, B. J. H. M.; Altenburg, W. J.; Oohora, K.; Hirayama, S.; Hayashi, T.; van Hest, J. C. M.; Brunsveld, L. DNA‐Mediated Protein Shuttling between Coacervate‐Based Artificial Cells. \u003cem\u003eAngew. Chem. Int. Ed. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e61\u003c/em\u003e, e202115041. https://doi.org/10.1002/anie.202115041.\u003c/li\u003e\n\u003cli\u003eYim, W.; Jin, Z.; Chang, Y.-C.; Brambila, C.; Creyer, M. N.; Ling, C.; He, T.; Li, Y.; Retout, M.; Penny, W. F.; Zhou, J.; Jokerst, J. V. Polyphenol-Stabilized Coacervates for Enzyme-Triggered Drug Delivery. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e15\u003c/em\u003e, 7295. https://doi.org/10.1038/s41467-024-51218-8.\u003c/li\u003e\n\u003cli\u003eIwata, T.; Hirose, H.; Sakamoto, K.; Hirai, Y.; Vincent, J.; Misao Akishiba; Imanishi, M.; Shiroh Futaki. Liquid Droplet Formation and Facile Cytosolic Translocation of IgG in the Presence of Attenuated Cationic Amphiphilic Lytic Peptides. \u003cem\u003eAngew. Chem. Int. Ed.\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e60\u003c/em\u003e, 198 04\u0026ndash;19812. https://doi.org/10.1002/anie.202105527.\u003c/li\u003e\n\u003cli\u003eun, Y.; Xu, X.; Chen, L.; Chew, W. L.; Ping, Y.; Miserez, A. Redox-Responsive Phase-Separating Peptide as a Universal Delivery Vehicle for CRISPR/Cas9 Genome Editing Machinery. \u003cem\u003eACS Nano\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e17\u003c/em\u003e, 16597\u0026ndash;16606. https://doi.org/10.1021/acsnano.3c02669.\u003c/li\u003e\n\u003cli\u003eSun, Y.; Wu, X.; Li, J.; Milad Radiom; Raffaele Mezzenga; Verma, C. S.; Yu, J.; Miserez, A. Phase-Separating Peptide Coacervates with Programmable Material Properties for Universal Intracellular Delivery of Macromolecules. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e15\u003c/em\u003e, 10094. https://doi.org/10.1038/s41467-024-54463-z.\u003c/li\u003e\n\u003cli\u003eCerrato, C. P.; Krko\u0026scaron;ka, M.; Sun, Y.; Lia\u0026ntilde;o-Pons, J.; Neo, Q. Y.; Vosselman, T.; Alzrigat, M.; Vojtě\u0026scaron;ek, B.; Lane, D. P.; Arsenian Henriksson, M.; Miserez, A.; Landreh, M. Engineered Peptide Coacervates Enable Efficient Intracellular Delivery of the MYC Inhibitor OmoMYC. \u003cem\u003eMol. Pharms.\u003c/em\u003e \u003cstrong\u003e2025\u003c/strong\u003e, \u003cem\u003e22\u003c/em\u003e, 3479\u0026ndash;3490. https://doi.org/10.1021/acs.molpharmaceut.5c00468.\u003c/li\u003e\n\u003cli\u003eSun, Y.; Wu, X.; Shen, K.; Guo, K.; Lim, D. S.; Chew, W. L.; Yu, J.; Miserez, A. Polyethylene-Glycol-Conjugated Peptide Coacervates with Tunable Size for Intracellular MRNA Delivery. \u003cem\u003eACS Nano\u003c/em\u003e \u003cstrong\u003e2025\u003c/strong\u003e, \u003cem\u003e10.1021/acsnano.4c16908\u003c/em\u003e, Advance online publication. https://doi.org/10.1021/acsnano.4c16908.\u003c/li\u003e\n\u003cli\u003eShebanova, A.; Perrin, Q. M.; Zhu, K.; Sushanth Gudlur; Chen, Z.; Sun, Y.; Huang, C.; Lim, Z. W.; Mondarte, E. A.; Sun, R.; Lim, S.; Yu, J.; Miao, Y.; Parikh, A. N.; Ludwig, A.; Miserez, A. Cellular Uptake of Phase‐Separating Peptide Coacervates. \u003cem\u003eAdv. Sci.\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e11\u003c/em\u003e, e2402652. https://doi.org/10.1002/advs.202402652.\u003c/li\u003e\n\u003cli\u003eXie, X.; Li, T.; Ma, L.; Wu, J.; Qi, Y.; Yang, B.; Li, Z.; Yang, Z.; Zhang, K.; Chu, Z.; Ngai, T.; Xia, J.; Wang, Y.; Zhao, P.; Bian, L. A Designer Minimalistic Model Parallels the Phase-Separation-Mediated Assembly and Biophysical Cues of Extracellular Matrix. \u003cem\u003eNat. Chem.\u003c/em\u003e \u003cstrong\u003e2025\u003c/strong\u003e, \u003cem\u003e10.1038/s41557-025-01837-5\u003c/em\u003e, Advance online publication. https://doi.org/10.1038/s41557-025-01837-5.\u003c/li\u003e\n\u003cli\u003eNasr, S. S.; Lee, S.; Durairaj Thiyagarajan; Boese, A.; Loretz, B.; Lehr, C.-M. Co-Delivery of MRNA and PDNA Using Thermally Stabilized Coacervate-Based Core-Shell Nanosystems. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e13\u003c/em\u003e, 1924\u0026ndash;1924. https://doi.org/10.3390/pharmaceutics13111924.\u003c/li\u003e\n\u003cli\u003eLu, T.; Liese, S.; Ludo Schoenmakers; Weber, C. A.; Suzuki, H.; Wilhelm; Spruijt, E. Endocytosis of Coacervates into Liposomes. \u003cem\u003eJ. Am. Chem. Soc.\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e144\u003c/em\u003e, 13451\u0026ndash;13455. https://doi.org/10.1021/jacs.2c04096.\u003c/li\u003e\n\u003cli\u003eIaneselli, A.; Damla Tetiker; Stein, J.; K\u0026uuml;hnlein, A.; Mast, C. B.; Braun, D.; T-Y Dora Tang. Non-Equilibrium Conditions inside Rock Pores Drive Fission, Maintenance and Selection of Coacervate Protocells. \u003cem\u003eNat. Chem.\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e14\u003c/em\u003e, 32\u0026ndash;39. https://doi.org/10.1038/s41557-021-00830-y.\u003c/li\u003e\n\u003cli\u003eChen, H.; Bao, Y.; Li, X.; Chen, F.; Sugimura, R.; Zeng, X.; Xia, J. Cell Surface Engineering by Phase‐Separated Coacervates for Antibody Display and Targeted Cancer Cell Therapy. \u003cem\u003eAngew. Chem. Int. Ed.\u003c/em\u003e \u003cstrong\u003e2024\u003c/strong\u003e, \u003cem\u003e63\u003c/em\u003e, e202410566. https://doi.org/10.1002/anie.202410566.\u003c/li\u003e\n\u003cli\u003eWelling, G. W.; Fries, H. Choice of peptide and peptide length for the generation of antibodies reactive with the intact protein. \u003cem\u003eFEBS Lett.\u003c/em\u003e \u003cstrong\u003e1985\u003c/strong\u003e, \u003cem\u003e182\u003c/em\u003e, 81-4. https://doi.org/10.1016/0014-5793(85)81158-3.\u003c/li\u003e\n\u003cli\u003eLiew, O. W.; Ling, S. S. M.; Lilyanna, S.; Zhou, Y.; Wang, P.; Chong, J. P. C.; Ng, Y. X.; Lim, A. E. S.; Leong, E. R. Y.; Lin, Q.; Lim, T. K.; Lin, Q.; Ng, E. M. W.; Ng, T. W.; Richards, A. M. Epitope-directed monoclonal antibody production using a mixed antigen cocktail facilitates antibody characterization and validation. \u003cem\u003eCommun Biol\u003c/em\u003e. \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e4\u003c/em\u003e, 441. https://doi.org/10.1038/s42003-021-01965-x.\u003c/li\u003e\n\u003cli\u003eWang, C.; You, J.; Gao, M.; Zhang, P.; Xu, G.; Dou, H. Bio-Inspired Gene Carriers with Low Cytotoxicity Constructed via the Assembly of Dextran Nanogels and Nano-Coacervates. \u003cem\u003eNanomedicine\u003c/em\u003e \u003cstrong\u003e2020\u003c/strong\u003e, \u003cem\u003e15\u003c/em\u003e, 1285\u0026ndash;1296. https://doi.org/10.2217/nnm-2020-0065.\u003c/li\u003e\n\u003cli\u003eKetting, R. F. Dicer Functions in RNA Interference and in Synthesis of Small RNA Involved in Developmental Timing in C. Elegans. \u003cem\u003eGene Dev.\u003c/em\u003e \u003cstrong\u003e2001\u003c/strong\u003e, \u003cem\u003e15\u003c/em\u003e, 2654\u0026ndash;2659. https://doi.org/10.1101/gad.927801.\u003c/li\u003e\n\u003cli\u003eFriedrich, M.; Aigner, A. Therapeutic SiRNA: State-of-The-Art and Future Perspectives. \u003cem\u003eBioDrugs\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e36\u003c/em\u003e, 549\u0026ndash;571. https://doi.org/10.1007/s40259-022-00549-3.\u003c/li\u003e\n\u003cli\u003ePrazeres, D. M. F.; Monteiro, G. A. Plasmid Biopharmaceuticals. \u003cem\u003eMicrobiol. Spectr.\u003c/em\u003e \u003cstrong\u003e2014\u003c/strong\u003e, \u003cem\u003e2\u003c/em\u003e, 10.1128/microbiolspec.PLAS0022-2014. https://doi.org/10.1128/microbiolspec.plas-0022-2014.\u003c/li\u003e\n\u003cli\u003ePeng, Z. Current Status of Gendicine in China: Recombinant Human Ad-P53 Agent for Treatment of Cancers. \u003cem\u003eHum. Gene Ther. \u003c/em\u003e\u003cstrong\u003e2005\u003c/strong\u003e, \u003cem\u003e16\u003c/em\u003e, 1016\u0026ndash;1027. https://doi.org/10.1089/hum.2005.16.1016.\u003c/li\u003e\n\u003cli\u003eRennick, J. J.; Johnston, A. P. R.; Parton, R. G. Key Principles and Methods for Studying the Endocytosis of Biological and Nanoparticle Therapeutics. \u003cem\u003eNat. Nanotechnol.\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e16\u003c/em\u003e, 266\u0026ndash;276. https://doi.org/10.1038/s41565-021-00858-8.\u003c/li\u003e\n\u003cli\u003eMendon\u0026ccedil;a, M. C. P.; Radaic, A.; Garcia-Fossa, F.; da Cruz-H\u0026ouml;fling, M. A.; Vinolo, M. A. R.; de Jesus, M. B. The in Vivo Toxicological Profile of Cationic Solid Lipid Nanoparticles. \u003cem\u003eDrug Deliv. Transl. Res.\u003c/em\u003e \u003cstrong\u003e2019\u003c/strong\u003e, \u003cem\u003e10\u003c/em\u003e, 34\u0026ndash;42. https://doi.org/10.1007/s13346-019-00657-8.\u003c/li\u003e\n\u003cli\u003eHusain, H.; Latief, U.; Ahmad, R. Pomegranate Action in Curbing the Incidence of Liver Injury Triggered by Diethylnitrosamine by Declining Oxidative Stress via Nrf2 and NF\u0026kappa;B Regulation. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e8\u003c/em\u003e, 8606. https://doi.org/10.1038/s41598-018-26611-1.\u003c/li\u003e\n\u003cli\u003eYoon, H. Y.; Son, S.; Lee, S. J.; You, D. G.; Yhee, J. Y.; Park, J. H.; Swierczewska, M.; Lee, S.; Kwon, I. C.; Kim, S. H.; Kim, K.; Pomper, M. G. Glycol Chitosan Nanoparticles as Specialized Cancer Therapeutic Vehicles: Sequential Delivery of Doxorubicin and Bcl-2 SiRNA. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e2014\u003c/strong\u003e, \u003cem\u003e4\u003c/em\u003e, 6878. https://doi.org/10.1038/srep06878.\u003c/li\u003e\n\u003cli\u003eYasmin, F.; Najeeb, H.; Naeem, U.; Moeed, A.; Atif, A. R.; Asghar, M. S.; Nimri, N.; Saleem, M.; Bandyopadhyay, D.; Krittanawong, C.; Fadelallah Eljack, M. M.; Tahir, M. J.; Waqar, F. Adverse Events Following COVID-19 MRNA Vaccines: A Systematic Review of Cardiovascular Complication, Thrombosis, and Thrombocytopenia. \u003cem\u003eImmun. Inflamm. Dis.\u003c/em\u003e \u003cstrong\u003e2023\u003c/strong\u003e, \u003cem\u003e11\u003c/em\u003e, e807. https://doi.org/10.1002/iid3.807.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Liquid-liquid phase separation, peptide coacervates, mRNA therapeutics, transfection, cancer immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-7559034/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7559034/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Nucleic acid therapeutics, such as mRNA vaccines, require effective delivery vehicles; yet widely used systems like lipid nanoparticles or cationic polymers are often associated with adverse responses. Developing low-molecular-weight compounds as delivery vehicles for nucleic acids is promising but remains challenging. Here, we identify a disulfide-bond-linked, six-residue peptide, WWKssKWW (WWK), which forms coacervates through liquid-liquid phase separation (LLPS) in aqueous solutions. The WWK coacervates encapsulate diverse nucleic acids—including single-stranded DNA, siRNA, mRNA, and plasmids—and deliver them into cells with efficiencies equivalent to or exceeding conventional agents (lipofectamine, InstantFECT, PEI, and lipid nanoparticles). Subcutaneous and intramuscular injection of mRNA-loaded WWK coacervates in mice results in robust protein expression and a favorable safety profile. Importantly, when human PBMCs were transfected with various mRNA formulations, WWK/mRNA induced significantly lower levels of the inflammatory cytokine IL-1β compared to liposomes and lipid nanoparticles, similar to naked mRNA, suggesting reduced toxicity risk. WWK coacervates also enabled efficient intramuscular delivery of SARS-CoV-2 spike mRNA, producing spike-specific cellular responses comparable to the commercial Comirnaty vaccine. Furthermore, in a B16-OVA cancer model, WWK/OVA mRNA coacervates exhibited potent anti-tumor effects and significantly enhanced tumor-free survival. Overall, our findings demonstrate that peptide coacervates are promising vehicles for mRNA delivery, offering improved safety and efficacy for gene therapy and vaccine development.","manuscriptTitle":"Phase-Separated Peptide Coacervates as Delivery Vehicles for mRNA Vaccines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 08:00:48","doi":"10.21203/rs.3.rs-7559034/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cdf6cdca-bb7f-473b-8f89-51d83a34c298","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55114027,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanostructures"},{"id":55114028,"name":"Physical sciences/Chemistry/Supramolecular chemistry"}],"tags":[],"updatedAt":"2025-11-11T10:51:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 08:00:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7559034","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7559034","identity":"rs-7559034","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}