Polymeric nanoparticle-loaded extracellular vesicles as biomimetic nucleic acid vaccines

Polymeric nanoparticle-loaded extracellular vesicles as biomimetic nucleic acid 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 Polymeric nanoparticle-loaded extracellular vesicles as biomimetic nucleic acid vaccines Cristina Fornaguera, Jennifer Fernandez Alarcon, Alessandro Masoero, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7030394/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 vaccination has expanded the traditional use of vaccines, ranging from infectious disease prophylaxis to therapeutic applications in oncology. However, nucleic acids require protection from nuclease degradation in vivo by using selective delivery vectors capable of transporting the cargo into targeted cells such as antigen-presenting cells (APCs) for efficient genetic material translation into proteins and immune system activation. Polymeric nanoparticles (NPs), particularly poly(beta aminoesters) (pBAEs), have demonstrated efficiency in transfecting cells in vitro and have also demonstrated promising results in vivo. Despite numerous attempts to target NPs to specific cells, selectivity still remains a challenge. In this study, we address this limitation by developing biomimetic nanosystems composed of pBAE NP-embedded in extracellular vesicles (EVs). These small vesicles, released by all cell types, facilitate intercellular communication among cells of the same lineage, making them ideal to be use as natural targeting moieties. Effective complexes were ensembled using a freeze-thaw method to efficiently entrap pBAE NPs loaded with nucleic acids in monocyte-derived EVs. Their in vitro safety, transfection, and monocyte activation capacity of these complexes outperformed the individual components of the nanosystem, demonstrating their suitability for parenteral use in vivo . Our findings confirm the potential of constructing efficacious biomimetic pBAE NPs-EVs nanocomplexes for nucleic acid vaccination for the first time. Biological sciences/Biotechnology/Biomaterials/Biomaterials – vaccines Biological sciences/Cancer/Cancer therapy/Tumour vaccines Physical sciences/Chemistry/Materials chemistry/Biomaterials/Drug delivery Polymeric nanoparticles extracellular vesicles nucleic acid vaccination targeted gene delivery systems biomimetic nanosystems Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Prophylactic vaccination is considered to be the public health milestone of 20th -century modern medicine, eradicating many infectious diseases 1 – 3 . While traditional vaccines commonly comprise attenuated, inactivated, or fractions of the infectious microorganisms, innovative vaccine designs are needed to extend beyond their prophylactic use and tackle therapeutic applications for cancer. The challenge of antigen selection needs robust and versatile vaccine formulations 4 . mRNA has emerged as a promising solution, particularly following the success of COVID-19 vaccination strategies. Its common structural features, regardless of the encoded antigen, eliminates the need for complete reformulation in personalized cancer therapeutics. However, nucleic acids present a major difficulty as their stability in physiological media is compromised by nucleases that cause their degradation, and they cannot penetrate plasmatic membranes. Therefore, their direct application in vivo requires protection through the use of biomaterials, which can be natural, such as extracellular vesicles (EVs) or synthetic delivery systems like lipid nanoparticles (LNPs), as used in COVID-19 vaccines, or polymeric NPs. Polymeric NPs offer greater stability in vivo than LNPs, and provide a wider range of functionalization options. Polymeric NPs have been extensively studied as advanced delivery systems for a wide range of diseases, with a particular focus on cancer treatments. Regarding nucleic acid vaccination, we 5 , 6 and others 1 , 7 , 8 have described the key features of effective delivery systems. In this context, we demonstrated that poly(beta aminoester) polymers (pBAEs) outperform others in efficiently complexing nucleic acids into small nanometric particles. These can then be safely administered in vivo , delivering mRNA and translating it into proteins in specific target cells and organs. pBAEs are biocompatible and biodegradable polymers with a common backbone that can be easily modified to taylor pBAEs with different molecules or functional groups, such as targeting moieties, hydrophobic monomers, and stimulus-sensitive groups. They can be synthesized through a two-step Michael addition of amines to acrylate terminal groups in a production process that is both scalable and compliant with GMP-like standards 4 , 5 , 9 , 10 . However, the use of pBAE NPs in vivo requires an active targeting moiety—a feature that continues to pose a challenge. In our previous works 5 , 11 , 12 and by others 13 – 16 , the addition of a targeting moiety is often a bottleneck due to opsonization of the functional groups once systemically administered or insufficient exposure of molecules on the NPs surface. This addition leads to only limited changes in the balance between target cells and liver accumulation, thereby indicating that targeting efficiency can be significantly improved. These limitations may be attributed to difficulties in identifying specific cell type receptors, which show distinct expression in different cell lineages 17 . Thus, there is a need to enhance the targeting efficiency and selectivity of the nanosystems. Bioengineering strategies that combine synthetic NPs with cell membrane coatings, which show have natural tropism and homing potential to target cells, could provide a solution 18 . In this context, EVs have gained relevance as promising natural delivery systems. EVs are nanometric vesicles composed of phospholipid bilayers, released by all cell types. These organelle-free vesicles carry biomacromolecules and play a role in cell-to-cell communication in both physiological and pathological conditions, particularly among cells of the same lineage. EVs can sometimes evade immune system recognition and cross major physiological barriers 19 – 21 . Despite the rapid expansion of our understanding of EV biology, function, and translational potential, the heterogeneous nature of EVs and the challenges in efficiently separating exosomal subpopulations have hindered the characterization of their molecular composition and biogenesis 22 . Nevertheless, the International Society of Extracellular Vesicles (ISEV) does not recommend subgrouping EVs due to technical difficulties regarding their purification 23 . EVs play a significant role in cancer diagnosis and treatment 20 , as they can selectively recruit proteins, lipids, metabolites, antigens and genetic material (i.e. mRNA, miRNA or DNA) and transfer this cargo into the targeting cells 18 . Thereby, EVs can be used as vehicles to encapsulate active principles for intercellular communication in pathological conditions such as cancer 17 , 18 . Additionally, EVs serve as selective targeting nanodevices, leveraging their plasma membrane to overcome current targeting issues in cancer vaccines 24 . However, the encapsulation efficiency of nucleic acids in EVs remains limited 25 – 28 . Here we harness the directed targeting capacity of EV membranes to coat pBAE NPs, creating biomimetic nanosystems naturally targeted to monocytes. To address the limitations of synthetic polymeric NPs, we aim to demonstrate the effectiveness of these nanosystems in maturing monocytes into dendritic cells (DCs). While numerous review articles highlight the potential of EVs as advanced delivery systems, and some suggest their use for vaccination 29 , a comprehensive study of their therapeutic applications is lacking. Moreover, the low encapsulation capacity of EVs in exogenous cargo loading can be effectively overcome by the superior encapsulation efficiency of pBAE NPs, allowing for a selective and effective delivery. Therefore, our ultimate objective herein is to design innovative biomimetic smart multicomponent nucleic acid vaccines that are both safe and functional. Our hypothsis drives that combining the strenghs of both delivery systems, we will obtain an enhanced biomimetic mRNA nanocarrier. MATERIALS AND METHODS 1. Materials Bovine serum albumin (BSA), sodium acetate (AcONa), Loading Buffer, Tween-80, and PBS were purchased from Sigma-Aldrich®, and Cyanine 5 NHS ether dye was supplied by Lumiprobe. Fetal Bovine Serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), glutamine, penicillin, and streptomycin were obtained from Gibco®. RPMI-1640 medium was purchased from Biowest (L0501-500). Recombinant human interleukin 4 (rhIL-4) and recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CFS) were supplied by PeproTech®. CellMask™Orange Plasma Membrane Stain was purchased from Thermo Fisher Scientific. Arginine and histidine end-modified poly(β)-amino ester (pBAE, named in the following as R and H) polymers were prepared by the GEMAT group following a two-step procedure described previously 5 , 30 . An acrylate-terminated polymer, C6, was first synthesized by addition reaction of primary amines with diacrylates (at 1:1.2 M ratio of amine:diacrylate). pBAEs were then obtained by end-capping modification of the resulting acrylate-terminated polymer with arginine or histidine at each end. When necessary, fluorescent pBAEs were also used. To this end, R-pBAEs were labelled with Cyanine 5 (Cy5), as described previously 31 , 32 . Anti-human CD11c-FITC, CD86-PE, and CD209-PE, as well as purified anti-chicken ovalbumin and APC anti-mouse/human CD11b, were purchased from BioLegend. The goat anti-mouse IgG (H + L) secondary antibody, HRP, was supplied by Thermo Fisher Scientific, and Ovalbumin-Fluorescein Isothiocyanate (OVA-FITC) by Santa Cruz Biotechnology. 2. Cell culture THP-1 cell lines were maintained in RMPI-1640 supplemented with FBS (10%, v/v), penicillin G (100 units mL − 1 ), streptomycin (100 µg mL − 1 ), and L-glutamine (2 mmol L − 1 ). HEK-293 cell lines were maintained in DMEM supplemented with FBS (10%, v/v), penicillin G (100 units mL − 1 ), streptomycin (100 µg mL − 1 ), and L-glutamine (2 mmol L − 1 ). All cells were cultured at 37°C under a 5% CO 2 with 95% humidity air atmosphere and passaged when they reached 80–90% confluence. THP-1 cells were differentiated to obtain immature dendritic cells (imDCs), as previously described 33 . Briefly, THP-1 cells were cultured in medium supplemented with recombinant human interleukin 4 (rhIL-4) (20 ng mL − 1 ) and phorbol 12-myristate 13-acetate (PMA) (20 ng mL − 1 ) for 4 days to trigger differentiation 33 . To verify differentiation, flow cytometer analysis was performed by staining the surface markers CD11b, CD11c, and CD209 and quantifying the extent of differentiation. Briefly, 2.5 x 10 5 of cells per well were retrieved from each cell culture 34 and fixed in formalin (4%) for 20 min at 4°C. The samples were then resuspended in a blocking buffer containing FBS in PBS (2%, v/v) for 20 min at 4°C. After another washing step in PBS, the samples were incubated for 1 h at RT with the primary antibodies anti-human CD11b conjugated with APC fluorophore, anti-human CD11c conjugated with FITC, and anti-human CD209 conjugated with PE, following the manufacturer's instructions. The unbound antibodies were washed away with FBS in PBS (2%, v/v), and the cells were resuspended in PBS and analyzed using a ACEA Flow Cytometer (NovoCyte, Santa Clara, USA). 3. Methods 3.1. Synthesis of the pBAE nanoparticles NPs were prepared following a well-established protocol in our lab as previously described 5 , 35 , 36 . Briefly, a mixture containing 60% C 6 CK 3 and 40% C 6 CH 3 pBAEs (12.5 mg/mL) were diluted in a sodium acetate solution (12.5 mM, pH = 5.2) with the same volume of plasmid (0.5 mg mL − 1 ) at a ratio 25:1. pBAE polymers were synthesized by a previously described protocol 5 , 9 , 10 . Plasmids were produced using Gigaprep kits, following the suppliers’ instructions. pPAX (a model plasmid not coding for any fluorescent protein), pMaxGFP (coding for GFP), and pOVA (coding for ovalbumin antigen) were used. The mixture was incubated for 30 min at 25°C. The KH NPs were then precipitated in the same volume of Milli-Q H 2 O and an equivalent volume of Hepes (20 mM, 4% w/v sucrose). For all experiments, fresh KH NPs were used. 3.2. Isolation and purification of EVs Culture samples were collected by mild centrifugation of the medium at 300 g for 5 min after 24 h incubation without FBS, and supernatants were stored at -80°C. EVs were isolated by sequential high-speed centrifugation, as described previously 37 , and stored at -80°C for a maximum of six months. EVs from undifferentiated THP-1 and HEK293 cells were produced and labelled using NBD-PE, a phospholipid tagged on the head group with the NBD fluorophore, which can intercalate into the EV phospholipidic bilayer by passive incubation. A ratio of NBD-PE (1 mg mL − 1 in PBS, 5 µL) for EVs (100 µL, at a concentration of approximately 5 x 10 10 EV mL − 1 ) was mixed and incubated for 1 h at 37°C under agitation. Later, the solution was filtered using a 100 kDa cutoff Amicon®Ultra Centrifugal Filter Unit (Merck Millipore, USA) to remove the free label. Isolated EVs were characterized by three distinct techniques, including Western blotting, NP tracking analysis, and dynamic light scattering, following MISEV guidelines 23 . The same method was used for Cyanine-3 (Cy3) labelling, at a ratio of Cy3 (1 mg mL − 1 in DMSO, 10 µL) for EVs (100 µL), and with CD81-FITC (1:400). 3.3. Characterization of EVs by Western blot The concentration of proteins in the samples was determined using the Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA; 23225), following the manufacturer’s instructions. EV protein content was evaluated by Western blot. Briefly, isolated EVs were lysed in reducing sample buffer [Tris-HCl (0.25 M, pH 6.8), glycerol (40%), SDS (8%), 2-mercaptoethanol (5%) and bromophenol blue (0.04%)] or non-reducing sample buffer (without 2-mercaptoethanol) and boiled for 10 min at 65°C. Protein samples were resolved by SDS-PAGE (10%, SDS-polyacrylamide gel electrophoresis, for CD63, TSG101, CD81, BSA, respectively), transferred to polyvinylidene fluoride membranes, blocked in 5% non-fat powdered milk in PBS-T (0.5% Tween-20), and probed with antibodies. Purified anti-TSG101 antibody (Cat. 934301, Biolegend) and Bovine Serum Albumin Polyclonal Antibody (BSA; Cat. A11133, Invitrogen) were applied to the reduced samples. CD81 antibody (1.3.3.22; sc-7637, Santa Cruz Biotechnology) and CD63 antibody (MX-49.129.5; sc-5275, Santa Cruz Biotechnology) were applied to non-reduced samples. For detection, goat polyclonal antibody to MS IgG (HRP, Mouse, GR3219929, Abcam), HRP goat anti-rat IgG (minimal x-reactivity; Cat 405405, Biolegend), goat anti-rabbit, rat IgG (H + L) secondary (NB7160, Novus Biologicals) and Pierce® ECL Western Blotting–substrate (Thermo Fischer Scientific, Rockford, IL, USA) were used. The membrane was examined using an Amersham ImageQuant™800 biomolecular imager (Cytiva Life Sciences, USA). 3.4. Biophysical characterization of complexes by dynamic light scattering, nanoparticle tracking analysis, and electron microscopy Hydrodynamic size, polydispersity index (PDI), surface charge (ζ-potential), size distribution, and sample concentration were analyzed by both dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). To determine the hydrodynamic size and PDI of NPs (50 µL) prepared as previously described, or thawed EV solution (50 µL) diluted 1:5 in PBS was put in a DLS micro-cuvette and analyzed in a Zetasizer Nano ZS with Zetasizer Software (DLS; Malvern Instruments, Worcestershire, UK). To measure ζ-potential, samples were diluted in Milli-Q water (1:100) to a final volume of 1 mL and samples were put in a Disposable Capillary cell (DTS1060, Malvern Instruments, Worcestershire, England) and analyzed by DLS. To determine size distribution and sample concentration, samples were diluted 1:100 in PBS (or Milli-Q water for NPs) in a final volume of 1 mL and run with the automated syringe pump in an NTA Nanosight NS300 (Malvern Panalytics, United Kingdom). A PDI was also calculated from the NTA, PDI = (σ/µ) 2 , where σ and µ are NTA standard deviation and mean size, respectively. Encapsulation efficiency was calculated using the RiboGreen® colorimetric test. 3.5. Nanoparticle encapsulation in EVs We tested the capacity of several methods to encapsulate pBAEs into EVs, namely sonication, extrusion, and freeze-thaw (summarized in Fig. 1 ). As pBAE NPs, in this study we only use KH NPs which encapsulated pGFP or mRNA OVA for the differentiation study in monocytes. EVs were collected from three different cell lines, human monocytic THP-1 cells, murine monocytic JAWS cells and murine lung cancer LLC1 cells, in order to probe the tropism of the EVs towards homing cells and the complexes between the KH NPs and the different EVs were highlighted as KH NP@THP-1 EV, KH NP@JAWS EV and KH NP@LLC1 EV, respectively. Extrusion was performed with an Avanti Polar Lipids mini-extruder using a 200 nm pore membrane to passthrough the EVs for 10 times, while for sonication, KH NPs were put in a sonication bath for 10 and 20 min. EVs were disrupted using fast freezing-thawing cycles combined with incubation with KH NPs, followed by a recovery period. Briefly, an appropriate amount of EV solution was frozen at -80°C in a mixture of acetone and dry- ice and allowed to thaw at RT for 10 min. These cycles were repeated 3 times. After the last cycle, the NP solution was rapidly added to the EV solution at different NP:EV volume ratios and gently pipetted to obtain a homogenous solution of the two nanosystems. The mixture was then incubated for 1 h at RT. This formulation is referred to as KH NP@EV complexes followed by an indication of the ratio (for example KH NP@EV 1:2, to indicate a 1:2 ratio of NP:EV). The ratio was calculated from the concentration of the samples of EVs and KH NPs, as determined by the NTA. The formulation was always used freshly prepared. 3.6. Confocal microscopy of the NP@EV complexes Confocal microscopy was used to verify the colocalization of KH NPs and THP-1 EVs after the encapsulation. Briefly, the THP-1 EVs stained with N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (NBD-PE) and the KH NPs labelled with Sulfo-Cyanine 5 (Cy5) (1%) were used to build the KH NP@THP-1 EV complexes. To this end, the sample (10 µL) was deposited onto the glass slide, covered with a coverslip, sealed, and analyzed with a Leica DMi8 confocal microscope. Image analysis was performed using the software Fiji /ImageJ 1.52v (Wayne Rasband, National Institutes of Health, USA). 3.7. Hyperspectral microscope and dark-field imaging Glass slides and coverslips were cleaned with Milli-Q water, EtOH, and acetone for 10 min before observations. Samples were visualized using Exponent 7 software and mapped from their hyperspectral images with a Cytoviva© high-resolution dark-field condenser (Auburn, AL, USA) coupled to an Olympus BX-43 optical microscope. Hyperspectral imaging (HSI) analysis was recorded using ENVI 4.8 software in which the hyperspectral camera operated in the visible-near infrared range (VNIR) of 400 to 1000 nm. A spectral library with a representative hyperspectral image was obtained from single components (EVs and NPs) separately by adding 10 µL of sample to a microscope glass slide. The freshly prepared NP@EV sample was placed on a microscope slide (10 µL) covered with a coverslip and observed. Hyperspectral images of the NP@EV complexes were obtained and spectral similarities that matched the spectral library of EVs and NPs facilitated the mapping process. 3.8. Fluorescence Resonance Energy Transfer (FRET) analysis FRET measurements were performed using an Infinite M Plex microplate reader from TECAN. Each sample had a final volume of 110 µL in PBS media and was run in triplicate in a flat black 96-well plate. Each component of the complexes was prepared at the same labelling concentration. The polymer was labelled with Cy5 or Sulfo-Cyanine 3 (Cy3), the plasmid with Cy5, and EVs with Cy3. As a negative control, the complexes were measured with only one of the components labelled, and PBS wells with no labelling were also included. The excitation wavelength was set to 535 nm for Cy3, and the emission wavelength to 675 nm to detect the emission of Cy5. Measurements were carried out at different times and temperatures (25 and 37°C) over 24 h. 3.9. Encapsulation efficiency Nucleic acid encapsulation efficiency was quantitatively assessed using the Quanti-It Pico-Green DNA assay kit (Thermo Fisher Scientific), following the manufacturer’s instructions. A qualitative analysis of the encapsulated genetic material was also performed using agarose gel electrophoresis (Sub-CellR GT Agarose Gel Electrophoresis System BIORAD). Briefly, an agarose solution (1.5%, 65 mL) in TAE buffer (1x) was prepared, then GelRed was added (1 uL). The mixture was allowed to polymerize in the appropriate mold for 30 min. Subsequently, the samples of interest, including the marker containing different DNA base pairs of varying molecular weights, KH NPs, KH NP@EV complexes, and the free plasmid at the same concentration as the NPs, were loaded and run on the gel. The resulting image was analyzed using ImageJ software. 4. In vitro biocompatibility 10.000 cells per well were seeded in a 96-well plate at 90% confluence 24 h before starting the experiment. They were then incubated with increasing amounts of the KH NP@EV complexes. Non-treated cells were used as negative controls. In vitro cell viability was evaluated through quantification of cell metabolic activity using the MTT colorimetric assay, as described previously 5 , 36 . Briefly, cells were incubated with the samples for 48 h, the media was removed and replaced with MTT (0.5 mg mL − 1 ) in complete media and cells were further incubated for around 1 h. Next, this medium was removed, and formazan crystals were dissolved in DMSO (100 µL). Absorbance was quantified at 570 nm using a plate reader (SpectraMax M5, Molecular Devices). Results are expressed as a percentage of viable cells relative to non-treated cells. 5. In vitro uptake experiments To assess the capacity of the engineered nanosystems to penetrate the cells, in vitro analyses were conducted using Cy5-labelled NPs (0.2%) and NBD-PE-labelled EVs. Thus, the uptake could be measured by flow cytometry or studied by confocal microscopy. The flow cytometer quantitative analysis was conducted on THP-1 and HEK-293 cell lines, while subcellular qualitative evaluation was conducted only on differentiated THP-1 cells (iDCs) by confocal microscopy. For the latter, THP-1 cells were cultured with rhIL-4 (recombinant human interleukin 4, 100 ng mL − 1 ) and rhGM-CFS (recombinant human granulocyte-macrophage colony-stimulating factor, 100 ng mL − 1 ) for 5 days, replacing the medium with fresh cytokine supplement after 3 days to induce direct differentiation to dendritic cells (iDCs) 32 , 36 . This approach allowed us to then culture cells on gelatin-coated coverslips and therefore visualize the samples under a confocal microscope. For each cell line, EVs isolated from each cell culture were used. For flow cytometry, cells were seeded on 96-well plates at a concentration of 2 x 10 4 cells per well. Cells were incubated at 37°C in a 5% CO 2 atmosphere at different time points (24, 36, and 48 h for THP-1 cells and 4, 24, and 48 h for HEK-293 cells) with the corresponding treatments. The treatment dose was calculated to obtain a final concentration of 0.3 µg of nucleic acids per well. After the incubations, the cells were washed with PBS and fixed in formalin (10%). As the cell lines were cultured in suspension, each washing step was done with a previous centrifugation step at 310 g for 5 min to sediment the cells at the bottom of the plate. For confocal experiments, cells were seeded in 24-well plates containing a cover glass with 500 µL of gelatin 0.1%, gelatin was incubated for 30 min and the excess was removed by aspiration. iDCs cells were seeded at a concentration of 3 x 10 4 cells per well and incubated for 24 h at 37°C. After short (6 h) and long (48 h) incubations with the treatments (with the same condition as in the previous protocol), the cells were fixed in formalin (10%) for 20 min and permeabilized with PBS-Tween80 (0.1%), washing the coverslip with PBS in each step. Additionally, the cell nucleus was stained (DAPI, 1:10.000 in PBS for 10 min at RT), as was the membrane (CellMask™ Orange, 1:1.000 in PBS for 10 min at RT), again washing the coverslip with PBS in each step. Cover glass was then placed onto the microscope slide and the cellular uptake of the NPs was observed using a Leica DMi8 S confocal microscope. Images were analyzed with the software Fiji /ImageJ 1.52v (Wayne Rasband, National Institutes of Health, USA). 6. Transfection of THP-1 cells with mRNA-OVA complexes Naïve THP-1 monocytes (undifferentiated cells) and immature DCs (iDCs) (THP-1 cells left to differentiate to DCs for 4 days with PMA and IL-4) 33 , 38 , 39 were transfected with the mRNA-OVA KH NPs@THP-1 EVs complexes (resulting in DCs). Also, eGFP mRNA-loaded complexes were tested by flow cytometry to confirm uptake and transfection efficiency. Briefly, cells were seeded in a 24-well or 12-well plate at a concentration of \(\:1\times\:{10}^{5}\) cells mL − 1 and incubated with the treatment at 37°C in a 5% CO 2 atmosphere for 24 h. A final concentration of 0.6 µg per well of ribonucleic acid was used. Since undifferentiated THP-1 cells were cultured in suspension, transfection was performed following a centrifugation step at 310 g for 5 min to sediment the cells at the bottom of the plate. To reach the desired concentration of genetic material, half of the medium volume was removed and replaced with half of the treated medium containing the samples. After the incubation, the cells were fixed in formalin (4%). To verify cell stimulation by the OVA antigen, changes in several differentiation markers (i.e. CD86, CD209, CD11c, and CD11b) were analyzed by flow cytometer. Various controls were used, including untreated monocytes, mRNA-OVA KH NP-treated and mRNA-OVA lipofectamine-treated. 7. Biodistribution studies in tumor-bearing mice The in vivo experiment was performed at the Centre d’Investigació i Desenvolupament (CID-CSIC), which adheres to the principles set out in the laws, regulations, and policies governing the care and use of laboratory animals by the Generalitat of Catalunya and following protocol number 11707, approved by the Direcció General del Medi Natural. Eight-week-old female C57BL6J mice were purchased from Envigo (Spain) and maintained at a constant temperature of 21 ± 1°C and humidity of 55 ± 10% under a 12 h light/dark cycle, and with ad libitum access to food and water. All mice were regularly monitored for animal welfare and adherence to the experimental protocol. The animals were randomly divided into 5 groups receiving the KH NPs, JAWS EVs, KH NP@JAWS EVS, KH NP@LLC1 EVs and CTRL containing mRNA luciferase (n = 4 for each experimental group). Because THP-1 EVs are human, for the in vivo study we selected the equivalent murine EVs from murine-monocyte derived macrophages, JAWS, which could be considered comparable to THP-1 human EVs. As a negative CTRL, we selected murine lung cancer LLC1 EVs to prove the tropism of JAWS EVs in vivo to DCs and macrophages. The mice were injected subcutaneously in the back with B16F10 cells (2.5 × 10 5 cells per mouse, 50 µL PBS 1x). Tumor volume and mouse weight were measured five times per week. Two weeks later, they were injected with the different complexes intravenously through the tail. Six hours later after injection, animals were anesthetized with ketamine (87 mg kg − 1 ) and xylazine (12.5 mg kg − 1 ) and firefly luciferine (150 µL) was intraperitoneally administered. Fifteen minutes later, mice were euthanized, organs of interest collected and ex vivo bioluminescence was assessed through radiance measurement. Negative control (non-injected mice) were used to subtract the background signal. Images were treated and analysed using Hokawo v3.0 software. Markers of cell toxicity such as serum aspartate aminotransferase activity (sAST), hepatocellular injury/toxicity such as serum alanine aminotransferase activity (sALT) or lactate dehydrogenase (sLDH) were monitored after hydrogel exposure by the IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) optimized kinetic UV method in an Aries chemical analyzer (Werfen Instrumentation Laboratory S.p.A., Italy) and expressed as U L − 1 . Each analysis was validated by a biochemical chemist and hematologist using quality control serums (CQI), in the San Raffaele Mouse Clinic. 8. Protein corona 8.1. Hard Corona (HC) and Soft Corona (SC) obtention To study protein composition and biological functions of the different EVs and complexes, we designed an experiment to mimic real bloodstream conditions. We assume that plasma represents 80% of the total interacting volume as it was previously described by Monopoli et al 40 – 42 . The concentration of EVs used was 1.04 x 10 9 particles mL − 1 . Human plasma (160 µL) and PBS (40 µL) were introduced into low protein binding 1.5 mL microtubes. The total sample volume used for the study was 40 µL. The solution was incubated at 37°C for 1 h under continuous agitation at 300 rpm. After incubation, the NP corona complex was pelleted from excess proteins by centrifugation at 15000 rpm, 4°C for 15 min. The supernatant was discarded, and the pellet was resuspended in PBS (500 µL) and centrifuged again. This washing procedure, repeated three times, removed unbound and loosely bound proteins from the NPs. The pellet was then resuspended in SDS (10%, 100 µL), heated at 95°C for 10 min, and centrifuged at 15000 rpm, 4°C for 15 min. The NP-SC complexes were obtained from the supernatant and the NP-HC complexes wer obtained by resuspending the pellet in PBS (100 µL), incubating with DTT (5 µL, 20 mM) to break disulfide bonds, and then this fraction was centrifuged again at 15000 rpm for 15 min. This fraction was centrifuged again to separate the more tightly bound proteins form the NPs. The proteins were stored at -80°C until further use. 8.2. Liquid Chromatography Mass Spectrometry (LC-MS) analysis For total protein purification, the pellets were resuspended in ammonium bicarbonate buffer (AB buffer, 50 mM). Phase separation was achieved by adding methanol (400 µL), chloroform (100 µL) and RNAse-free water (300 µL) was added to the reaction in a 4:1:3 ratio. The mixture was vigorously shaken for 15 sec, and incubated for 5 min at RT. After the incubation, samples were centrifuged at 14.000 g for 1 min at RT. The interphase with the proteins was extracted and precipitated, dried and resuspended in AB buffer (50 mM). A 1:80 ratio of the protein concentration from each sample was digested using a mixture of trypsin and lysoenzsime C. To determine the protein composition of the EVs, SC and HC complexes, the samples were analyzed using a M5 MicroLC system coupled to ZenoTOF™ 7600 System (SCIEX, USA) with positive electrospray ionization (ESI) with OptiFlow 1–50 µL Micro/MicroCal Turbo V Ion Source. Chromatographic separation was performed with FA water solution (MP A, 0.1%) and FA acetonitrile solution (MP B, 0.1%), with a linear gradient applied at a constant flow rate (7 mL min − 1 ). The gradient started at 5% B, and increased to 25% at 38 min, 32% at 43 min, 40% at 48 min, and 80% at 50 min. The gradient was then returned to initial conditions and held until 65 minutes for re-equilibration. The chromatographic column used was a microLC Luna Omega Polar (H24-06911, Phenomenex) with full loop injections set at 5 µL. MS data were acquired in data-dependent acquisition (DDA) mode. MS1 spectra were collected over an m/z range of 400–1400. For MS2, a peptide workflow was used, selecting up to 20 precursor ions per cycle with Zeno Pulsing enabled (threshold set above 100.000 cps), and an m/z range of 100–1700. All samples were injected in triplicates. A merged database search for IDA runs was performed using MSFragger against Mus Musculus and human proteome from UniProtKB after appending decoy sequences (reverse contruction) and common contaminant sequences (UP000005640 contains 40.900 entries and 20.450 decoys and UP000000589 with 34.681 entries and 17.340 decoys). The XTandem! algorithm was used to obtain the peptide spectrum matches. The PTMs enabled were methionine oxidation and N-terminal acetilations. False discovery rate (FDR) analysis was enabled and we used MSBooster, Percolator and Protein Prophet as protein hits validation tools and rescoring 43 – 46 . Only proteins identified with 1% global FDR were considered true identification. Perseus statistical software was used to analyse the LFQ intensities (MSFragger IonQuant), and data was log-transformed and imputed by Fragpipe Analyst R (v1.0.4). Data processing was performed using SCIEX OS 2.2 acquisition software, MSFragger (v22.0) software, and PDV viewer and Fragpipe-Analyst for proteome last analysis and data visualization. 9. Statistical analysis All data presented, unless otherwise stated, represent the mean value ± standard deviation (SD) of at least a three independent samples. Statistical differences were evaluated using GraphPad Prism®, by performing an ANOVA comparisons between the different groups. P values lower than 0.05 were considered statistically significant. For confocal imaging analysis, JACoP (Just Another Colocalization Plug-in) was used 47 . Images were generated using BioRender. RESULTS AND DISCUSSION 1. Efficient encapsulation of pBAE NPs inside EVs The natural tropism of EVs to target cells of the same lineage from which they originated can be used for the directed targeting of pBAE NPs to DCs. Although we previously demonstrated that pBAE NPs can passively accumulate in DCs through the selection of an appropriate oligopeptide-end modification, thereby confirming their suitability as nucleic acid vaccines 6 , here our goal was to enhance their selectivity. As this is the first attempt to encapsulate pBAE NPs within EVs, various EV encapsulation methods were evaluated and subsequently, the resulting complexes were analyzed both biophysically and in vitro . The pBAE NPs used were formed by a peptide sequence of Lys-Lys-Lys, combined with another of His-His-His, named KH NPs, as it has demonstrated a high transfection in cells due to proton sponge effect 48 . KH NPs were loaded with reporter plasmid GFP as a model for the firsts experiments, since in our previous studies no differences in the physicochemical properties of pBAE NPs were found when shifting from plasmid to mRNA encapsulation 35 , 48 , 49 . Various approaches to encapsulate macromolecules or NPs of different sizes into EVs have been reported in the bibliography (Fig. 1 A) 18 , 22 , 25 . Incubation is a widely used method 26 , 50 . It consists of incubating the molecules with a cargo of interest and it is particularly efficient for hydrophobic interfaces. To increase the encapsulation efficiency, sonication and extrusion strategies have been also described 26 , 51 , 52 . These two techniques are borrowed from liposomal formulations that can cause changes or twisting in the EV membrane, helping to introduce the cargo. Saponin-assisted treatment for EV permeabilization can further enhance cargo internalization. Saponin is an active compound that can generate complexes with cholesterol on the EV surface and create pores, thereby increasing membrane permeability. However, in contrast to cell membranes, EVs have more rigid lipid bilayers due to aggregation of sphingomyelin, cholesterol, and ganglioside. This feature can thus hinder the insertion of hydrophobic substances into the vesicles 20 . Finally, the freeze–thaw method is a straightforward technique used to load drugs into EVs, as active compounds mixed with EVs undergo a few cycles of freezing at -80°C in a dry ice bath with acetone and thawing at RT 22 , 53 . Nowadays, most of the NPs encapsulated into EVs have been metallic such as gold 54 , platinum 25 , iron oxide 55 or zinc oxide 50 , 56 . However, few studies have addressed the encapsulation of soft nanomaterials like poly(lactic-co-glycolic acid) (PLGA) NPs 57 or metal-organic framework (MOF) NPs 58 . Importantly, encapsulation of pBAEs NPs into EVs has not been yet described in bibliography. One of the challenges to successfully coat soft nanomaterials with EVs consists in designing fast and novel coating protocols that are NP material-specific, since soft NPs are not stable for long periods of time under harsh conditions required by many encapsulation protocols. For example, PLGA NPs coated with EVs or cell-membrane-derived vesicles exploited the use of the sonication and extrusion methods 18 , 59 , 60 . Given the lack of previous studies reporting pBAE NPs encapsulation into EVs, we assessed various techniques and analyzed their potential impact in the nanomaterial functionality. Three different techniques were selected, sonication, extrusion, and freeze-thaw, as these methods were previously proposed for soft organic NPs encapsulation 51 , 52 , 61 . Sonication of pBAE NPs for 10 min slightly increased the mean size of the particles from a 130 ± 3.3 nm to 154 ± 2.1 nm, while the PDI value remained unchanged. However, longer periods of sonication of 20 min increased the mean size to more than 200 nm and the PDI over 0.3, thereby indicating partial degradation of the nanomaterial morphology (Fig. 1 B-C). Thus, we evaluated the functionality only with sonicated pBAE NPs using an in vitro transfection assay with ARPE-19 cell line as a suitable transfection host. pGFP transfection confirmed only a slight reduction in transfection efficiency after 20 min of sonication (Fig. 1 D). By using extrusion, the pBAE NPs presented a high PDI and very polydisperse size distributions, demonstrating the NPs degradation, and thus, discarding this method. Finally, for the freeze-thaw method only the EVs underwent freeze-thawing and pBAE NPs were added during the recovery time since previous experiments by the group discouraged the repeated freeze-thaw of pBAE NPs 62 . The advantages of the freeze-thaw method include its simplicity and loading capacity (regarding encapsulation of compounds such as active molecules, proteins, or genetic material) 39 , especially for lipid structures like EVs. Freeze-thaw cycles promote lipid mixing in the lipidic bilayer, pointing to conformational changes, breakdown, and the rearrangement of lipid vesicles during repeated fast freezing and thawing processes 63 . Additionally, negatively charged liposomes can entrap cationic NPs of various natures (i.e. polyplexes or mesoporous silica NPs) by a spontaneous charge interaction, a process also called membrane fusion 28 , 58 , 64 , 65 . Triggered by electrostatic interactions, these vesicles fuse to build a lipidic double-layer coating. To assess this technique, three cycles of freeze-thaw were analyzed after recovery times of 0, 20, 40, 60, and 90 min in THP-1 EVs (Fig. 1 E). As expected, the mean size decreased immediately after the freeze-thaw cycles, thereby indicating that the fast-freezing process breaks down EVs, which rapidly reassemble into smaller vesicles that regain their initial sizes after a recovery time (Fig. 1 E). Moreover, the PDI values increased rapidly after the freeze-thaw cycles and decreased after 60 min of recovery time. However, the 60 min recovery time was the chosen one as the size distribution graphs showed EV aggregation after 90 min (Fig. 1 F). 2. Physico-chemical characterization of the biomimetic nanosystems After selecting the freeze-thaw method as the most suitable approach to combine EVs and pBAE NPs, we conducted a more detailed characterization of KH NP@THP-1 EV complexes (Fig. 2 A). While KH NPs presented a size of around 130 ± 3.3 nm, THP-1 EVs were slightly larger, around 152 ± 1.2 nm nm (Fig. 2 B). Three different formulations of KH NP@THP-1 EV complexes with different NP:EV ratios (1:1, 1:2, 1:10) were prepared and the distribution size followed by NTA (Fig. 2 B). Only the complexes formed with a ratio 1:2 presented a monodisperse system (PDI = 0.15) with a significantly increased mean size with respect to EVs and KH NPs separately (Figure S1 ). Lower ratios resulted in the aggregation of the nanosystem by an excess of KH NPs that cannot be fully coated and may tend to aggregate with other THP-1 EVs driven by electrostatic forces. In contrast, with higher ratios there was an excess of free EVs that did not interact with NPs, as the size remained around 100 nm, as found for naked EVs. By zeta potential THP-1 EVs and KH NPs alone revealed a surface net charge of -29.0 ± 1.9 mV and 19.9 ± 1.2 mV respectively, while different values were obtained for the KH NP@THP-1 EV complexes, as a function of the ratio used. Lower ratios 1:1 presented a slight positive charge, consistent with the notion that not all the NPs were fully coated, due to an insufficient amount of THP-1 EVs (Fig. 2 C). The 1:10 ratio was discontinued due to high instability, which impeded reliable measures of the surface charge. Thus, the ratio 1:2 was the best proportion to obtain monodispersed and small KH NP@EVs with a negative surface net charge, indicating the EV bilayer surrounding the KH NPs. To have a further insight into the structure of the complexes, the EVs were analyzed by cryo-transmission electron microscopy (cryo-TEM) to analyze their shape and size. We demonstrated the presence of physical interactions between KH NPs and THP-1 EVs (Fig. 2 D, Figure S2). Despite interference from excess polymer, KH NP@THP-1 EV 1:2 complexes elucidated the encapsulation dynamics, emphasizing the presence of NPs within EVs. Following MISEV2018 and 2023 guidelines 25 – 27 , we further characterized the biochemical features of empty and loaded THP-1 EVs (Fig. 2 E-F). Western blot was used to characterize the presence of three types of proteins: 1) a transmembrane protein to demonstrate the presence of a lipid bilayer, TGS101; 2) a tetraspanin to demonstrate that the nanosystems are EVs, CD63, or CD81; and 3) BSA, to demonstrate the lack of contaminants in the samples. These markers were analyzed in THP-1 cell lysates, THP-1- derived EVs, KH NP@THP-1 EV complexes, and a sample containing only KH NPs. As expected, BSA levels were significantly reduced in the EV samples compared to the cell lysates. The BSA residual presence could be attributed to proteins from the FBS used for cell maintenance. Predictably, CD63, CD81, and TGS101 were present in all the samples containing EVs, although their concentration appeared to decrease after the encapsulation process. The maintenance of the high encapsulation efficiency of the plasmid after building the biomimetic nanosystems must be highlighted (Fig. 2 G). KH NPs achieved more than 90% of nucleic acid encapsulation, while pBAE NPs complexed with EVs were over 80%. The efficiency was considerably higher than previous reports for engineered hybrid EV-liposome systems through freeze-thaw method for siRNA loading 17 . To confirm the interaction between THP-1 EVs and KH NPs, we performed confocal and hyperspectral microscopy. The two nanosystems were labelled with two distinct fluorophores, KH NPs (Cy5, in red) and THP-1 EVs (NBD-PE, in green) (Fig. 2 H). By confocal, the KH NP@EV complex presented a colocalization of the two signals by giving a yellow spot (Fig. 2 H, Figure S3-S4). To estimate Pearson’s (PC) and Manders’s coefficients (M1, green fraction overlapping with red; M2, red fraction overlapping with green) 33 , were calculated using a different threshold to exclude background noise. Both coefficients reported an average between 40 and 50% colocalization between EVs and KH NPs, confirming that the sample contained both uncoated NPs and EVs without any NPs inside, together with biomimetic systems. Details are provided in Explanation S1 in the SI. To visualize the motion in real-time dispersion 66 , dark-field hyperspectral (HSI) microscopy was performed, including optical and spectral images (Fig. 2 I, Figure S5). The mapping of THP-1 EVs (in green) and KH NPs (in red) for the KH NP@EV (1:2) sample, showed that both materials colocalized when they were combined and KH NP@EV complexes were spherical as observed by cryoTEM. Next, the formation and stability of the biomimetic systems was assessed by using FRET, previously demonstrated by us for pBAE NPs 48 , and others 67 , for different hybrid systems. FRET signal depends on the lateral distance between the fluorophores labelling distinct structures 17 . Therefore, the higher the FRET signal, the closer the two labelled molecules are. The signals exceeded the lower control signal of each pair of components (Figure S6), thus indicating the presence of FRET signals between all pairs (Fig. 2 J). At 25°C, even that the plasmid and the polymer showed a strong interaction during the first hours, it decreased over time going from 0.65 to 0.37 AU, pointing the instability and gradual dissociation of the polymer-plasmid complex. However, at 37°C, the FRET signal remained relatively constant, a higher temperature appeared to enhance the interactions, possibly due to an increased molecular mobility, even that the signal was less stable than at 25°C. Regarding the EV-pBAE interaction, after an initial fluctuation, the FRET signal remained relatively stable around 0.88 AU at both temperatures. Compared to our previous stability studies addressing pBAE NPs 31 , 48 , it is also reasonable to attribute the increased long-term stability to the EV shell. As naturally stable structures, these vesicles might provide additional protection or a shell that promotes closer proximity between the plasmid and the polymer. Finally, and as expected, the EV-plasmid interaction was the lowest at both temperatures, as the plasmid was encapsulated inside the polymer, presenting the highest distance. 3. KH NP@EV selective targeting to DCs and further immune cell activation Before evaluating immunogenicity, we performed a preliminary toxicological and functional study in vitro using human THP-1 cells. At a concentration of 0.1 µg mL − 1 of KH NP@EV complex and its components (THP-1 EVs and KH NPs) did not result in citotoxicity (Figure S7), as reported in previous studies 5 , 31 . Consequently, the capacity of this complex to safely penetrate the targeting cells was studied and compared to THP-1 EVs and KH NPs alone. Two different cell lines were used, undifferentiated THP-1 as the targeting cell and HEK293, as a permissive cell line (Fig. 3 A). THP-1 EVs were labelled with NBD-PE fluorophore and KH NPs with Cy5 (Fig. 3 B-E). Both cell lines presented no signal when treated with empty EVs by flow cytometry, which was expected in HEK293 cells but not in THP-1 cells. Even EV membranes facilitate receptor-mediated endocytosis—they may be limited by membrane fusion efficiency 68 . Fusion is often pH or receptor-dependent, and without triggering conditions such as acidic endosomes, EVs may be internalized and degraded rather than delivering the cargo along with the fluorescent dye, creating a false impression of low uptake. However, KH NP@THP-1 EV complexes present a high fluorescent signal, thanks to the proton sponge effect of KH NPs, allowing for the release of the decomplexed biomimetic nanosystems inside the cell's cytoplasm. The internalization capacity of the nanosystems was studied at 4, 24, and 48 h (Fig. 3 B-E). THP-1 cells, as target model cell line for our vaccine, presented a higher and faster internalization rate of the complex in comparison to HEK293 cells. Regarding the analysis in HEK293 cells (Fig. 3 C-E), the internalization pattern of naked KH NPs and KH NP@THP-1 EVs differed. The uptake of the complexed KH NPs was significantly higher than naked NPs at 4 h, with around 30% and 20%, respectively. Meanwhile KH NPs@THP-1 EVs level remained steady during the 48 h of the experiment, naked KH NPs signal decayed over time. This reduction could be due to the rapid doubling time of this cell line (24–36 h) 69 , which leads to clearance of the internalized NPs, as well as a reduction of the fraction of cells that had taken up this nanomaterial. In contrast, KH NP@THP-1 EVs signal did not decrease over time, which might indicate enhanced stability in the culture medium conferred by the THP-1 EV coating and, consequently, less uptake, due to a difficulted disassembly of the biomimetic complexes. In this regard, the FRET analysis demonstrated that the complexes presented greater stability over an extended period. However, it is important to note that FRET analysis was performed in FBS-free medium. Overall, the THP-1 EV coating increased the internalization of KH NPs maintaining a higher level of internalized EV-coated NPs for a longer period. This can be explained by the fact that this formulation is optimized for cell transfection and antigen presentation 2 and the elevated internalization is attributed to the higher duplication time of the THP-1 cell line (around 60–70 h) 70 . However, the uptake of naked THP-1 EVs into THP-1 cells, the same cell lineage they come from, was surprisingly low, not in accordance with previous bibliography indicating a certain tropism of EVs to be internalized by the parental cell lineage 22 , 23 , 71 . This finding could be explained by the limited membrane fusion efficiency. Nevertheless, our findings indicate that the complexes enhanced the internalization capacity of both THP-1 EVs and KH NPs. This unexpected advantage of the complex nanosystem could be attributed to an increased surface charge towards a cationic range when the cationic pBAE NPs were coated with THP-1 EVs. As in fact, strongly negatively charged lipid vesicles have a lower tendency to be internalized by cells because the cell membrane already has a negative charge 65 . To validate the engineered nanosystems for immunization purposes, an in vitro model, comparing the expression levels of specific markers on cells stimulated with the antigen with those of naïve THP-1 cells was used. This approach provides insights into the phenotypic profiles of cells. Significant increase in the expression of CD86, CD11b, CD11c and CD209, on those cells treated with the cytokine cocktail of IL-4 and PMA, confirmed the differentiation to iDCs 34 (Fig. 3 G, Figure S8-S9). To confirm the selectivity of our complex, we used fluorescence microscopy to qualitatively study the internalization of KH NP@THP-1 EVs in THP-1 cells directly differentiated to iDCs, as a more representative model of in vivo target cells (Figure S10). THP-1 EVs achieved minimal penetration, meanwhile KH NP@THP-1 EVs presented a higher uptake of the biomimetic nanosystem. Although the cell membrane appeared to be slightly damaged in the cells treated with the complexes, especially after 48 h, biocompatibility experiments confirmed no cytotoxicity at the concentrations tested (Figure S7). Then, we examined the same markers after transfection with our biomimetic nanosystems. After determining the optimal concentration of OVA mRNA in non-differentiated THP-1 cells (Figure S11), we transfected the cells using both KH NPs and KH NP@THP-1 EV complexes (Fig. 3 H, Figure S12). We analyzed the surface markers CD86, CD11b, CD209 and CD11c, which increase when an immune response is induced 33 . Of note, there was no statistically significant difference between control iDCs and those transfected with KH NPs, thereby indicating an insignificant effect of the NPs on the cells. However, all markers showed changes when cells were transfected with the KH NP@THP-1 EV complexes, thus confirming the need for the EV coating for the selective transfection and functionality of the antigenic mRNA. Upon closer examination of the differentiation markers, CD86 and CD11c showed a decrease, indicating possible incomplete cell maturation. The mRNA-OVA administered may stimulate the cells through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), however, the adjuvant effect did not promote the complete maturation of DCs. Furthermore, THP-1 cells differentiated into DCs and then induced to mature may have a different phenotypic profile compared to DCs derived from primary sources such as human peripheral blood 33 , 72 . An increase in CD11b indicated maturation to DCs 33 , thus confirming the potential immunization properties of the KH NP@EV complexes. Indeed, despite the significant expression of CD11b and CD86, the cells resulting from the maturation of THP-1 cells through the iDC state cannot be considered fully mature because they lack the expression of CD83, another essential marker 73 . 4. EVs maintain similar proteomic content and cellular functions across species Because the EVs in this study were designed to enhance accumulation in antigen-presenting cells (APCs) such as DCs and macrophages, we initially selected EVs derived from human THP-1 cells. For in vivo experiments, however, we used murine EVs derived from JAWS cells—a murine monocyte-derived macrophage line considered functionally comparable to the human THP-1 model. To characterize the EVs and evaluate the conservation of their proteomic composition across species, we performed label-free MS on EVs isolated from both THP-1 and JAWS cells. Given their species-specific origins, we used the human protein database to analyze THP-1 EVs and the murine database for JAWS EVs. We cross-referenced the identified proteins across both datasets to assess whether the EV protein composition was comparable despite species differences. This comparative analysis enabled us to evaluate the degree of conservation in EV protein cargo and potential functional similarities between the two species. A total of 383 proteins were identified in THP-1 EVs (178 with more than 2 peptides) and 144 proteins in JAWS EVs (61 with more than 2 peptides). Additionally, unique proteins were detected in each EV subtype (Fig. 4 A), suggesting unique entities released by cells rather than debris. To identify the signature proteins in the EVs, we performed statistical analysis on the expression levels of proteins identified in these data sets. We pinpointed a top 31 proteins, with a false discovery rate (FDR) of < 0.1 (Fig. 4 B). Histones, cytoskeleton proteins (actins and tubulins), small ribosomes and heat shock proteins (HSP) ranked as the most abundant proteins in both EVs. Interestingly, HSP90AB1 and HSPA8 were preferentially packaged in THP-1 EVs, which are involved in intercellular communication between EVs and other cells for the delivery of the EV cargo. Other proteins relatively enriched in THP-1 EVs included annexin A4 (ANXA4), complement C3 (C3), α-2-macroglobulin (A2M) and proteins involved in metabolism, such as receptor-type tyrosine-protein phosphatase (PTPRC) and vitamin D-binding protein (GC). The expression of C3 in EVs it has been proved to be involved in the activation of the inflammatory response, as immune cells such as macrophages or DCs in the TME, can taken up EVs containing C3 and repolarize macrophages into an immunosuppresive phenotype 74 . However, the formation of the biomimetic system can turn this disadvantage as an advantage by selectively targeting macrophages or DCs. Once internalized, the system can release the mRNA cargo inside the cells, thereby activating the immune system and promoting a pro-inflammatory phenotype response. They also presented some glycoproteins such as immunoglobulin kappa constant (IGKC) and immunoglobulin heavy constant gamma 1 (IGHG1), which play a crucial role in immune system activation. JAWS EVs presented phosphoglycerate mutase 1 (PGAM1), ATP synthase subunit alpha (ATP5F1A), inter-α-trypsin inhibitor heavy chain 2 family members (ITIH2), and peroxiredoxin-1 (PRDX1), all involved in glycolysis and cancer metabolism. Remarkably, all EVs were significantly enriched in proteins involved in transport, including clathrin heavy chain 1 (CLTC), lactotransferrin (LTF), hemopexin (HPX) and serrotransferin (TF). Significant differentially expressed protein-coding genes are presented in the volcano plot (Fig. 4 C). In particular, proteins associated with multivesicular bodies, plasma membrane, extracellular matrix and microtubule/cytoskeleton were enriched. Meanwhile, proteins related to cell motility, adhesion and membrane trafficking were downregulated. The protein composition of both EVs was remarkably similar, which was represented in the biological functions of the detected proteins (Fig. 4 D). THP-1 EVs presented a higher amount of structural proteins with a 42% in comparison with the 24% found in JAWS EVs. However, JAWS EVs had around a 10% more of immune and inflammatory proteins in comparison with THP-1 EVs. The other protein portions remained very alike. To gain insight into the function of the EVs, we conducted GSEA using the Gene Ontology (GO) and Hallmark databases. Strikingly, genes encoding proteins demonstrated that EV-specific proteins were selectively enriched in MYC targeting and mTORC1 signalling (Fig. 4 E, Figure S13). They also were involved in metabolic processes such as complement activation, G2-M checkpoint or unfolded protein response. Collectively, these bioinformatic analyses of the proteomic content of both EVs revealed that even coming from different species, both monocytic cell lines produced EVs with highly conserved core proteome and signalling transduction pathways. 5. KH NP@JAWS EVs drives trafficking to macrophages A biodistribution study was carried out in mice to determine if the differences found in vitro prevail in vivo . We first induced the tumor formation subcutaneously using murine melanoma cell line, B16F10 (Fig. 5 A). Since these tumors present a great amount of immune infiltrating cells, including different types of APCs, we tested the complex selectivity and accumulation in WT tumours with similar tumor volumes. Mice were followed up and treated when the tumor volumes were similar for all the groups (Fig. 5 B-C). Murine EVs collected from JAWS cell line were used as the targeting EVs model for the in vivo biodistribution study. JAWS EVs were labelled with a fluorescent dye Cy3 and KH NPs were labelled with Cy5. Mice were intravenously administered with the complex KH NP@JAWS EVs and KH NPs, both encapsulating a reporter mRNA of luciferase. As CTRL we had non-treated mice, mice treated with JAWS EVs without mRNA encapsulated and mice treated with KH NPs@LLC1 EVs containing luciferase mRNA. LLC1 EVs were used as negative CTRL because they should not target APCs or neither the tumor as they are not homing cells, they were derived from murine lung cancer. After 6 h, mice organs and tumors were collected and in vivo and ex vivo imaging were performed to examine the complex biodistribution in B16F10 tumor model. The short time point of 6 h was chosen because it was previously observed in other studies that we have the maximum level of expression of luciferase 5 , 11 (Figure S14). As expected, mice treated with KH NP@JAWS EVs and KH NPs presented accumulation in the liver, spleen, and lungs. KH NPs had negligible luciferase expression in the tumor (Fig. 5 D). In contrast, KH NP@JAWS EVs tumor-bearing mice showed significant increases in luciferase signal in the tumor compared with the other treated groups. Instead, KH NP@LLC1 EVs even being composed by the same NPs but with EVs non-targeting antigen-presenting cells, did not express luciferase in the tumor, liver, or lungs, but only in the spleen. This result strikingly demonstrates the superior and successful transfection capacity of KH NP@JAWS EVs concerning the CTRL samples, i.e. KH NPs without EVs shell, or the non-targeting KH NP@LLC1 EVs complexes. Through the Cy3 signal, we also demonstrated that JAWS EVs and KH NP@JAWS EVs mostly accumulated in the liver and spleen, as those organs contain high amounts of DCs and macrophages such as Kupffer cells or splenic macrophages (Fig. 5 E-F). Fluorescence was also detected in the tumor and lungs (Fig. 5 G, Figure S15). By Cy5 signal KH NP@LLC1 EVs were found significantly accumulated in the lung but also in the spleen (Fig. 5 H, Figure S14). Fluorescent signal was also observed in the liver for KH NPs, KH NP@JAWS EVs and KH NP@LLC1 EVs (Figure S16). No signal was detected in the tumors (Figure S16). We also demonstrated that liver enzyme levels across different groups were similar for ALT and AST (Fig. 5 I-J), indicating that the accumulation in the liver did not result in differences in liver damage among the different groups. However, a significant increase in LDH was observed in both mice treated with KH NP@JAWS EVs and KH NP@LLC1 EVs (Fig. 5 K). These increased levels reflect that the complexes were able to stimulate the immune system like macrophages or T cells. KH NP@JAWS EVs presented an increased uptake by Kupffer cells or splenic macrophages and an enhanced delivery to tumor cells. 6. The uptake of KH NP@THP-1 EVs by cells relies on EVs surface adhesion molecules Since we have demonstrated physical interactions between EVs and KH NPs, it is possible that the uptake of KH NP@JAWS EVs by antigen presenting cells is mediated by specific surface molecules on EVs. To determine if there is a correlation between EVs’ in vitro and in vivo behaviours in patients, as well as their biological processes and protein corona compositions, soft (SC) and hard corona (HC) proteins identified by LC–MS were subjected to human protein databases (UniProt) analysis. We conducted proteomic profiling of THP-1 EVs alone and the SC and HC of THP-1 EVs and KH NP@THP-1 EVs after incubation with human plasma. A total of 371 proteins were identified in THP-1 EVs, 147 in THP-1 EVs HC and 172 in KH NP@THP-1 EVs HC (Fig. 6 A). Meanwhile in the SC it was detected a 428 proteins in THP-1 EVs SC and 430 in KH NP@THP-1 EVs SC (Fig. 6 B). Principal component analysis (PCA) demonstrated a closer correlation between the HC protein expression for THP-1 EVs and KH NP@THP-1 EVs compared to THP-1 EVs alone (Fig. 6 C). According to PCA and consensus clustering analysis, complex and EVs presented a higher degree of similarity in their protein coronas. The score plot displayed a difference between THP-1 EVs alone, HCs and SCs, presenting three different populations. Interestingly, no difference was observed between the HC and SC compositions of THP-1 EVs and KH NP@THP-1 EVs, confirming that the complex formation did not change the EVs structural and biological properties (Figure S17-S18). Similar results were observed by comparing the most abundant proteins displayed as a relative protein abundance in the heatmap (Fig. 6 D, Figure S19). The proteome of THP-1 EVs presented a completely opposite protein enrichment in comparison to the HC of THP-1 EVs and KH NP@THP-1 EVs. The most abundant proteins in the HC were serum albumin (ALB) and actin cytoplasmic 1 (ACTB) which are secreted proteins and subcellular components. Also, glycoproteins such as immunoglobulin lambda (IGLC2), kappa (IGKC) and gamma 1 (IGHG1) were detected. These proteins are part of the genetic machinery that produces the structural components of antibodies, which are crucial for the immune system's defense against foreign bodies (Fig. 6 E). In contrast, the proteins observed in the SC presented a more similar composition to THP-1 EVs. The SC main proteins were serum albumin (ALB), fibrinogen α (FGA), β (FGB) and γ (FGG), actin cytoplasmic 1 (ACTB), and immunoglobulin heavy constant gamma (IGHG1) (Fig. 6 F). The higher abundance of IGHG1 protein in the protein corona favoured their engulfment by phagocytes and induced complement activation (classical pathway) as interpreted from associated protein functions. An influential protein deleted in HC was sialic acid- binding Ig-like lectin 16 (SIGLEC16), which plays a major role in immune balance by activating DCs and macrophages response. Previous reports have demonstrated that Siglec receptors can impact in DC function, including increased antigen presentation, cytokine production, and migration to lymph nodes to prime tumor-specific T cells 75 – 79 . Relative protein abundance of SIGLEC16 demonstrated an upregulation in the HC of both, THP-1 EVs and KH NP@THP-1 EVs (Fig. 6 G). To gain insight into the HC protein functions using the Gene Ontology (GO) database, it demonstrated enrichment in specific metabolic pathways such as MYC targeting, protein secretion and mTORC1 signaling (Fig. 6 H, Figure S20). Meanwhile the protein content in the SC is related to angiogenesis (Figure S21). The expression of SIGLEC 16 can activate DCs and macrophages thanks to the interaction with the adaptor protein DAP12, an immunoreceptor tyrosine-based receptor (Fig. 6 I). Immune cells are a major source of EVs 21 . Also, the ability of DC precursors to interact with T cells is beneficial in terms of obtaining EVs loaded with immunomodulatory molecules, which would enhance vaccination potential. Some reports have associated DC-derived EVs with APCs due to the presence of MHC-II molecules on their surface 24 . Unlike previous studies that explored other aspects, our study specifically focuses on immune system activation in the context of vaccination applications. Taken together, this data demonstrates that the expression of SIGLEC 16 on our NPs@EVs complexes could potentially be involved in the activation of DCs and immune response regulation. CONCLUSION Although mRNA-based vaccines for SARS-CoV-2 have been successful, there are still many unresolved issues that could be addressed by developing safer, more efficient, and more selective nano-delivery systems. Here, we combined the physico-chemical properties of synthetic polymeric NPs with the natural biointerface functions of EV membranes to engineer hybrid smart multifunctional biomimetic nanocarriers. Until now, only a few studies have explored the use of biomimetic lipid/polymeric nanosystems or cell membrane-coating of synthetic nanoplatforms to enhance controlled delivery features. With our results, we conclude that these biomimetic EV-coated pBAE NPs outperform the functionality of the individual systems in vitro and in vivo . Undoubtedly, these discoveries will open avenues for translational studies of these novel nanosystems that hold potential for their use in nucleic-acid-based vaccination strategies. Declarations ACKNOWLEDGEMENTS We thank Dario Castellana, Giacomo Sansone, Jonas Reckmann, and Jordi Guixeras for performing preliminary and complementary experiments. We also thank Maria Stampa Lopez-Pinto and Antoni Torres-Coll for polymer synthesis. Funding from AGAUR-Generalitat de Catalunya (2021 SGR 00537), from MICIN/AEI (PID2021-125910OB-I00, MCIN/AEI /10.13039/501100011033 / FEDER, UE), from the Institute of Health Carlos III (ISCIII) (AC22/00042), and from FCAECC (TRNSC213882FORN), both from the Joint Transnational Initiative 2021 ERA-NET TRANSCAN-3, European Commission is acknowledged. Funding from CA21154, CA21135, CA CIG 17104 COST ACTIONS is also acknowledged. CF acknowledges the support of the Departament de Recerca i Universitats of the Generalitat de Catalunya through the ICREA Acadèmia 2024 programme. AM thanks POLITO for funding his research internship. References Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. 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Supplementary Files Proteomicsdata.txt Proteomics data SIpBAEEVsfinal.docx Supplementary information TABLEOFCONTENTS.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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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A) \u003c/strong\u003eScheme of the tested encapsulation methods freeze-thaw, extrusion, sonication and membrane fusion. The selected method was freeze-thawing of THP-1 EVs followed by incubation with cationic KH NPs. \u003cstrong\u003eB)\u003c/strong\u003e Hydrodynamic diameter of KH NPs as mean and polydispersity index (PDI) after sonication and extrusion. \u003cstrong\u003eC)\u003c/strong\u003eNTA size distribution of KH NPs after sonication and extrusion methods \u003cstrong\u003eD)\u003c/strong\u003eTransfection efficiency of KH NPs with pGFP after 48 h incubation with ARPE-19 cells. \u003cstrong\u003eE)\u003c/strong\u003e Hydrodynamic diameter of THP-1 EVs after three freeze-thaw cycles as mean and PDI at different recovery times. \u003cstrong\u003eF)\u003c/strong\u003e NTA size distribution of THP-1 EVs at 0 and 90 min after three freeze-thaw cycles.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7030394/v1/df81529f615c795d960d6c5b.png"},{"id":86004583,"identity":"7c35694e-2916-41de-997c-aae958e192bf","added_by":"auto","created_at":"2025-07-04 08:19:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":487953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysicochemical characterization of complex. A)\u003c/strong\u003e Scheme of the KH NP@EV. \u003cstrong\u003eB)\u003c/strong\u003e NTA size distribution of THP-1 EVs, KH NPs, and KH NP@THP-1 EV complexes at different ratios. Normalized data to 1, to compare size distribution profiles. \u003cstrong\u003eC) \u003c/strong\u003eζ−potential of THP-1 EVs, KH NPs, and KH NP@THP-1 EV complexes at different ratios. \u003cstrong\u003eD) \u003c/strong\u003eCryoTEM analysis of naked THP-1 EVs and KH NP@THP-1 EVs (additional images are shown in Figure S2). \u003cstrong\u003eE)\u003c/strong\u003e Western blot analysis of THP-1 cells, THP-1 derived EVs, pristine KH NPs, and KH NP@THP-1 EVs complex at a ratio of 1:2. \u003cstrong\u003eF)\u003c/strong\u003e Quantification of the Western blot signals, normalized by cell signals. NP signals not quantified, tested just as negative controls. \u003cstrong\u003eG)\u003c/strong\u003e Encapsulation efficiency of KH NPs and KH NP@THP-1 EVs complex at a ratio of 1:2. \u003cstrong\u003eH)\u003c/strong\u003e Confocal microscopy of fluorescently labelled THP-1 EVs and KH NPs. Scale bar = 10 mm. \u003cstrong\u003eI) \u003c/strong\u003eHyperspectral mapping obtained from the dark-field images – extracted libraries. \u003cstrong\u003eJ)\u003c/strong\u003e FRET analysis of the complexes over time, including all the components with differential labelling, as indicated in the legend; at 25 °C and 37 °C.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7030394/v1/2b3cab324e4e268abed2716b.png"},{"id":86004346,"identity":"34d2b9b1-8566-4547-af44-06e7057a31bd","added_by":"auto","created_at":"2025-07-04 08:11:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":328455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e selectivity of complex nanosystem.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e Scheme of the uptake in THP-1 cells and HEK293 cells by using KH NP@THP-1 EVs labelling the polymers with Cy5 and EVs with NBD-PE. \u003cstrong\u003eB-E)\u003c/strong\u003e Uptake in undifferentiated THP-1 (B-D) and HEK293 (C-E) cells, measuring the Cy5-KH NP (B-C) and NBD-PE-EV (D-E). \u003cstrong\u003eF)\u003c/strong\u003e Scheme of the successive states of monocyte differentiation into immature DCs (iDCs) via cytokines\u003csup\u003e66\u003c/sup\u003e, and further into mature DCs via antigen uptake, with distinctive markers for each state.\u0026nbsp; \u003cstrong\u003eG)\u003c/strong\u003e Expression levels of CD86, CD11b, and CD11c markers in undifferentiated THP-1 cells and iDCs treated with cytokine cocktails.\u003cstrong\u003e H) \u003c/strong\u003eExpression levels of CD86, CD11b, and CD11c markers after transfection of undifferentiated THP-1 monocytes with KH NPs and KH NP@THP-1 EVs encapsulating OVA mRNA. Data are presented as mean ± SEM of n = 3. P\u0026nbsp;values were determined by one-way ANOVA with Bonferroni’s correction *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7030394/v1/cf9029c0838e73d7216b9d40.png"},{"id":86004369,"identity":"e9b72985-33e6-4015-9972-30c5df2db648","added_by":"auto","created_at":"2025-07-04 08:11:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":213611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomic profiling of EVs from THP-1 and JAWS derived from cancer cells.\u003c/strong\u003e \u003cstrong\u003eA) \u003c/strong\u003eVenn diagram of proteins identified in each subset of EVs. \u003cstrong\u003eB) \u003c/strong\u003eHeatmap illustration of unique proteins associated with EVs. Scale shown is intensity (area) subtracted by mean and divided by row standard deviation (that is, Δ(area − mean)/s.d.). \u003cstrong\u003eC)\u003c/strong\u003e Volcano plot of differentially expressed proteins in THP-1 EVs vs. JAWS EVs. \u003cstrong\u003eD) \u003c/strong\u003eProtein composition was determined by LC–MS/MS. Change in the components of EVs proteins were classified by biological function. Pie-chart depicts the relative abundance of identified proteins belonging to each of seven categories, including cell motion/apoptosis, transport, cell adhesion, signaling, immune and inflammatory process, biosynthesis and structural. Protein compositions are identified by manual searching of the Human Database via the UniProt website. \u003cstrong\u003eE)\u003c/strong\u003e Variable importance in projection (VIP) scores of all the proteins contributing to the clustering pattern.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7030394/v1/3757460f24c228c87b5c76cf.png"},{"id":86004374,"identity":"05c277ed-0965-43d0-9636-19a7a9588e4e","added_by":"auto","created_at":"2025-07-04 08:11:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":210857,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiodistribution of complex in tumor-bearing mice.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003e Scheme of the experimental setup. Mice were injected subcutaneously with B16F10 cells at day 0 and after tumor formation \u0026nbsp;were treated with KH NP@JAWS EV complex on day 10 and the biodistribution studied after 6 h. \u003cstrong\u003eB)\u003c/strong\u003e Tumor volume of female mice. \u003cstrong\u003eC) \u003c/strong\u003eBody weight of female tumor-bearing mice during the experiment. \u003cstrong\u003eD)\u003c/strong\u003e Luciferase expression in all organs of tumor-bearing mice treated with the different complexes. \u003cstrong\u003eE)\u003c/strong\u003e Cy3-MFI of liver of tumor-bearing mice treated with the CTRL, JAWS EVs, KH NP@JAWs EVs, KH NPs and KH NP@LLC1 EVs groups.\u003cstrong\u003e F) \u003c/strong\u003eCy3-MFI of spleen of tumor-bearing mice treated with the CTRL, JAWS EVs, KH NP@JAWs EVs, KH NPs and KH NP@LLC1 EVs groups. \u003cstrong\u003eG) \u003c/strong\u003eCy3-MFI of tumor of tumor-bearing mice treated with the CTRL, JAWS EVs, KH NP@JAWs EVs, KH NPs and KH NP@LLC1 EVs groups. \u003cstrong\u003eH)\u003c/strong\u003e Cy5-MFI of lung of tumor-bearing mice treated with the CTRL, JAWS EVs, KH NP@JAWs EVs, KH NPs and KH NP@LLC1 EVs groups. \u003cstrong\u003eI-K)\u003c/strong\u003e Hepatic transaminase levels in serum of \u003cstrong\u003eI) \u003c/strong\u003eALT, \u003cstrong\u003eJ) \u003c/strong\u003eAST and \u003cstrong\u003eK)\u003c/strong\u003e LDH in tumor-bearing mice mice treated with the CTRL, JAWS EVs, KH NP@JAWs EVs, KH NPs and KH NP@LLC1 EVs groups. Dashed lines indicated the upper value of normality of each variable (70 U L\u003csup\u003e−1\u003c/sup\u003e for ALT, 83 U L\u003csup\u003e−1\u003c/sup\u003e for AST, and 750 U L\u003csup\u003e-1\u003c/sup\u003e for LDH). \u003cstrong\u003eL-M)\u003c/strong\u003e Cytokines quantification in plasma from tumor-bearing mice \u003cstrong\u003eL)\u003c/strong\u003e IL-1β and \u003cstrong\u003eM)\u003c/strong\u003e TNF-α treated with with the CTRL, JAWS EVs, KH NP@JAWs EVs, KH NPs and KH NP@LLC1 EVs groups.\u0026nbsp; Data are presented as mean ± SEM of n = 4 mice. P values were determined by two-way ANOVA with Bonferroni’s correction *p \u0026lt; 0.05, **p \u0026lt; 0.01 and ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7030394/v1/6539f7d96796fb5878c5bb59.png"},{"id":86004371,"identity":"897d0d95-b2c5-47d0-9ece-2966a5d9a050","added_by":"auto","created_at":"2025-07-04 08:11:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":331735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein corona of KH NPs@THP-1 EVs in human plasma.\u003c/strong\u003e \u003cstrong\u003eA-B) \u003c/strong\u003eVenn diagram of proteins identified in \u003cstrong\u003eA) \u003c/strong\u003eHC and \u003cstrong\u003eB)\u003c/strong\u003eSC. \u003cstrong\u003eC) \u003c/strong\u003ePrincipal clustering analysis of proteome data in THP-1 EVs without protein corona and THP-1 EVs and KH NP@THP-1 EVs protein coronas.\u003cstrong\u003e D) \u003c/strong\u003eHeatmap illustration of unique proteins associated with EVs. Scale shown is intensity (area) subtracted by mean and divided by row standard deviation (that is, Δ(area − mean)/s.d.). \u003cstrong\u003eE-F)\u003c/strong\u003e Volcano plot of differentially expressed proteins in \u003cstrong\u003eE)\u003c/strong\u003e THP-1 EVs HC vs. KH NP@THP-1 EVs HC and \u003cstrong\u003eF)\u003c/strong\u003e THP-1 EVs SC vs. KH NP@THP-1 EVs SC. \u003cstrong\u003eG) \u003c/strong\u003eRelative abundance of Siglec-16. \u003cstrong\u003eH)\u003c/strong\u003e Variable importance in projection (VIP) scores of all the proteins contributing to the clustering pattern in HC. \u003cstrong\u003eI) \u003c/strong\u003eSchematic representation of the activation of DCs via siglec-16 receptor from THP-1 EVs.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7030394/v1/72cef0bef4c838889454e802.png"},{"id":86362702,"identity":"6e1a4c31-0afd-493c-a2aa-204ba2e39877","added_by":"auto","created_at":"2025-07-09 19:33:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3485247,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7030394/v1/6c27530e-55f5-4a32-8360-1a20acf6bf4d.pdf"},{"id":86004347,"identity":"8f08635a-f35a-42ce-90a8-bbb9580aca97","added_by":"auto","created_at":"2025-07-04 08:11:10","extension":"txt","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":210669,"visible":true,"origin":"","legend":"Proteomics data","description":"","filename":"Proteomicsdata.txt","url":"https://assets-eu.researchsquare.com/files/rs-7030394/v1/1e16e8e056f92dfe32c0fbdf.txt"},{"id":86004355,"identity":"9d9eb856-b006-4e08-b7da-2648c3d1aa53","added_by":"auto","created_at":"2025-07-04 08:11:10","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11977856,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"SIpBAEEVsfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-7030394/v1/e1ab625fbaccc31cd9ffdb17.docx"},{"id":86004359,"identity":"f8bc293d-5d92-40bb-a915-ee00b4cec0f3","added_by":"auto","created_at":"2025-07-04 08:11:10","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":120268,"visible":true,"origin":"","legend":"","description":"","filename":"TABLEOFCONTENTS.docx","url":"https://assets-eu.researchsquare.com/files/rs-7030394/v1/6646774dc608f39a75aa5d95.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Polymeric nanoparticle-loaded extracellular vesicles as biomimetic nucleic acid vaccines","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eProphylactic vaccination is considered to be the public health milestone of 20th -century modern medicine, eradicating many infectious diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. While traditional vaccines commonly comprise attenuated, inactivated, or fractions of the infectious microorganisms, innovative vaccine designs are needed to extend beyond their prophylactic use and tackle therapeutic applications for cancer. The challenge of antigen selection needs robust and versatile vaccine formulations\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. mRNA has emerged as a promising solution, particularly following the success of COVID-19 vaccination strategies. Its common structural features, regardless of the encoded antigen, eliminates the need for complete reformulation in personalized cancer therapeutics. However, nucleic acids present a major difficulty as their stability in physiological media is compromised by nucleases that cause their degradation, and they cannot penetrate plasmatic membranes. Therefore, their direct application \u003cem\u003ein vivo\u003c/em\u003e requires protection through the use of biomaterials, which can be natural, such as extracellular vesicles (EVs) or synthetic delivery systems like lipid nanoparticles (LNPs), as used in COVID-19 vaccines, or polymeric NPs. Polymeric NPs offer greater stability \u003cem\u003ein vivo\u003c/em\u003e than LNPs, and provide a wider range of functionalization options.\u003c/p\u003e \u003cp\u003ePolymeric NPs have been extensively studied as advanced delivery systems for a wide range of diseases, with a particular focus on cancer treatments. Regarding nucleic acid vaccination, we\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e and others\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e have described the key features of effective delivery systems. In this context, we demonstrated that poly(beta aminoester) polymers (pBAEs) outperform others in efficiently complexing nucleic acids into small nanometric particles. These can then be safely administered \u003cem\u003ein vivo\u003c/em\u003e, delivering mRNA and translating it into proteins in specific target cells and organs. pBAEs are biocompatible and biodegradable polymers with a common backbone that can be easily modified to taylor pBAEs with different molecules or functional groups, such as targeting moieties, hydrophobic monomers, and stimulus-sensitive groups. They can be synthesized through a two-step Michael addition of amines to acrylate terminal groups in a production process that is both scalable and compliant with GMP-like standards\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, the use of pBAE NPs \u003cem\u003ein vivo\u003c/em\u003e requires an active targeting moiety\u0026mdash;a feature that continues to pose a challenge. In our previous works\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and by others\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, the addition of a targeting moiety is often a bottleneck due to opsonization of the functional groups once systemically administered or insufficient exposure of molecules on the NPs surface. This addition leads to only limited changes in the balance between target cells and liver accumulation, thereby indicating that targeting efficiency can be significantly improved. These limitations may be attributed to difficulties in identifying specific cell type receptors, which show distinct expression in different cell lineages\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Thus, there is a need to enhance the targeting efficiency and selectivity of the nanosystems. Bioengineering strategies that combine synthetic NPs with cell membrane coatings, which show have natural tropism and homing potential to target cells, could provide a solution\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In this context, EVs have gained relevance as promising natural delivery systems.\u003c/p\u003e \u003cp\u003eEVs are nanometric vesicles composed of phospholipid bilayers, released by all cell types. These organelle-free vesicles carry biomacromolecules and play a role in cell-to-cell communication in both physiological and pathological conditions, particularly among cells of the same lineage. EVs can sometimes evade immune system recognition and cross major physiological barriers\u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Despite the rapid expansion of our understanding of EV biology, function, and translational potential, the heterogeneous nature of EVs and the challenges in efficiently separating exosomal subpopulations have hindered the characterization of their molecular composition and biogenesis\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the International Society of Extracellular Vesicles (ISEV) does not recommend subgrouping EVs due to technical difficulties regarding their purification\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. EVs play a significant role in cancer diagnosis and treatment\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, as they can selectively recruit proteins, lipids, metabolites, antigens and genetic material (i.e. mRNA, miRNA or DNA) and transfer this cargo into the targeting cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Thereby, EVs can be used as vehicles to encapsulate active principles for intercellular communication in pathological conditions such as cancer\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Additionally, EVs serve as selective targeting nanodevices, leveraging their plasma membrane to overcome current targeting issues in cancer vaccines\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, the encapsulation efficiency of nucleic acids in EVs remains limited\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere we harness the directed targeting capacity of EV membranes to coat pBAE NPs, creating biomimetic nanosystems naturally targeted to monocytes. To address the limitations of synthetic polymeric NPs, we aim to demonstrate the effectiveness of these nanosystems in maturing monocytes into dendritic cells (DCs). While numerous review articles highlight the potential of EVs as advanced delivery systems, and some suggest their use for vaccination\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, a comprehensive study of their therapeutic applications is lacking. Moreover, the low encapsulation capacity of EVs in exogenous cargo loading can be effectively overcome by the superior encapsulation efficiency of pBAE NPs, allowing for a selective and effective delivery. Therefore, our ultimate objective herein is to design innovative biomimetic smart multicomponent nucleic acid vaccines that are both safe and functional. Our hypothsis drives that combining the strenghs of both delivery systems, we will obtain an enhanced biomimetic mRNA nanocarrier.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\n\u003ch3\u003e1. Materials\u003c/h3\u003e\n\u003cp\u003eBovine serum albumin (BSA), sodium acetate (AcONa), Loading Buffer, Tween-80, and PBS were purchased from Sigma-Aldrich\u0026reg;, and Cyanine 5 NHS ether dye was supplied by Lumiprobe. Fetal Bovine Serum (FBS), Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM), glutamine, penicillin, and streptomycin were obtained from Gibco\u0026reg;. RPMI-1640 medium was purchased from Biowest (L0501-500). Recombinant human interleukin 4 (rhIL-4) and recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CFS) were supplied by PeproTech\u0026reg;. CellMask\u0026trade;Orange Plasma Membrane Stain was purchased from Thermo Fisher Scientific. Arginine and histidine end-modified poly(β)-amino ester (pBAE, named in the following as R and H) polymers were prepared by the GEMAT group following a two-step procedure described previously\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. An acrylate-terminated polymer, C6, was first synthesized by addition reaction of primary amines with diacrylates (at 1:1.2 M ratio of amine:diacrylate). pBAEs were then obtained by end-capping modification of the resulting acrylate-terminated polymer with arginine or histidine at each end. When necessary, fluorescent pBAEs were also used. To this end, R-pBAEs were labelled with Cyanine 5 (Cy5), as described previously\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Anti-human CD11c-FITC, CD86-PE, and CD209-PE, as well as purified anti-chicken ovalbumin and APC anti-mouse/human CD11b, were purchased from BioLegend. The goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) secondary antibody, HRP, was supplied by Thermo Fisher Scientific, and Ovalbumin-Fluorescein Isothiocyanate (OVA-FITC) by Santa Cruz Biotechnology.\u003c/p\u003e\n\u003ch3\u003e2. Cell culture\u003c/h3\u003e\n\u003cp\u003eTHP-1 cell lines were maintained in RMPI-1640 supplemented with FBS (10%, v/v), penicillin G (100 units mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), streptomycin (100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and L-glutamine (2 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). HEK-293 cell lines were maintained in DMEM supplemented with FBS (10%, v/v), penicillin G (100 units mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), streptomycin (100 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and L-glutamine (2 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). All cells were cultured at 37\u0026deg;C under a 5% CO\u003csub\u003e2\u003c/sub\u003e with 95% humidity air atmosphere and passaged when they reached 80\u0026ndash;90% confluence. THP-1 cells were differentiated to obtain immature dendritic cells (imDCs), as previously described\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Briefly, THP-1 cells were cultured in medium supplemented with recombinant human interleukin 4 (rhIL-4) (20 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and phorbol 12-myristate 13-acetate (PMA) (20 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 4 days to trigger differentiation\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. To verify differentiation, flow cytometer analysis was performed by staining the surface markers CD11b, CD11c, and CD209 and quantifying the extent of differentiation. Briefly, 2.5 x 10\u003csup\u003e5\u003c/sup\u003e of cells per well were retrieved from each cell culture\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e and fixed in formalin (4%) for 20 min at 4\u0026deg;C. The samples were then resuspended in a blocking buffer containing FBS in PBS (2%, v/v) for 20 min at 4\u0026deg;C. After another washing step in PBS, the samples were incubated for 1 h at RT with the primary antibodies anti-human CD11b conjugated with APC fluorophore, anti-human CD11c conjugated with FITC, and anti-human CD209 conjugated with PE, following the manufacturer's instructions. The unbound antibodies were washed away with FBS in PBS (2%, v/v), and the cells were resuspended in PBS and analyzed using a ACEA Flow Cytometer (NovoCyte, Santa Clara, USA).\u003c/p\u003e\n\u003ch3\u003e3. Methods\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Synthesis of the pBAE nanoparticles\u003c/h2\u003e \u003cp\u003eNPs were prepared following a well-established protocol in our lab as previously described\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Briefly, a mixture containing 60% C\u003csub\u003e6\u003c/sub\u003eCK\u003csub\u003e3\u003c/sub\u003e and 40% C\u003csub\u003e6\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e pBAEs (12.5 mg/mL) were diluted in a sodium acetate solution (12.5 mM, pH\u0026thinsp;=\u0026thinsp;5.2) with the same volume of plasmid (0.5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at a ratio 25:1. pBAE polymers were synthesized by a previously described protocol\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Plasmids were produced using Gigaprep kits, following the suppliers\u0026rsquo; instructions. pPAX (a model plasmid not coding for any fluorescent protein), pMaxGFP (coding for GFP), and pOVA (coding for ovalbumin antigen) were used. The mixture was incubated for 30 min at 25\u0026deg;C. The KH NPs were then precipitated in the same volume of Milli-Q H\u003csub\u003e2\u003c/sub\u003eO and an equivalent volume of Hepes (20 mM, 4% w/v sucrose). For all experiments, fresh KH NPs were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Isolation and purification of EVs\u003c/h2\u003e \u003cp\u003eCulture samples were collected by mild centrifugation of the medium at 300 \u003cem\u003eg\u003c/em\u003e for 5 min after 24 h incubation without FBS, and supernatants were stored at -80\u0026deg;C. EVs were isolated by sequential high-speed centrifugation, as described previously\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, and stored at -80\u0026deg;C for a maximum of six months. EVs from undifferentiated THP-1 and HEK293 cells were produced and labelled using NBD-PE, a phospholipid tagged on the head group with the NBD fluorophore, which can intercalate into the EV phospholipidic bilayer by passive incubation. A ratio of NBD-PE (1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in PBS, 5 \u0026micro;L) for EVs (100 \u0026micro;L, at a concentration of approximately 5 x 10\u003csup\u003e10\u003c/sup\u003e EV mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was mixed and incubated for 1 h at 37\u0026deg;C under agitation. Later, the solution was filtered using a 100 kDa cutoff Amicon\u0026reg;Ultra Centrifugal Filter Unit (Merck Millipore, USA) to remove the free label. Isolated EVs were characterized by three distinct techniques, including Western blotting, NP tracking analysis, and dynamic light scattering, following MISEV guidelines\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The same method was used for Cyanine-3 (Cy3) labelling, at a ratio of Cy3 (1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in DMSO, 10 \u0026micro;L) for EVs (100 \u0026micro;L), and with CD81-FITC (1:400).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Characterization of EVs by Western blot\u003c/h2\u003e \u003cp\u003eThe concentration of proteins in the samples was determined using the Pierce\u0026trade; BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA; 23225), following the manufacturer\u0026rsquo;s instructions. EV protein content was evaluated by Western blot. Briefly, isolated EVs were lysed in reducing sample buffer [Tris-HCl (0.25 M, pH 6.8), glycerol (40%), SDS (8%), 2-mercaptoethanol (5%) and bromophenol blue (0.04%)] or non-reducing sample buffer (without 2-mercaptoethanol) and boiled for 10 min at 65\u0026deg;C. Protein samples were resolved by SDS-PAGE (10%, SDS-polyacrylamide gel electrophoresis, for CD63, TSG101, CD81, BSA, respectively), transferred to polyvinylidene fluoride membranes, blocked in 5% non-fat powdered milk in PBS-T (0.5% Tween-20), and probed with antibodies. Purified anti-TSG101 antibody (Cat. 934301, Biolegend) and Bovine Serum Albumin Polyclonal Antibody (BSA; Cat. A11133, Invitrogen) were applied to the reduced samples. CD81 antibody (1.3.3.22; sc-7637, Santa Cruz Biotechnology) and CD63 antibody (MX-49.129.5; sc-5275, Santa Cruz Biotechnology) were applied to non-reduced samples. For detection, goat polyclonal antibody to MS IgG (HRP, Mouse, GR3219929, Abcam), HRP goat anti-rat IgG (minimal x-reactivity; Cat 405405, Biolegend), goat anti-rabbit, rat IgG (H\u0026thinsp;+\u0026thinsp;L) secondary (NB7160, Novus Biologicals) and Pierce\u0026reg; ECL Western Blotting\u0026ndash;substrate (Thermo Fischer Scientific, Rockford, IL, USA) were used. The membrane was examined using an Amersham ImageQuant\u0026trade;800 biomolecular imager (Cytiva Life Sciences, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Biophysical characterization of complexes by dynamic light scattering, nanoparticle tracking analysis, and electron microscopy\u003c/h2\u003e \u003cp\u003eHydrodynamic size, polydispersity index (PDI), surface charge (ζ-potential), size distribution, and sample concentration were analyzed by both dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). To determine the hydrodynamic size and PDI of NPs (50 \u0026micro;L) prepared as previously described, or thawed EV solution (50 \u0026micro;L) diluted 1:5 in PBS was put in a DLS micro-cuvette and analyzed in a Zetasizer Nano ZS with Zetasizer Software (DLS; Malvern Instruments, Worcestershire, UK). To measure ζ-potential, samples were diluted in Milli-Q water (1:100) to a final volume of 1 mL and samples were put in a Disposable Capillary cell (DTS1060, Malvern Instruments, Worcestershire, England) and analyzed by DLS. To determine size distribution and sample concentration, samples were diluted 1:100 in PBS (or Milli-Q water for NPs) in a final volume of 1 mL and run with the automated syringe pump in an NTA Nanosight NS300 (Malvern Panalytics, United Kingdom). A PDI was also calculated from the NTA, PDI = (σ/\u0026micro;)\u003csup\u003e2\u003c/sup\u003e, where σ and \u0026micro; are NTA standard deviation and mean size, respectively. Encapsulation efficiency was calculated using the RiboGreen\u0026reg; colorimetric test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.5. \u003cb\u003eNanoparticle encapsulation in EVs\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eWe tested the capacity of several methods to encapsulate pBAEs into EVs, namely sonication, extrusion, and freeze-thaw (summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As pBAE NPs, in this study we only use KH NPs which encapsulated pGFP or mRNA OVA for the differentiation study in monocytes. EVs were collected from three different cell lines, human monocytic THP-1 cells, murine monocytic JAWS cells and murine lung cancer LLC1 cells, in order to probe the tropism of the EVs towards homing cells and the complexes between the KH NPs and the different EVs were highlighted as KH NP@THP-1 EV, KH NP@JAWS EV and KH NP@LLC1 EV, respectively. Extrusion was performed with an Avanti Polar Lipids mini-extruder using a 200 nm pore membrane to passthrough the EVs for 10 times, while for sonication, KH NPs were put in a sonication bath for 10 and 20 min. EVs were disrupted using fast freezing-thawing cycles combined with incubation with KH NPs, followed by a recovery period. Briefly, an appropriate amount of EV solution was frozen at -80\u0026deg;C in a mixture of acetone and dry- ice and allowed to thaw at RT for 10 min. These cycles were repeated 3 times. After the last cycle, the NP solution was rapidly added to the EV solution at different NP:EV volume ratios and gently pipetted to obtain a homogenous solution of the two nanosystems. The mixture was then incubated for 1 h at RT. This formulation is referred to as KH NP@EV complexes followed by an indication of the ratio (for example KH NP@EV 1:2, to indicate a 1:2 ratio of NP:EV). The ratio was calculated from the concentration of the samples of EVs and KH NPs, as determined by the NTA. The formulation was always used freshly prepared.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Confocal microscopy of the NP@EV complexes\u003c/h2\u003e \u003cp\u003eConfocal microscopy was used to verify the colocalization of KH NPs and THP-1 EVs after the encapsulation. Briefly, the THP-1 EVs stained with N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (NBD-PE) and the KH NPs labelled with Sulfo-Cyanine 5 (Cy5) (1%) were used to build the KH NP@THP-1 EV complexes. To this end, the sample (10 \u0026micro;L) was deposited onto the glass slide, covered with a coverslip, sealed, and analyzed with a Leica DMi8 confocal microscope. Image analysis was performed using the software Fiji /ImageJ 1.52v (Wayne Rasband, National Institutes of Health, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Hyperspectral microscope and dark-field imaging\u003c/h2\u003e \u003cp\u003eGlass slides and coverslips were cleaned with Milli-Q water, EtOH, and acetone for 10 min before observations. Samples were visualized using Exponent 7 software and mapped from their hyperspectral images with a Cytoviva\u0026copy; high-resolution dark-field condenser (Auburn, AL, USA) coupled to an Olympus BX-43 optical microscope. Hyperspectral imaging (HSI) analysis was recorded using ENVI 4.8 software in which the hyperspectral camera operated in the visible-near infrared range (VNIR) of 400 to 1000 nm. A spectral library with a representative hyperspectral image was obtained from single components (EVs and NPs) separately by adding 10 \u0026micro;L of sample to a microscope glass slide. The freshly prepared NP@EV sample was placed on a microscope slide (10 \u0026micro;L) covered with a coverslip and observed. Hyperspectral images of the NP@EV complexes were obtained and spectral similarities that matched the spectral library of EVs and NPs facilitated the mapping process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Fluorescence Resonance Energy Transfer (FRET) analysis\u003c/h2\u003e \u003cp\u003eFRET measurements were performed using an Infinite M Plex microplate reader from TECAN. Each sample had a final volume of 110 \u0026micro;L in PBS media and was run in triplicate in a flat black 96-well plate. Each component of the complexes was prepared at the same labelling concentration. The polymer was labelled with Cy5 or Sulfo-Cyanine 3 (Cy3), the plasmid with Cy5, and EVs with Cy3. As a negative control, the complexes were measured with only one of the components labelled, and PBS wells with no labelling were also included. The excitation wavelength was set to 535 nm for Cy3, and the emission wavelength to 675 nm to detect the emission of Cy5. Measurements were carried out at different times and temperatures (25 and 37\u0026deg;C) over 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Encapsulation efficiency\u003c/h2\u003e \u003cp\u003eNucleic acid encapsulation efficiency was quantitatively assessed using the Quanti-It Pico-Green DNA assay kit (Thermo Fisher Scientific), following the manufacturer\u0026rsquo;s instructions. A qualitative analysis of the encapsulated genetic material was also performed using agarose gel electrophoresis (Sub-CellR GT Agarose Gel Electrophoresis System BIORAD). Briefly, an agarose solution (1.5%, 65 mL) in TAE buffer (1x) was prepared, then GelRed was added (1 uL). The mixture was allowed to polymerize in the appropriate mold for 30 min. Subsequently, the samples of interest, including the marker containing different DNA base pairs of varying molecular weights, KH NPs, KH NP@EV complexes, and the free plasmid at the same concentration as the NPs, were loaded and run on the gel. The resulting image was analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e\u003ch3\u003e4. \u003cem\u003eIn vitro\u003c/em\u003e biocompatibility\u003c/h3\u003e\n\u003cp\u003e10.000 cells per well were seeded in a 96-well plate at 90% confluence 24 h before starting the experiment. They were then incubated with increasing amounts of the KH NP@EV complexes. Non-treated cells were used as negative controls. \u003cem\u003eIn vitro\u003c/em\u003e cell viability was evaluated through quantification of cell metabolic activity using the MTT colorimetric assay, as described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Briefly, cells were incubated with the samples for 48 h, the media was removed and replaced with MTT (0.5 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in complete media and cells were further incubated for around 1 h. Next, this medium was removed, and formazan crystals were dissolved in DMSO (100 \u0026micro;L). Absorbance was quantified at 570 nm using a plate reader (SpectraMax M5, Molecular Devices). Results are expressed as a percentage of viable cells relative to non-treated cells.\u003c/p\u003e\n\u003ch3\u003e5. \u003cem\u003eIn vitro\u003c/em\u003e uptake experiments\u003c/h3\u003e\n\u003cp\u003eTo assess the capacity of the engineered nanosystems to penetrate the cells, \u003cem\u003ein vitro\u003c/em\u003e analyses were conducted using Cy5-labelled NPs (0.2%) and NBD-PE-labelled EVs. Thus, the uptake could be measured by flow cytometry or studied by confocal microscopy. The flow cytometer quantitative analysis was conducted on THP-1 and HEK-293 cell lines, while subcellular qualitative evaluation was conducted only on differentiated THP-1 cells (iDCs) by confocal microscopy. For the latter, THP-1 cells were cultured with rhIL-4 (recombinant human interleukin 4, 100 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and rhGM-CFS (recombinant human granulocyte-macrophage colony-stimulating factor, 100 ng mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) for 5 days, replacing the medium with fresh cytokine supplement after 3 days to induce direct differentiation to dendritic cells (iDCs)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. This approach allowed us to then culture cells on gelatin-coated coverslips and therefore visualize the samples under a confocal microscope. For each cell line, EVs isolated from each cell culture were used.\u003c/p\u003e\n\u003cp\u003eFor flow cytometry, cells were seeded on 96-well plates at a concentration of 2 x 10\u003csup\u003e4\u003c/sup\u003e cells per well. Cells were incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at different time points (24, 36, and 48 h for THP-1 cells and 4, 24, and 48 h for HEK-293 cells) with the corresponding treatments. The treatment dose was calculated to obtain a final concentration of 0.3 \u0026micro;g of nucleic acids per well. After the incubations, the cells were washed with PBS and fixed in formalin (10%). As the cell lines were cultured in suspension, each washing step was done with a previous centrifugation step at 310 \u003cem\u003eg\u003c/em\u003e for 5 min to sediment the cells at the bottom of the plate.\u003c/p\u003e\n\u003cp\u003eFor confocal experiments, cells were seeded in 24-well plates containing a cover glass with 500 \u0026micro;L of gelatin 0.1%, gelatin was incubated for 30 min and the excess was removed by aspiration. iDCs cells were seeded at a concentration of 3 x 10\u003csup\u003e4\u003c/sup\u003e cells per well and incubated for 24 h at 37\u0026deg;C. After short (6 h) and long (48 h) incubations with the treatments (with the same condition as in the previous protocol), the cells were fixed in formalin (10%) for 20 min and permeabilized with PBS-Tween80 (0.1%), washing the coverslip with PBS in each step. Additionally, the cell nucleus was stained (DAPI, 1:10.000 in PBS for 10 min at RT), as was the membrane (CellMask\u0026trade; Orange, 1:1.000 in PBS for 10 min at RT), again washing the coverslip with PBS in each step. Cover glass was then placed onto the microscope slide and the cellular uptake of the NPs was observed using a Leica DMi8 S confocal microscope. Images were analyzed with the software Fiji /ImageJ 1.52v (Wayne Rasband, National Institutes of Health, USA).\u003c/p\u003e\n\u003ch3\u003e6. Transfection of THP-1 cells with mRNA-OVA complexes\u003c/h3\u003e\n\u003cp\u003eNa\u0026iuml;ve THP-1 monocytes (undifferentiated cells) and immature DCs (iDCs) (THP-1 cells left to differentiate to DCs for 4 days with PMA and IL-4)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e were transfected with the mRNA-OVA KH NPs@THP-1 EVs complexes (resulting in DCs). Also, eGFP mRNA-loaded complexes were tested by flow cytometry to confirm uptake and transfection efficiency. Briefly, cells were seeded in a 24-well or 12-well plate at a concentration of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:1\\times\\:{10}^{5}\\)\u003c/span\u003e\u003c/span\u003ecells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and incubated with the treatment at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere for 24 h. A final concentration of 0.6 \u0026micro;g per well of ribonucleic acid was used. Since undifferentiated THP-1 cells were cultured in suspension, transfection was performed following a centrifugation step at 310 \u003cem\u003eg\u003c/em\u003e for 5 min to sediment the cells at the bottom of the plate. To reach the desired concentration of genetic material, half of the medium volume was removed and replaced with half of the treated medium containing the samples. After the incubation, the cells were fixed in formalin (4%). To verify cell stimulation by the OVA antigen, changes in several differentiation markers (i.e. CD86, CD209, CD11c, and CD11b) were analyzed by flow cytometer. Various controls were used, including untreated monocytes, mRNA-OVA KH NP-treated and mRNA-OVA lipofectamine-treated.\u003c/p\u003e\n\u003ch3\u003e7. Biodistribution studies in tumor-bearing mice\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e experiment was performed at the Centre d\u0026rsquo;Investigaci\u0026oacute; i Desenvolupament (CID-CSIC), which adheres to the principles set out in the laws, regulations, and policies governing the care and use of laboratory animals by the Generalitat of Catalunya and following protocol number 11707, approved by the Direcci\u0026oacute; General del Medi Natural.\u003c/p\u003e\n\u003cp\u003eEight-week-old female C57BL6J mice were purchased from Envigo (Spain) and maintained at a constant temperature of 21\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;10% under a 12 h light/dark cycle, and with ad libitum access to food and water. All mice were regularly monitored for animal welfare and adherence to the experimental protocol. The animals were randomly divided into 5 groups receiving the KH NPs, JAWS EVs, KH NP@JAWS EVS, KH NP@LLC1 EVs and CTRL containing mRNA luciferase (n\u0026thinsp;=\u0026thinsp;4 for each experimental group). Because THP-1 EVs are human, for the \u003cem\u003ein vivo\u003c/em\u003e study we selected the equivalent murine EVs from murine-monocyte derived macrophages, JAWS, which could be considered comparable to THP-1 human EVs. As a negative CTRL, we selected murine lung cancer LLC1 EVs to prove the tropism of JAWS EVs \u003cem\u003ein vivo\u003c/em\u003e to DCs and macrophages. The mice were injected subcutaneously in the back with B16F10 cells (2.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per mouse, 50 \u0026micro;L PBS 1x). Tumor volume and mouse weight were measured five times per week. Two weeks later, they were injected with the different complexes intravenously through the tail. Six hours later after injection, animals were anesthetized with ketamine (87 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and xylazine (12.5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and firefly luciferine (150 \u0026micro;L) was intraperitoneally administered. Fifteen minutes later, mice were euthanized, organs of interest collected and \u003cem\u003eex vivo\u003c/em\u003e bioluminescence was assessed through radiance measurement. Negative control (non-injected mice) were used to subtract the background signal. Images were treated and analysed using Hokawo v3.0 software.\u003c/p\u003e\n\u003cp\u003eMarkers of cell toxicity such as serum aspartate aminotransferase activity (sAST), hepatocellular injury/toxicity such as serum alanine aminotransferase activity (sALT) or lactate dehydrogenase (sLDH) were monitored after hydrogel exposure by the IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) optimized kinetic UV method in an Aries chemical analyzer (Werfen Instrumentation Laboratory S.p.A., Italy) and expressed as U L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Each analysis was validated by a biochemical chemist and hematologist using quality control serums (CQI), in the San Raffaele Mouse Clinic.\u003c/p\u003e\n\u003ch3\u003e8. Protein corona\u003c/h3\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e8.1. Hard Corona (HC) and Soft Corona (SC) obtention\u003c/h2\u003e\n \u003cp\u003eTo study protein composition and biological functions of the different EVs and complexes, we designed an experiment to mimic real bloodstream conditions. We assume that plasma represents 80% of the total interacting volume as it was previously described by Monopoli et al\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. The concentration of EVs used was 1.04 x 10\u003csup\u003e9\u003c/sup\u003e particles mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Human plasma (160 \u0026micro;L) and PBS (40 \u0026micro;L) were introduced into low protein binding 1.5 mL microtubes. The total sample volume used for the study was 40 \u0026micro;L. The solution was incubated at 37\u0026deg;C for 1 h under continuous agitation at 300 rpm. After incubation, the NP corona complex was pelleted from excess proteins by centrifugation at 15000 rpm, 4\u0026deg;C for 15 min. The supernatant was discarded, and the pellet was resuspended in PBS (500 \u0026micro;L) and centrifuged again. This washing procedure, repeated three times, removed unbound and loosely bound proteins from the NPs. The pellet was then resuspended in SDS (10%, 100 \u0026micro;L), heated at 95\u0026deg;C for 10 min, and centrifuged at 15000 rpm, 4\u0026deg;C for 15 min. The NP-SC complexes were obtained from the supernatant and the NP-HC complexes wer obtained by resuspending the pellet in PBS (100 \u0026micro;L), incubating with DTT (5 \u0026micro;L, 20 mM) to break disulfide bonds, and then this fraction was centrifuged again at 15000 rpm for 15 min. This fraction was centrifuged again to separate the more tightly bound proteins form the NPs. The proteins were stored at -80\u0026deg;C until further use.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e8.2. Liquid Chromatography Mass Spectrometry (LC-MS) analysis\u003c/h2\u003e\n \u003cp\u003eFor total protein purification, the pellets were resuspended in ammonium bicarbonate buffer (AB buffer, 50 mM). Phase separation was achieved by adding methanol (400 \u0026micro;L), chloroform (100 \u0026micro;L) and RNAse-free water (300 \u0026micro;L) was added to the reaction in a 4:1:3 ratio. The mixture was vigorously shaken for 15 sec, and incubated for 5 min at RT. After the incubation, samples were centrifuged at 14.000 g for 1 min at RT. The interphase with the proteins was extracted and precipitated, dried and resuspended in AB buffer (50 mM). A 1:80 ratio of the protein concentration from each sample was digested using a mixture of trypsin and lysoenzsime C.\u003c/p\u003e\n \u003cp\u003eTo determine the protein composition of the EVs, SC and HC complexes, the samples were analyzed using a M5 MicroLC system coupled to ZenoTOF\u0026trade; 7600 System (SCIEX, USA) with positive electrospray ionization (ESI) with OptiFlow 1\u0026ndash;50 \u0026micro;L Micro/MicroCal Turbo V Ion Source. Chromatographic separation was performed with FA water solution (MP A, 0.1%) and FA acetonitrile solution (MP B, 0.1%), with a linear gradient applied at a constant flow rate (7 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The gradient started at 5% B, and increased to 25% at 38 min, 32% at 43 min, 40% at 48 min, and 80% at 50 min. The gradient was then returned to initial conditions and held until 65 minutes for re-equilibration. The chromatographic column used was a microLC Luna Omega Polar (H24-06911, Phenomenex) with full loop injections set at 5 \u0026micro;L. MS data were acquired in data-dependent acquisition (DDA) mode. MS1 spectra were collected over an m/z range of 400\u0026ndash;1400. For MS2, a peptide workflow was used, selecting up to 20 precursor ions per cycle with Zeno Pulsing enabled (threshold set above 100.000 cps), and an m/z range of 100\u0026ndash;1700. All samples were injected in triplicates.\u003c/p\u003e\n \u003cp\u003eA merged database search for IDA runs was performed using MSFragger against Mus Musculus and human proteome from UniProtKB after appending decoy sequences (reverse contruction) and common contaminant sequences (UP000005640 contains 40.900 entries and 20.450 decoys and UP000000589 with 34.681 entries and 17.340 decoys). The XTandem! algorithm was used to obtain the peptide spectrum matches. The PTMs enabled were methionine oxidation and N-terminal acetilations. False discovery rate (FDR) analysis was enabled and we used MSBooster, Percolator and Protein Prophet as protein hits validation tools and rescoring\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Only proteins identified with 1% global FDR were considered true identification. Perseus statistical software was used to analyse the LFQ intensities (MSFragger IonQuant), and data was log-transformed and imputed by Fragpipe Analyst R (v1.0.4). Data processing was performed using SCIEX OS 2.2 acquisition software, MSFragger (v22.0) software, and PDV viewer and Fragpipe-Analyst for proteome last analysis and data visualization.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e9. Statistical analysis\u003c/h3\u003e\n\u003cp\u003eAll data presented, unless otherwise stated, represent the mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) of at least a three independent samples. Statistical differences were evaluated using GraphPad Prism\u0026reg;, by performing an ANOVA comparisons between the different groups. P values lower than 0.05 were considered statistically significant. For confocal imaging analysis, JACoP (Just Another Colocalization Plug-in) was used\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Images were generated using BioRender.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003ch3\u003e1. Efficient encapsulation of pBAE NPs inside EVs\u003c/h3\u003e\n\u003cp\u003eThe natural tropism of EVs to target cells of the same lineage from which they originated can be used for the directed targeting of pBAE NPs to DCs. Although we previously demonstrated that pBAE NPs can passively accumulate in DCs through the selection of an appropriate oligopeptide-end modification, thereby confirming their suitability as nucleic acid vaccines\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, here our goal was to enhance their selectivity. As this is the first attempt to encapsulate pBAE NPs within EVs, various EV encapsulation methods were evaluated and subsequently, the resulting complexes were analyzed both biophysically and \u003cem\u003ein vitro\u003c/em\u003e. The pBAE NPs used were formed by a peptide sequence of Lys-Lys-Lys, combined with another of His-His-His, named KH NPs, as it has demonstrated a high transfection in cells due to proton sponge effect\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. KH NPs were loaded with reporter plasmid GFP as a model for the firsts experiments, since in our previous studies no differences in the physicochemical properties of pBAE NPs were found when shifting from plasmid to mRNA encapsulation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eVarious approaches to encapsulate macromolecules or NPs of different sizes into EVs have been reported in the bibliography (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Incubation is a widely used method\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. It consists of incubating the molecules with a cargo of interest and it is particularly efficient for hydrophobic interfaces. To increase the encapsulation efficiency, sonication and extrusion strategies have been also described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. These two techniques are borrowed from liposomal formulations that can cause changes or twisting in the EV membrane, helping to introduce the cargo. Saponin-assisted treatment for EV permeabilization can further enhance cargo internalization. Saponin is an active compound that can generate complexes with cholesterol on the EV surface and create pores, thereby increasing membrane permeability. However, in contrast to cell membranes, EVs have more rigid lipid bilayers due to aggregation of sphingomyelin, cholesterol, and ganglioside. This feature can thus hinder the insertion of hydrophobic substances into the vesicles\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Finally, the freeze\u0026ndash;thaw method is a straightforward technique used to load drugs into EVs, as active compounds mixed with EVs undergo a few cycles of freezing at -80\u0026deg;C in a dry ice bath with acetone and thawing at RT\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNowadays, most of the NPs encapsulated into EVs have been metallic such as gold\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, platinum\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, iron oxide\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e or zinc oxide\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. However, few studies have addressed the encapsulation of soft nanomaterials like poly(lactic-co-glycolic acid) (PLGA) NPs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e or metal-organic framework (MOF) NPs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Importantly, encapsulation of pBAEs NPs into EVs has not been yet described in bibliography. One of the challenges to successfully coat soft nanomaterials with EVs consists in designing fast and novel coating protocols that are NP material-specific, since soft NPs are not stable for long periods of time under harsh conditions required by many encapsulation protocols. For example, PLGA NPs coated with EVs or cell-membrane-derived vesicles exploited the use of the sonication and extrusion methods\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Given the lack of previous studies reporting pBAE NPs encapsulation into EVs, we assessed various techniques and analyzed their potential impact in the nanomaterial functionality. Three different techniques were selected, sonication, extrusion, and freeze-thaw, as these methods were previously proposed for soft organic NPs encapsulation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Sonication of pBAE NPs for 10 min slightly increased the mean size of the particles from a 130\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 nm to 154\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1 nm, while the PDI value remained unchanged. However, longer periods of sonication of 20 min increased the mean size to more than 200 nm and the PDI over 0.3, thereby indicating partial degradation of the nanomaterial morphology (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). Thus, we evaluated the functionality only with sonicated pBAE NPs using an \u003cem\u003ein vitro\u003c/em\u003e transfection assay with ARPE-19 cell line as a suitable transfection host. pGFP transfection confirmed only a slight reduction in transfection efficiency after 20 min of sonication (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD). By using extrusion, the pBAE NPs presented a high PDI and very polydisperse size distributions, demonstrating the NPs degradation, and thus, discarding this method.\u003c/p\u003e\n\u003cp\u003eFinally, for the freeze-thaw method only the EVs underwent freeze-thawing and pBAE NPs were added during the recovery time since previous experiments by the group discouraged the repeated freeze-thaw of pBAE NPs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. The advantages of the freeze-thaw method include its simplicity and loading capacity (regarding encapsulation of compounds such as active molecules, proteins, or genetic material)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, especially for lipid structures like EVs. Freeze-thaw cycles promote lipid mixing in the lipidic bilayer, pointing to conformational changes, breakdown, and the rearrangement of lipid vesicles during repeated fast freezing and thawing processes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAdditionally, negatively charged liposomes can entrap cationic NPs of various natures (i.e. polyplexes or mesoporous silica NPs) by a spontaneous charge interaction, a process also called membrane fusion\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Triggered by electrostatic interactions, these vesicles fuse to build a lipidic double-layer coating. To assess this technique, three cycles of freeze-thaw were analyzed after recovery times of 0, 20, 40, 60, and 90 min in THP-1 EVs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). As expected, the mean size decreased immediately after the freeze-thaw cycles, thereby indicating that the fast-freezing process breaks down EVs, which rapidly reassemble into smaller vesicles that regain their initial sizes after a recovery time (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE). Moreover, the PDI values increased rapidly after the freeze-thaw cycles and decreased after 60 min of recovery time. However, the 60 min recovery time was the chosen one as the size distribution graphs showed EV aggregation after 90 min (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\n\u003ch3\u003e2. Physico-chemical characterization of the biomimetic nanosystems\u003c/h3\u003e\n\u003cp\u003eAfter selecting the freeze-thaw method as the most suitable approach to combine EVs and pBAE NPs, we conducted a more detailed characterization of KH NP@THP-1 EV complexes (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). While KH NPs presented a size of around 130\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 nm, THP-1 EVs were slightly larger, around 152\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 nm nm (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Three different formulations of KH NP@THP-1 EV complexes with different NP:EV ratios (1:1, 1:2, 1:10) were prepared and the distribution size followed by NTA (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Only the complexes formed with a ratio 1:2 presented a monodisperse system (PDI\u0026thinsp;=\u0026thinsp;0.15) with a significantly increased mean size with respect to EVs and KH NPs separately (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Lower ratios resulted in the aggregation of the nanosystem by an excess of KH NPs that cannot be fully coated and may tend to aggregate with other THP-1 EVs driven by electrostatic forces. In contrast, with higher ratios there was an excess of free EVs that did not interact with NPs, as the size remained around 100 nm, as found for naked EVs. By zeta potential THP-1 EVs and KH NPs alone revealed a surface net charge of -29.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 mV and 19.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mV respectively, while different values were obtained for the KH NP@THP-1 EV complexes, as a function of the ratio used. Lower ratios 1:1 presented a slight positive charge, consistent with the notion that not all the NPs were fully coated, due to an insufficient amount of THP-1 EVs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). The 1:10 ratio was discontinued due to high instability, which impeded reliable measures of the surface charge. Thus, the ratio 1:2 was the best proportion to obtain monodispersed and small KH NP@EVs with a negative surface net charge, indicating the EV bilayer surrounding the KH NPs.\u003c/p\u003e\n\u003cp\u003eTo have a further insight into the structure of the complexes, the EVs were analyzed by cryo-transmission electron microscopy (cryo-TEM) to analyze their shape and size. We demonstrated the presence of physical interactions between KH NPs and THP-1 EVs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD, Figure S2). Despite interference from excess polymer, KH NP@THP-1 EV 1:2 complexes elucidated the encapsulation dynamics, emphasizing the presence of NPs within EVs. Following MISEV2018 and 2023 guidelines\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, we further characterized the biochemical features of empty and loaded THP-1 EVs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE-F). Western blot was used to characterize the presence of three types of proteins: 1) a transmembrane protein to demonstrate the presence of a lipid bilayer, TGS101; 2) a tetraspanin to demonstrate that the nanosystems are EVs, CD63, or CD81; and 3) BSA, to demonstrate the lack of contaminants in the samples. These markers were analyzed in THP-1 cell lysates, THP-1- derived EVs, KH NP@THP-1 EV complexes, and a sample containing only KH NPs. As expected, BSA levels were significantly reduced in the EV samples compared to the cell lysates. The BSA residual presence could be attributed to proteins from the FBS used for cell maintenance. Predictably, CD63, CD81, and TGS101 were present in all the samples containing EVs, although their concentration appeared to decrease after the encapsulation process.\u003c/p\u003e\n\u003cp\u003eThe maintenance of the high encapsulation efficiency of the plasmid after building the biomimetic nanosystems must be highlighted (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG). KH NPs achieved more than 90% of nucleic acid encapsulation, while pBAE NPs complexed with EVs were over 80%. The efficiency was considerably higher than previous reports for engineered hybrid EV-liposome systems through freeze-thaw method for siRNA loading\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo confirm the interaction between THP-1 EVs and KH NPs, we performed confocal and hyperspectral microscopy. The two nanosystems were labelled with two distinct fluorophores, KH NPs (Cy5, in red) and THP-1 EVs (NBD-PE, in green) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH). By confocal, the KH NP@EV complex presented a colocalization of the two signals by giving a yellow spot (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH, Figure S3-S4). To estimate Pearson\u0026rsquo;s (PC) and Manders\u0026rsquo;s coefficients (M1, green fraction overlapping with red; M2, red fraction overlapping with green)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, were calculated using a different threshold to exclude background noise. Both coefficients reported an average between 40 and 50% colocalization between EVs and KH NPs, confirming that the sample contained both uncoated NPs and EVs without any NPs inside, together with biomimetic systems. Details are provided in Explanation S1 in the SI. To visualize the motion in real-time dispersion\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, dark-field hyperspectral (HSI) microscopy was performed, including optical and spectral images (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI, Figure S5). The mapping of THP-1 EVs (in green) and KH NPs (in red) for the KH NP@EV (1:2) sample, showed that both materials colocalized when they were combined and KH NP@EV complexes were spherical as observed by cryoTEM.\u003c/p\u003e\n\u003cp\u003eNext, the formation and stability of the biomimetic systems was assessed by using FRET, previously demonstrated by us for pBAE NPs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and others\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, for different hybrid systems. FRET signal depends on the lateral distance between the fluorophores labelling distinct structures\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Therefore, the higher the FRET signal, the closer the two labelled molecules are. The signals exceeded the lower control signal of each pair of components (Figure S6), thus indicating the presence of FRET signals between all pairs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eJ). At 25\u0026deg;C, even that the plasmid and the polymer showed a strong interaction during the first hours, it decreased over time going from 0.65 to 0.37 AU, pointing the instability and gradual dissociation of the polymer-plasmid complex. However, at 37\u0026deg;C, the FRET signal remained relatively constant, a higher temperature appeared to enhance the interactions, possibly due to an increased molecular mobility, even that the signal was less stable than at 25\u0026deg;C. Regarding the EV-pBAE interaction, after an initial fluctuation, the FRET signal remained relatively stable around 0.88 AU at both temperatures. Compared to our previous stability studies addressing pBAE NPs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, it is also reasonable to attribute the increased long-term stability to the EV shell. As naturally stable structures, these vesicles might provide additional protection or a shell that promotes closer proximity between the plasmid and the polymer. Finally, and as expected, the EV-plasmid interaction was the lowest at both temperatures, as the plasmid was encapsulated inside the polymer, presenting the highest distance.\u003c/p\u003e\n\u003ch3\u003e3. KH NP@EV selective targeting to DCs and further immune cell activation\u003c/h3\u003e\n\u003cp\u003eBefore evaluating immunogenicity, we performed a preliminary toxicological and functional study \u003cem\u003ein vitro\u003c/em\u003e using human THP-1 cells. At a concentration of 0.1 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of KH NP@EV complex and its components (THP-1 EVs and KH NPs) did not result in citotoxicity (Figure S7), as reported in previous studies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Consequently, the capacity of this complex to safely penetrate the targeting cells was studied and compared to THP-1 EVs and KH NPs alone. Two different cell lines were used, undifferentiated THP-1 as the targeting cell and HEK293, as a permissive cell line (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). THP-1 EVs were labelled with NBD-PE fluorophore and KH NPs with Cy5 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB-E). Both cell lines presented no signal when treated with empty EVs by flow cytometry, which was expected in HEK293 cells but not in THP-1 cells. Even EV membranes facilitate receptor-mediated endocytosis\u0026mdash;they may be limited by membrane fusion efficiency\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Fusion is often pH or receptor-dependent, and without triggering conditions such as acidic endosomes, EVs may be internalized and degraded rather than delivering the cargo along with the fluorescent dye, creating a false impression of low uptake. However, KH NP@THP-1 EV complexes present a high fluorescent signal, thanks to the proton sponge effect of KH NPs, allowing for the release of the decomplexed biomimetic nanosystems inside the cell\u0026apos;s cytoplasm. The internalization capacity of the nanosystems was studied at 4, 24, and 48 h (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB-E). THP-1 cells, as target model cell line for our vaccine, presented a higher and faster internalization rate of the complex in comparison to HEK293 cells.\u003c/p\u003e\n\u003cp\u003eRegarding the analysis in HEK293 cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC-E), the internalization pattern of naked KH NPs and KH NP@THP-1 EVs differed. The uptake of the complexed KH NPs was significantly higher than naked NPs at 4 h, with around 30% and 20%, respectively. Meanwhile KH NPs@THP-1 EVs level remained steady during the 48 h of the experiment, naked KH NPs signal decayed over time. This reduction could be due to the rapid doubling time of this cell line (24\u0026ndash;36 h)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, which leads to clearance of the internalized NPs, as well as a reduction of the fraction of cells that had taken up this nanomaterial. In contrast, KH NP@THP-1 EVs signal did not decrease over time, which might indicate enhanced stability in the culture medium conferred by the THP-1 EV coating and, consequently, less uptake, due to a difficulted disassembly of the biomimetic complexes. In this regard, the FRET analysis demonstrated that the complexes presented greater stability over an extended period. However, it is important to note that FRET analysis was performed in FBS-free medium.\u003c/p\u003e\n\u003cp\u003eOverall, the THP-1 EV coating increased the internalization of KH NPs maintaining a higher level of internalized EV-coated NPs for a longer period. This can be explained by the fact that this formulation is optimized for cell transfection and antigen presentation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and the elevated internalization is attributed to the higher duplication time of the THP-1 cell line (around 60\u0026ndash;70 h)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. However, the uptake of naked THP-1 EVs into THP-1 cells, the same cell lineage they come from, was surprisingly low, not in accordance with previous bibliography indicating a certain tropism of EVs to be internalized by the parental cell lineage\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. This finding could be explained by the limited membrane fusion efficiency. Nevertheless, our findings indicate that the complexes enhanced the internalization capacity of both THP-1 EVs and KH NPs. This unexpected advantage of the complex nanosystem could be attributed to an increased surface charge towards a cationic range when the cationic pBAE NPs were coated with THP-1 EVs. As in fact, strongly negatively charged lipid vesicles have a lower tendency to be internalized by cells because the cell membrane already has a negative charge\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo validate the engineered nanosystems for immunization purposes, an \u003cem\u003ein vitro\u003c/em\u003e model, comparing the expression levels of specific markers on cells stimulated with the antigen with those of na\u0026iuml;ve THP-1 cells was used. This approach provides insights into the phenotypic profiles of cells. Significant increase in the expression of CD86, CD11b, CD11c and CD209, on those cells treated with the cytokine cocktail of IL-4 and PMA, confirmed the differentiation to iDCs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG, Figure S8-S9). To confirm the selectivity of our complex, we used fluorescence microscopy to qualitatively study the internalization of KH NP@THP-1 EVs in THP-1 cells directly differentiated to iDCs, as a more representative model of \u003cem\u003ein vivo\u003c/em\u003e target cells (Figure S10). THP-1 EVs achieved minimal penetration, meanwhile KH NP@THP-1 EVs presented a higher uptake of the biomimetic nanosystem. Although the cell membrane appeared to be slightly damaged in the cells treated with the complexes, especially after 48 h, biocompatibility experiments confirmed no cytotoxicity at the concentrations tested (Figure S7).\u003c/p\u003e\n\u003cp\u003eThen, we examined the same markers after transfection with our biomimetic nanosystems. After determining the optimal concentration of OVA mRNA in non-differentiated THP-1 cells (Figure S11), we transfected the cells using both KH NPs and KH NP@THP-1 EV complexes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH, Figure S12). We analyzed the surface markers CD86, CD11b, CD209 and CD11c, which increase when an immune response is induced\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Of note, there was no statistically significant difference between control iDCs and those transfected with KH NPs, thereby indicating an insignificant effect of the NPs on the cells. However, all markers showed changes when cells were transfected with the KH NP@THP-1 EV complexes, thus confirming the need for the EV coating for the selective transfection and functionality of the antigenic mRNA.\u003c/p\u003e\n\u003cp\u003eUpon closer examination of the differentiation markers, CD86 and CD11c showed a decrease, indicating possible incomplete cell maturation. The mRNA-OVA administered may stimulate the cells through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), however, the adjuvant effect did not promote the complete maturation of DCs. Furthermore, THP-1 cells differentiated into DCs and then induced to mature may have a different phenotypic profile compared to DCs derived from primary sources such as human peripheral blood\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. An increase in CD11b indicated maturation to DCs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, thus confirming the potential immunization properties of the KH NP@EV complexes. Indeed, despite the significant expression of CD11b and CD86, the cells resulting from the maturation of THP-1 cells through the iDC state cannot be considered fully mature because they lack the expression of CD83, another essential marker\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003e4. EVs maintain similar proteomic content and cellular functions across species\u003c/h3\u003e\n\u003cp\u003eBecause the EVs in this study were designed to enhance accumulation in antigen-presenting cells (APCs) such as DCs and macrophages, we initially selected EVs derived from human THP-1 cells. For \u003cem\u003ein vivo\u003c/em\u003e experiments, however, we used murine EVs derived from JAWS cells\u0026mdash;a murine monocyte-derived macrophage line considered functionally comparable to the human THP-1 model. To characterize the EVs and evaluate the conservation of their proteomic composition across species, we performed label-free MS on EVs isolated from both THP-1 and JAWS cells. Given their species-specific origins, we used the human protein database to analyze THP-1 EVs and the murine database for JAWS EVs. We cross-referenced the identified proteins across both datasets to assess whether the EV protein composition was comparable despite species differences. This comparative analysis enabled us to evaluate the degree of conservation in EV protein cargo and potential functional similarities between the two species. A total of 383 proteins were identified in THP-1 EVs (178 with more than 2 peptides) and 144 proteins in JAWS EVs (61 with more than 2 peptides). Additionally, unique proteins were detected in each EV subtype (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), suggesting unique entities released by cells rather than debris.\u003c/p\u003e\n\u003cp\u003eTo identify the signature proteins in the EVs, we performed statistical analysis on the expression levels of proteins identified in these data sets. We pinpointed a top 31 proteins, with a false discovery rate (FDR) of \u0026lt;\u0026thinsp;0.1 (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Histones, cytoskeleton proteins (actins and tubulins), small ribosomes and heat shock proteins (HSP) ranked as the most abundant proteins in both EVs. Interestingly, HSP90AB1 and HSPA8 were preferentially packaged in THP-1 EVs, which are involved in intercellular communication between EVs and other cells for the delivery of the EV cargo. Other proteins relatively enriched in THP-1 EVs included annexin A4 (ANXA4), complement C3 (C3), \u0026alpha;-2-macroglobulin (A2M) and proteins involved in metabolism, such as receptor-type tyrosine-protein phosphatase (PTPRC) and vitamin D-binding protein (GC). The expression of C3 in EVs it has been proved to be involved in the activation of the inflammatory response, as immune cells such as macrophages or DCs in the TME, can taken up EVs containing C3 and repolarize macrophages into an immunosuppresive phenotype\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. However, the formation of the biomimetic system can turn this disadvantage as an advantage by selectively targeting macrophages or DCs. Once internalized, the system can release the mRNA cargo inside the cells, thereby activating the immune system and promoting a pro-inflammatory phenotype response. They also presented some glycoproteins such as immunoglobulin kappa constant (IGKC) and immunoglobulin heavy constant gamma 1 (IGHG1), which play a crucial role in immune system activation. JAWS EVs presented phosphoglycerate mutase 1 (PGAM1), ATP synthase subunit alpha (ATP5F1A), inter-\u0026alpha;-trypsin inhibitor heavy chain 2 family members (ITIH2), and peroxiredoxin-1 (PRDX1), all involved in glycolysis and cancer metabolism. Remarkably, all EVs were significantly enriched in proteins involved in transport, including clathrin heavy chain 1 (CLTC), lactotransferrin (LTF), hemopexin (HPX) and serrotransferin (TF).\u003c/p\u003e\n\u003cp\u003eSignificant differentially expressed protein-coding genes are presented in the volcano plot (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). In particular, proteins associated with multivesicular bodies, plasma membrane, extracellular matrix and microtubule/cytoskeleton were enriched. Meanwhile, proteins related to cell motility, adhesion and membrane trafficking were downregulated. The protein composition of both EVs was remarkably similar, which was represented in the biological functions of the detected proteins (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). THP-1 EVs presented a higher amount of structural proteins with a 42% in comparison with the 24% found in JAWS EVs. However, JAWS EVs had around a 10% more of immune and inflammatory proteins in comparison with THP-1 EVs. The other protein portions remained very alike.\u003c/p\u003e\n\u003cp\u003eTo gain insight into the function of the EVs, we conducted GSEA using the Gene Ontology (GO) and Hallmark databases. Strikingly, genes encoding proteins demonstrated that EV-specific proteins were selectively enriched in MYC targeting and mTORC1 signalling (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE, Figure S13). They also were involved in metabolic processes such as complement activation, G2-M checkpoint or unfolded protein response. Collectively, these bioinformatic analyses of the proteomic content of both EVs revealed that even coming from different species, both monocytic cell lines produced EVs with highly conserved core proteome and signalling transduction pathways.\u003c/p\u003e\n\u003ch3\u003e5. KH NP@JAWS EVs drives trafficking to macrophages\u003c/h3\u003e\n\u003cp\u003eA biodistribution study was carried out in mice to determine if the differences found \u003cem\u003ein vitro\u003c/em\u003e prevail \u003cem\u003ein vivo\u003c/em\u003e. We first induced the tumor formation subcutaneously using murine melanoma cell line, B16F10 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Since these tumors present a great amount of immune infiltrating cells, including different types of APCs, we tested the complex selectivity and accumulation in WT tumours with similar tumor volumes. Mice were followed up and treated when the tumor volumes were similar for all the groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). Murine EVs collected from JAWS cell line were used as the targeting EVs model for the \u003cem\u003ein vivo\u003c/em\u003e biodistribution study. JAWS EVs were labelled with a fluorescent dye Cy3 and KH NPs were labelled with Cy5. Mice were intravenously administered with the complex KH NP@JAWS EVs and KH NPs, both encapsulating a reporter mRNA of luciferase. As CTRL we had non-treated mice, mice treated with JAWS EVs without mRNA encapsulated and mice treated with KH NPs@LLC1 EVs containing luciferase mRNA. LLC1 EVs were used as negative CTRL because they should not target APCs or neither the tumor as they are not homing cells, they were derived from murine lung cancer. After 6 h, mice organs and tumors were collected and \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e imaging were performed to examine the complex biodistribution in B16F10 tumor model. The short time point of 6 h was chosen because it was previously observed in other studies that we have the maximum level of expression of luciferase\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e (Figure S14). As expected, mice treated with KH NP@JAWS EVs and KH NPs presented accumulation in the liver, spleen, and lungs. KH NPs had negligible luciferase expression in the tumor (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). In contrast, KH NP@JAWS EVs tumor-bearing mice showed significant increases in luciferase signal in the tumor compared with the other treated groups. Instead, KH NP@LLC1 EVs even being composed by the same NPs but with EVs non-targeting antigen-presenting cells, did not express luciferase in the tumor, liver, or lungs, but only in the spleen. This result strikingly demonstrates the superior and successful transfection capacity of KH NP@JAWS EVs concerning the CTRL samples, i.e. KH NPs without EVs shell, or the non-targeting KH NP@LLC1 EVs complexes.\u003c/p\u003e\n\u003cp\u003eThrough the Cy3 signal, we also demonstrated that JAWS EVs and KH NP@JAWS EVs mostly accumulated in the liver and spleen, as those organs contain high amounts of DCs and macrophages such as Kupffer cells or splenic macrophages (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE-F). Fluorescence was also detected in the tumor and lungs (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG, Figure S15). By Cy5 signal KH NP@LLC1 EVs were found significantly accumulated in the lung but also in the spleen (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH, Figure S14). Fluorescent signal was also observed in the liver for KH NPs, KH NP@JAWS EVs and KH NP@LLC1 EVs (Figure S16). No signal was detected in the tumors (Figure S16).\u003c/p\u003e\n\u003cp\u003eWe also demonstrated that liver enzyme levels across different groups were similar for ALT and AST (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eI-J), indicating that the accumulation in the liver did not result in differences in liver damage among the different groups. However, a significant increase in LDH was observed in both mice treated with KH NP@JAWS EVs and KH NP@LLC1 EVs (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eK). These increased levels reflect that the complexes were able to stimulate the immune system like macrophages or T cells. KH NP@JAWS EVs presented an increased uptake by Kupffer cells or splenic macrophages and an enhanced delivery to tumor cells.\u003c/p\u003e\n\u003ch3\u003e6. The uptake of KH NP@THP-1 EVs by cells relies on EVs surface adhesion molecules\u003c/h3\u003e\n\u003cp\u003eSince we have demonstrated physical interactions between EVs and KH NPs, it is possible that the uptake of KH NP@JAWS EVs by antigen presenting cells is mediated by specific surface molecules on EVs. To determine if there is a correlation between EVs\u0026rsquo; \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e behaviours in patients, as well as their biological processes and protein corona compositions, soft (SC) and hard corona (HC) proteins identified by LC\u0026ndash;MS were subjected to human protein databases (UniProt) analysis. We conducted proteomic profiling of THP-1 EVs alone and the SC and HC of THP-1 EVs and KH NP@THP-1 EVs after incubation with human plasma. A total of 371 proteins were identified in THP-1 EVs, 147 in THP-1 EVs HC and 172 in KH NP@THP-1 EVs HC (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Meanwhile in the SC it was detected a 428 proteins in THP-1 EVs SC and 430 in KH NP@THP-1 EVs SC (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003ePrincipal component analysis (PCA) demonstrated a closer correlation between the HC protein expression for THP-1 EVs and KH NP@THP-1 EVs compared to THP-1 EVs alone (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). According to PCA and consensus clustering analysis, complex and EVs presented a higher degree of similarity in their protein coronas. The score plot displayed a difference between THP-1 EVs alone, HCs and SCs, presenting three different populations. Interestingly, no difference was observed between the HC and SC compositions of THP-1 EVs and KH NP@THP-1 EVs, confirming that the complex formation did not change the EVs structural and biological properties (Figure S17-S18).\u003c/p\u003e\n\u003cp\u003eSimilar results were observed by comparing the most abundant proteins displayed as a relative protein abundance in the heatmap (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD, Figure S19). The proteome of THP-1 EVs presented a completely opposite protein enrichment in comparison to the HC of THP-1 EVs and KH NP@THP-1 EVs. The most abundant proteins in the HC were serum albumin (ALB) and actin cytoplasmic 1 (ACTB) which are secreted proteins and subcellular components. Also, glycoproteins such as immunoglobulin lambda (IGLC2), kappa (IGKC) and gamma 1 (IGHG1) were detected. These proteins are part of the genetic machinery that produces the structural components of antibodies, which are crucial for the immune system\u0026apos;s defense against foreign bodies (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE). In contrast, the proteins observed in the SC presented a more similar composition to THP-1 EVs. The SC main proteins were serum albumin (ALB), fibrinogen \u0026alpha; (FGA), \u0026beta; (FGB) and \u0026gamma; (FGG), actin cytoplasmic 1 (ACTB), and immunoglobulin heavy constant gamma (IGHG1) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF). The higher abundance of IGHG1 protein in the protein corona favoured their engulfment by phagocytes and induced complement activation (classical pathway) as interpreted from associated protein functions.\u003c/p\u003e\n\u003cp\u003eAn influential protein deleted in HC was sialic acid- binding Ig-like lectin 16 (SIGLEC16), which plays a major role in immune balance by activating DCs and macrophages response. Previous reports have demonstrated that Siglec receptors can impact in DC function, including increased antigen presentation, cytokine production, and migration to lymph nodes to prime tumor-specific T cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Relative protein abundance of SIGLEC16 demonstrated an upregulation in the HC of both, THP-1 EVs and KH NP@THP-1 EVs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eG). To gain insight into the HC protein functions using the Gene Ontology (GO) database, it demonstrated enrichment in specific metabolic pathways such as MYC targeting, protein secretion and mTORC1 signaling (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH, Figure S20). Meanwhile the protein content in the SC is related to angiogenesis (Figure S21). The expression of SIGLEC 16 can activate DCs and macrophages thanks to the interaction with the adaptor protein DAP12, an immunoreceptor tyrosine-based receptor (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eI).\u003c/p\u003e\n\u003cp\u003eImmune cells are a major source of EVs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Also, the ability of DC precursors to interact with T cells is beneficial in terms of obtaining EVs loaded with immunomodulatory molecules, which would enhance vaccination potential. Some reports have associated DC-derived EVs with APCs due to the presence of MHC-II molecules on their surface\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Unlike previous studies that explored other aspects, our study specifically focuses on immune system activation in the context of vaccination applications. Taken together, this data demonstrates that the expression of SIGLEC 16 on our NPs@EVs complexes could potentially be involved in the activation of DCs and immune response regulation.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eAlthough mRNA-based vaccines for SARS-CoV-2 have been successful, there are still many unresolved issues that could be addressed by developing safer, more efficient, and more selective nano-delivery systems. Here, we combined the physico-chemical properties of synthetic polymeric NPs with the natural biointerface functions of EV membranes to engineer hybrid smart multifunctional biomimetic nanocarriers. Until now, only a few studies have explored the use of biomimetic lipid/polymeric nanosystems or cell membrane-coating of synthetic nanoplatforms to enhance controlled delivery features. With our results, we conclude that these biomimetic EV-coated pBAE NPs outperform the functionality of the individual systems \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Undoubtedly, these discoveries will open avenues for translational studies of these novel nanosystems that hold potential for their use in nucleic-acid-based vaccination strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dario Castellana, Giacomo Sansone, Jonas Reckmann, and Jordi Guixeras for performing preliminary and complementary experiments. We also thank Maria Stampa Lopez-Pinto and Antoni Torres-Coll for polymer synthesis. Funding from AGAUR-Generalitat de Catalunya (2021 SGR 00537), from MICIN/AEI (PID2021-125910OB-I00, MCIN/AEI /10.13039/501100011033 / FEDER, UE), from the Institute of Health Carlos III (ISCIII) (AC22/00042), and from FCAECC (TRNSC213882FORN), both from the Joint Transnational Initiative 2021 ERA-NET TRANSCAN-3, European Commission is acknowledged. Funding from CA21154, CA21135, CA CIG 17104 COST ACTIONS is also acknowledged. CF acknowledges the support of the Departament de Recerca i Universitats of the Generalitat de Catalunya through the ICREA Acad\u0026egrave;mia 2024 programme. AM thanks POLITO for funding his research internship.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePardi, N., Hogan, M. J., Porter, F. W. \u0026amp; Weissman, D. mRNA vaccines \u0026mdash; a new era in vaccinology. \u003cem\u003eNat Rev Drug Discov\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 261\u0026ndash;279 (2018).\u003c/li\u003e\n\u003cli\u003eBarbier, A. J., Jiang, A. Y., Zhang, P., Wooster, R. \u0026amp; Anderson, D. G. 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A. \u003cem\u003eet al.\u003c/em\u003e Self-associated molecular patterns mediate cancer immune evasion by engaging Siglecs on T cells. \u003cem\u003eJournal of Clinical Investigation\u003c/em\u003e \u003cstrong\u003e128\u003c/strong\u003e, 4912\u0026ndash;4923 (2018).\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":"Polymeric nanoparticles, extracellular vesicles, nucleic acid vaccination, targeted gene delivery systems, biomimetic nanosystems","lastPublishedDoi":"10.21203/rs.3.rs-7030394/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7030394/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNucleic acid vaccination has expanded the traditional use of vaccines, ranging from infectious disease prophylaxis to therapeutic applications in oncology. However, nucleic acids require protection from nuclease degradation \u003cem\u003ein vivo\u003c/em\u003e by using selective delivery vectors capable of transporting the cargo into targeted cells such as antigen-presenting cells (APCs) for efficient genetic material translation into proteins and immune system activation. Polymeric nanoparticles (NPs), particularly poly(beta aminoesters) (pBAEs), have demonstrated efficiency in transfecting cells \u003cem\u003ein vitro\u003c/em\u003e and have also demonstrated promising results \u003cem\u003ein vivo.\u003c/em\u003e Despite numerous attempts to target NPs to specific cells, selectivity still remains a challenge. In this study, we address this limitation by developing biomimetic nanosystems composed of pBAE NP-embedded in extracellular vesicles (EVs). These small vesicles, released by all cell types, facilitate intercellular communication among cells of the same lineage, making them ideal to be use as natural targeting moieties. Effective complexes were ensembled using a freeze-thaw method to efficiently entrap pBAE NPs loaded with nucleic acids in monocyte-derived EVs. Their \u003cem\u003ein vitro\u003c/em\u003e safety, transfection, and monocyte activation capacity of these complexes outperformed the individual components of the nanosystem, demonstrating their suitability for parenteral use \u003cem\u003ein vivo\u003c/em\u003e. Our findings confirm the potential of constructing efficacious biomimetic pBAE NPs-EVs nanocomplexes for nucleic acid vaccination for the first time.\u003c/p\u003e","manuscriptTitle":"Polymeric nanoparticle-loaded extracellular vesicles as biomimetic nucleic acid vaccines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-04 08:11:05","doi":"10.21203/rs.3.rs-7030394/v1","editorialEvents":[{"type":"communityComments","content":1}],"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":"d569888b-6d3a-49b0-bcc4-1cf4b79e8f0d","owner":[],"postedDate":"July 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50973527,"name":"Biological sciences/Biotechnology/Biomaterials/Biomaterials \u0026#x2013; vaccines"},{"id":50973528,"name":"Biological sciences/Cancer/Cancer therapy/Tumour vaccines"},{"id":50973529,"name":"Physical sciences/Chemistry/Materials chemistry/Biomaterials/Drug delivery"}],"tags":[],"updatedAt":"2025-07-09T19:25:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-04 08:11:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7030394","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7030394","identity":"rs-7030394","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}