Harnessing extracellular vesicle membrane for gene therapy: EVs-biomimetic nanoparticles

Colloids and Surfaces B: Biointerfaces 239 (2024) 113951 Available online 7 May 2024 0927-7765/© 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/bync/4.0/). Harnessing extracellular vesicle membrane for gene therapy: EVs-biomimetic nanoparticles Erik Briffault a , b , Patricia Garcia-Garcia a , b , Rebeca Martinez-Borrajo c , Carmen Evora a , b , Araceli Delgado a , b , * , Patricia Diaz-Rodriguez b , c , ** a Department of Chemical Engineering and Pharmaceutical Technology, Universidad de La Laguna, La Laguna 38206, Spain b Institute of Biomedical Technologies (ITB), Universidad de La Laguna, La Laguna 38320, Spain c Department of Pharmacology, Pharmacy and Pharmaceutical Technology, I+D Farma Group (GI-1645), Facultad de Farmacia, Instituto de Materiales (iMATUS) and Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain ARTICLE INFO Keywords: Gene therapy GapmeR Biomimetic nanoparticles Extracellular vesicles Fluorescence cross-correlation spectroscopy ABSTRACT One of the main concerns in oligonucleotide-based therapeutics is achieving a successful cell targeting while avoiding drug degradation and clearance. Nanoparticulated drug delivery systems have emerged as a way of overcoming these issues. Among them, membrane-coated nanoparticles are of increasing relevance mainly due to their enhanced cellular uptake, immune evasion and biocompatibility. In this study, we designed and elaborated a simple and highly tuneable biomimetic drug delivery nanosystem based on a polymeric core surrounded by extracellular vesicles (EVs)-derived membranes. This strategy should allow the nanosystems to benefit from the properties conferred by the membrane proteins present in EVs membrane, key paracrine mediators. The developed systems were able to successfully encapsulate the required oligonucleotides. Also, their characterisation through already well standardised methods (dynamic light scattering, transmission electron microscopy and nanoparticle tracking analysis) and by fluorescence cross-correlation spectroscopy (FCCS) showed the desired core-shell structure. The cellular uptake using different cell types further confirmed the coating though an enhancement in cell internalisation of the developed biomimetic nanoparticles. This study brings up new possibilities for GapmeR delivery as it might be a base for the development of new delivery systems for gene therapy. 1. Introduction The interest and research on nanoscale delivery systems have been steadily increasing for the last couple of decades due to their potential to successfully cross biological barriers and accumulate in target organs and tissues [1]. This property allows for enhancing the effectivity while reducing the adverse effects of conventional therapies and protecting the drug from its environment [2]. However, despite all the improvements achieved, there are still issues to be addressed and refined, such as more specific targeting after systemic administration and higher immune evasion, biocompatibility, cell uptake and circulation time. These points become even more important for oligonucleotide-based therapeutics, considering the delivery challenges involved [3]. Among oligonucleotides, GapmeRs, antisense oligonucleotides comprised of a short specific DNA sequence flanked by locked sugar-modified nucleotides on both ends, have gained visibility over the last years as gene therapy agents characterized by their higher stability [4,5]. However, despite their potential utility, their structure supposes a hinder for their intracellular delivery [3,6]. Therefore, novel approaches such as complexation or conjugation with other components have been proposed to increase their therapeutic utility [3]. Other strategies to enhance intracellular oligonucleotide delivery have been already developed, including their encapsulation inside delivery systems such as lipidic [7] or hybrid nanoparticles (NPs) [8,9]. An alternative to achieve suitable internalisation and selectivity could be based on the functionalisation of nanoparticles in such a way that their * Corresponding author at: Department of Chemical Engineering and Pharmaceutical Technology, Universidad de La Laguna, La Laguna 38206, Spain. ** Corresponding author at: Department of Pharmacology, Pharmacy and Pharmaceutical Technology, I+D Farma Group (GI-1645), Facultad de Farmacia, Instituto de Materiales (iMATUS) and Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, Santiago de Compostela 15782, Spain. E-mail addresses: [email protected] (A. Delgado), [email protected] (P. Diaz-Rodriguez). Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb https://doi.org/10.1016/j.colsurfb.2024.113951 Received 7 January 2024; Received in revised form 28 April 2024; Accepted 4 May 2024 Colloids and Surfaces B: Biointerfaces 239 (2024) 113951 2 surface resembles a biological membrane, leading to biomimetic nanoparticles. However, elaborating these nanosystems following a classical perspective would entail to obtain a highly complex surface requiring different raw materials though a bottom-up approach [10]. This would be immensely complicated considering the variety of specific lipids, proteins and other molecules present in biological membranes [11]. An alternative top-bottom approach is to obtain biomimetic biological membrane-coated nanoparticles (BMNPs), which arise as a more biocompatible concept than completely artificial delivery systems, partially solving the aforementioned issues [12,13]. Regarding their general structure, these NPs unify the advantages of artificial nanoparticles and biological membranes and allow for a great variety of core-shell combinations [14–16]. The selection of the components of both parts of the structure is a key point conditioning the success of the designed nanocarrier. The NP core will be, at greater extend, responsible of the BMNP size and shape [17]. The selection of the core material (e.g. polymers, metals, silica, lipids, among other materials) is based on the purpose of the nanocarrier (therapy, imaging), capacity to load and release the desired cargo, biocompatibility and required size, shape and/or surface charge. Moreover, as a general rule, the surface charge of the BMNPs cores should be neutral or negative to allow for an adequate subsequent membrane coating. Conversely, positively charged cores could lead to disordered membrane coatings and even nanoparticle aggregation because of the strong interactions with the negatively charged biological membranes [18–20]. The biological coatings are, on the other side, responsible for the BMNPs interaction with the environment. The main purpose of the biological coating is to endow the NPs with a “self” marker by showing off specific membrane proteins, leading to the aforenoted immune evasion, circulation time prolongation, higher biocompatibility and better toxicity profile [19,21]. A specific surface composition would also grant them targeting capabilities and the ability to interact with specific cells and/or tissues [16]. In this regard, homologous targeting to the mimicked cells has been widely observed [22,23] while cross-targeting has also been reported [24]. Additionally, the biological coating acts as a modifier of the release profile of the cargoes [25]. Biomimetic nanoparticles have been widely explored in the recent years for several applications: drug delivery systems [26,27], tissue regeneration [18,22], clearance and/or neutralisation of substances [28] or imaging [29,30]. The coating membrane source is chosen in accordance with the intended functionality for the BMNPs. Different cell membranes sources have been used for coating nanoparticles, such as platelets, macrophages, stem cells, red blood cells, cancer cells or even bacteria [16,31]. Among them, mesenchymal stem cells (MSCs) stand out, been proven to be useful in the development of nanoparticles designed for tumour treatment, tissue regeneration and immunomodulation purposes [16,18]. Also, MSCs are easy to isolate and expand in vitro, making them a suitable source for BMNPs elaboration [32,33]. Extracellular vesicles (EVs) are nanometric-sized membrane vesicles released by cells. They possess unique membrane lipid and protein compositions which facilitate their uptake by cells and their intracellular trafficking [34,35], making them suitable carriers for drug delivery [34] while preserving the identity of their original cell type. To this regard, EVs of several origins, ranging from animal to bacterial [36] and vegetal [37–39], have been used for biomedical purposes with successful outputs [40]. Furthermore, unlike for cell membranes, which must undergo several processes for cell lysis, removal of the intracellular content and isolation for NPs coating; EVs are suitable for a more straightforward application in BMNPs elaboration, having led to promising results in several applications, as recently reported [24]. Fluorescence cross-correlation spectroscopy (FCCS) is a very sensitive technique that provides information related with biochemical interactions between two different fluorescently labelled species [41–43]. This technique can be employed to further explore interactions in biological compounds, cellular extracts, particles, or even live cells [41,43]. FCCS could be a good strategy to explore and to obtain more information about the attachment between different labelled components. In this scenario when the core and shell components of the BMNPs are fluorescently labelled with two different fluorophores, the analysis of the correlation signals of both fluorophores can confirm the core-shell structure of the systems and the coating effectiveness. Despite its utility, to the best of our knowledge this technique has not been used previously to characterise biomimetic nanoparticles. Herein we designed, developed and characterised a simple, versatile and highly tuneable biomimetic drug delivery nanosystem consisting of a PLGA nanocore coated with MSCs EVs membranes (BMNPs), able to load protamine-condensed GapmeRs for oligonucleotide delivery. Departing from previous results of our research group, we optimised the PLGA polymeric cores (PCs) and came up with a formulation able to efficiently load GapmeRs, while preserving a small size and negative surface charge. The EVs isolation method was optimised to obtain the EVs for coating the PCs and the BMNPs were elaborated by the coextrusion method. The obtained nanosystems were characterised in terms of physicochemical properties and the coating was confirmed by TEM and, for the first time for this purpose, by FCCS. Also, we demonstrated a higher cellular uptake rate for BMNPs in comparison with PCs. This work gives as a result a new BMNPs formulation that might be a starting point for further GapmeR delivery systems. 2. Materials and methods 2.1. Selection of an EVs isolation method To select the most suitable method for EVs isolation in terms of physicochemical characteristics of the obtained particles and purity (presence of non-EVs-associated proteins), four isolation methods were tested: ultrafiltration (UF), size exclusion chromatography (SEC; qEVoriginal/70 nm, Izon Science Ltd., New Zealand), precipitation with a commercial reagent (Total Exosome Isolation Reagent (from cell culture media), Invitrogen, USA) and ultracentrifugation (UC). In all cases EVs were isolated from murine mesenchymal stem cells (C3H10T1/2; ATCC CCL-226) cultures. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1 % penicillin/streptomycin and 1 % L-glutamine (complete DMEM) in a humidified atmosphere at 37ºC and 5 % CO 2 . When 80 % confluence was reached, cell monolayers were washed with DPBS (Dulbecco’s Phosphate Buffered Saline) and medium was changed to EVs isolation DMEM (same composition as the standard one, but with 5 % EVs-free FBS instead of 10 % FBS). This EVs-free FBS was obtained by ultrafiltration FBS through 10 kDa MWCO (Millipore Amicon, USA) filters at 3000 g, 55 min, 4ºC. Cells were cultured in this new medium for 48 h. Then, the EVs-containing medium was removed and treated correspondingly to the EVs isolation method described below, while EVs-producing cells were counted. For UF, 10 mL of EVs-containing medium were centrifuged at 2000 g and 4ºC for 30 min to remove dead cells and then ultrafiltered though 10 kDa MWCO filters (Millipore Amicon, USA) at 3000 g and 4ºC for 55 min. The concentrate was diluted in 10 mL DPBS and centrifuged again. The EVs-containing concentrate was finally diluted in 1 mL DPBS to obtain the EVs sample. For the precipitation method, 10 mL of EVscontaining medium were also centrifuged at 2000 g, 30 min, 4ºC to remove dead cells. The pellet was discarded and the isolation reagent was added to the supernatant in a 1:0.5 supernatant to reagent ratio, incubated for 18 h at 4ºC and centrifuged at 10,000 g for 1 h at 4ºC. The supernatant was discarded, and the EVs-containing pellet was resuspended in 1 mL DPBS. For SEC isolation, 10 mL of EVs-containing medium were sequentially centrifuged at 500 and 10,000 g for 10 min to remove dead cells and debris. Then, the supernatant was concentrated by centrifugation using 100 kDa MWCO filters (Millipore Amicon, USA) at 3000 g for 55 min and 4ºC and taken to a final volume of 1 mL with DPBS. This concentrated sample was eluted in qEVoriginal SEC columns and the EVs-corresponding fraction was recovered and centrifuged again E. Briffault et al. Colloids and Surfaces B: Biointerfaces 239 (2024) 113951 3 using 100 kDa MWCO filters (Millipore Amicon, USA) at 3000 g for 55 min and 4ºC. The concentrate was taken to 1 mL with DPBS to obtain the EVs sample. To isolate EVs by UC, EVs-containing medium was centrifuged consecutively at 2000 g and 4ºC for 30 min to pellet dead cells, at 10,000 g and 4ºC for 30 min to remove cell debris and at 100,000 g and 4ºC for 2 h to precipitate EVs. The EVs-containing pellet was resuspended in 1 mL DPBS. For all methods the resulting EVs were filtered through 0,22 µm filters (PES membranes Millex®-GP, Millipore, Ireland). 2.2. Synthesis of polymeric cores Two different Poly(D,L-lactide-co-glycolide) 50:50 (PLGA) of the same molecular weight were tested for the elaboration of the polymeric cores (PCs). PLGAs with different end group were used, one alkyl esterterminated (Resomer® RG 502, 0.16–0.24 dL/g, Evonik, Germany) and one carboxylic acid-terminated (Resomer® RG 502 H, 0.16–0.24 dL/g, Evonik, Germany). PCs were obtained by the nanoprecipitation method. Briefly, 50 µL of a 33,33 µg/mL protamine or a 50 µg/mL GapmeR/protamine complex aqueous solution were pipetted into 1 mL of an organic phase of PLGA at different concentrations in acetone or acetonitrile. This mixture was then poured into 6 mL of milli-Q water under constant magnetic stirring and left for one hour under agitation to allow the evaporation of the organic phase. Then, the resulting suspension was rotavaporated for 15 min at 30ºC for complete elimination of the organic solvent. The resulting PCs were filtered through 0.22 µm PES membranes. For GapmeR-loaded PCs (PCs-G), control GapmeR (Qiagen) was left to condensate with the protamine for 40 min. 1 µg of gapmer was added to the protamine solution in a 1:2 GapmeR to protamine mass ratio [9] in a total volume of 60 µL to form the 50 µg/mL GapmeR/protamine complex aqueous solution. 2.3. PCs coating with EVs membranes PCs or PCs-G were coated with EVs membranes to obtain biomimetic nanoparticles, BMNPs or BMNPs-G, respectively. Both PCs (or PCs-G) and EVs were filtered through 0,22 µm and extruded together using an Avanti Mini-Extruder (Avanti Polar Lipids, USA). PCs were mixed with EVs in a 2.5:1 no. ratio and filled up to 1 mL with DPBS. Then, this mixture was extruded 11 times through a 0.2 μ m pore size Nuclepore Track-Etch Membrane (Avanti Polar Lipids, USA) to coat the PCs or PCsG with the EVs membranes, similarly to previously reported [44]. Fig. 1 summarises the whole BMNPs-G elaboration process. 2.4. Characterisation of EVs, PCs and BMNPs All PCs, EVs and BMNPs were characterised in terms of size (average hydrodynamic diameter and polydispersity index –PdI–) and ζ-potential (ZP) by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively, using a Zetasizer Nano ZS (Malvern Panalytical Ltd., UK). For the analysis, formulations were conveniently diluted with Milli-Q water and loaded into disposable microcuvettes. Additionally, the stability of both PCs and BMNPs was assessed in DPBS and 10 % FBS. To this end, the obtained nanoparticles were 1.25-fold diluted with DPBS or 50 % FBS. Nanoparticles were then incubated at 37 ⁰C for 2, 6 and 24 h and, afterwards, characterized by DLS as previously described. Furthermore, the amount of total protein in the EVs isolates and the percentage of EV-associated protein were assessed by measuring total protein concentration with a Micro BCA Protein Assay Kit (Thermo Scientific, USA). To this end, the protein content in EVs samples was measured both with whole EVs and with lysed EVs, so that the percentage of EV-associated protein could be calculated from the difference between these two values. For lysis, EVs samples were diluted 1:25 with 0.2 % sodium dodecyl sulphate (SDS) in DPBS and subjected to six 30 s cycles of sonication in ice-cold water with 30 s intervals between cycles. EVs, PCs and BMNPs were also analysed by nanoparticle tracking analysis (NTA; Nanosight NS300, Malvern Panalytical Ltd., UK) and transmission electron microscopy (TEM; JEOL JEM-2100, 200 kV, Japan). NTA was used to confirm the particle sizes measured by DLS, as well as for quantifying and stablishing a particle number ratio between PCs and EVs to from BMNPs. Formulations were conveniently diluted with Milli-Q water to perform the measurements. TEM analysis was also Fig. 1. Schematic view of the elaboration process of GapmeR-loaded, MSCs EV-membrane coated biomimetic nanoparticles (BMNPs-G). Created with BioRender. E. Briffault et al. Colloids and Surfaces B: Biointerfaces 239 (2024) 113951 4 used to confirm the sizes measured by DLS and NTA as well as to verify the successful coating of the PCs. For TEM analysis, samples were stained with 1 % uranyl acetate for two minutes. 2.5. Fluorescence cross-correlation spectroscopy of BMNPs The core-shell structure of the nanoparticles was additionally confirmed by FCCS. To this end, PCs were stained with DiD (Molecular Probes® Invitrogen) and DOPE-Atto488 (Invitrogen) was added to the nanoparticles shell by hydrophobic insertion. FCCS experiments were carried out in a Laser Scanning Microscope Zeiss LSM780 (Carl Zeiss AG, Jena, Germany) employing a Zeiss C-Apochromat 40X, 1.2 NA water immersion objective with a correction collar. A drop of immersion oil (Immersol™ W with a η e=1.334) was placed in the objective and a 0.17 mm thick coverslip was located directly on the oil drop. The sample was placed on the coverslip and both fluorophores (DiD and ATTO 488) were excited. DiD fluorophore was excited with a 633 nm diode-pumped solid-state laser with a detection range of 635 nm to 692 nm, while ATTO 488 was excited employing an argon laser operating at 488 nm with a detection range of 490 nm to 621 nm. The fluorescence emission was collected and separated from the excitation fluorescence with a dichroic mirror (MBS 488/561/633). Before performing FCCS experiments, a pinhole alignment was carried out and laser intensities were adjusted to obtain the maximum counts per molecule (CPM), that is, the maximum quantity of brightness particles in the detection volume as well as adjusted to avoid saturation and photobleaching [41,43]. In FCCS experiments, fluorescence fluctuations of the labelled components that diffuse through the confocal volume can be analysed and represented with an autocorrelation function (G) (Eq. 1), where G ( τ ) represents average over time, F(t) denotes fluorescence intensity signal at a certain time, δF(t) =F(t)-〈F(t)〉represent the time-dependent fluctuations, and τ represents the lag time [43,45]. G( τ ) = 〈δF(t)δF(t+ τ )〉 〈F(t)〉2(1) 2.6. GapmeR encapsulation efficiency (EE) Oligonucleotide encapsulation efficiency inside the PCs-G was evaluated using a FAM-labelled GapmeR (Antisense LNA GapmeR Control, Quiagen). After rotavaporation, PCs-G were filtered through 100 kDa MWCO (Millipore Amicon, USA) at 10.000 rpm and free (non-encapsulated) GapmeR was quantified using a plate reader (Biotek, USA) at 485/528 nm. Furthermore, the amount of free GapmeR after PCs-G coating (GapmeR lost in the extrusion process) was also assessed following the same procedure. 2.7. Cellular uptake assay BMNPs and PCs cellular uptake was analysed in murine mesenchymal stem cells (MSCs, C3H10T1/2), murine fibroblasts (BALB/ 3T3A31) and murine macrophages (RAW 264.7). For this assay, fluorescently labelled systems were used. These PCs and BMNPs were prepared by adding 4 µg of coumarin-6 (Sigma-Aldrich, USA) to the PLGA organic phase during formulation. Cells were cultured in complete DMEM in a humidified atmosphere at 37ºC and 5 % CO 2 , seeded at a 15,000 cells per well for BALB and RAW or at 10,000 for MSCs in 96-well plates with complete DMEM and allowed to attach for 24 h. After this time, culture medium was removed, and wells were washed twice with DPBS. PCs and BMNPs were added to the cells and incubated for 2 h. Formulations were dosed based on PCs mass, adding the equivalent to 25 µg of PCs per well (300 µL) for BMNPs and PCs. Then, formulations were removed, wells were washed with DPBS and Triton-X 1 % was added to lyse the cells. The cell uptake rate was calculated by assessing the difference in fluorescence between after lysis vs right after adding the formulations. Cellular uptake was also analysed by confocal microscopy. MSCs, BALB and RAW were seeded in 8-well chamber slides (ThermoFisher Scientific, USA) at the same density as previously mentioned and allowed to attach for 24 h. Afterwards, cell monolayers were washed with DPBS and treated for two hours with DiD labelled PCs and BMNPs. After the treatment, cells were washed with DPBS and fixed with 4 % formaldehyde overnight. Monolayers were then washed thrice with DPBS, permeabilised with 0.1 % of Triton X-100, washed again thrice with DPBS and stained with Phalloidin-Alexa Fluor 488 (ThermoFisher Scientific, USA) for 40 min at room temperature. Stained cells were washed again with DPBS and mounted with DAPI Gold Antifade mounting solution. Cells were observed at 63X using a confocal microscope Stellaris (Leica). 2.8. Statistical analysis Statistical differences between two experimental groups were analysed by Student’s t-test. Statistical differences between multiple experimental groups were analysed by one-way ANOVA with post-hoc Tukey’s test for multiple comparisons. All tests were performed at a significance level of α =0.05. Data are presented as mean±SEM. 3. Results and discussion 3.1. EVs isolation methods to obtain adequate EVs suspensions To select the best method to isolate the EVs for PCs coating, four of the most used isolation methods were tested (UF, SEC, precipitation and UC). The evaluated parameters were EVs size (hydrodynamic diameter), PdI, rate of particles smaller than 400 nm, ZP and rate of EVs-associated proteins. EVs isolated by all methods showed an average size diameter over 200 nm, PdI over 0.4 and negative ZP. However, differences were observed between isolation methods mainly in size, rate of small particles and EVs-associated proteins rate, as shown in Fig. 2. Precipitation yielded EVs suspensions with the highest average diameter and variability (732.43 ±273.96 nm), while not being able to successfully isolate the lower size range of EVs (54.27 ±9.95 % of EVs <400 nm). Furthermore, the percentage of EV-associated protein for this method was the lowest (6.71 ±32.24 %), indicating high levels of residual proteins. On the other hand, the use of SEC, while yielding the highest percentage of EV-associated protein value (97.14 ±0.51 %), giving rise to high purity EVs suspensions, presented a relatively large size (523.57 ±116.20 nm) and a relatively low percentage of <400 nm EVs (51.95 ± 14.94 %). Moreover, UF, interestingly, yielded lower size EVs (342.57 ± 30.92 nm) than SEC and a lower percentage of <400 nm EVs (27.78 ± 7.24 %) than other methods. Also, the percentage of EV-associated proteins was low compared to other methods (38.45 ±23.43 %). Finally, the isolation of EVs using UC resulted in the smallest EVs size diameter (281.66 ±62.63 nm) and the highest percentage of EVs population below 400 nm (73.12 ±10.18 %), whilst performing well regarding purity (61.54 ±8.71 % EV-associated protein). Based on these data, the selected isolation method for obtaining EVs for coating the PCs was UC. 3.2. Suitable polymeric cores PCs elaborated with PLGAs with different terminal groups, ester (Resomer® RG 502) or carboxylic acid (Resomer® RG 502-H), from now on, 502 and 502 H, respectively, at different concentrations (1, 5 and 10 mg/mL) were prepared. Results show that PCs were bigger when obtained with any PLGA at 1 mg/mL, than at 5 or 10 mg/mL, and size was similar between these two latter concentrations for any of the polymers. Nonetheless, this decrease in size as polymer concentration increases is more notable in 502 PCs, moving from >2µm for 1 mg/mL, associated to nanoparticle aggregation due to neutral ZP, to 162.16 ± 1.02 and 167.74 ±0.65 nm for 5 and 10 mg/mL, respectively. However, the same effect is present in 502 H-based PCs (144.73 ±4.08 nm for E. Briffault et al. Colloids and Surfaces B: Biointerfaces 239 (2024) 113951 5 Fig. 2. EVs characterisation regarding the isolation method. A) Hydrodynamic size (bars) and ZP (diamonds) of EVs measured by DLS and ELS, respectively, B) percentage of EVs population below 400 nm and C) percentage of EV-associated protein. & indicates differences vs. UF, * indicates differences vs. precipitation, # indicates differences vs. pre-isolation. Fig. 3. Hydrodynamic diameter (bars) and ZP (diamonds) measured by DLS and ELS, respectively, of PCs elaborated at 1, 5 and 10 mg/mL with (A) PLGA 502 or 502 H, (B) PLGA 502 H using acetone (Ac) or acetonitrile (ACN), and (C) 5 mg/mL PLGA 502 H/acetone PCs and PCs-G. * indicates differences in size, §indicates differences in size compared to all other formulations, # indicates differences in ZP, †indicates exclusion from statistical analysis due to aggregation. E. Briffault et al. Colloids and Surfaces B: Biointerfaces 239 (2024) 113951 6 1 mg/mL and 120.16 ±1.05 nm and 124.57 ±1.52 nm for 5 and 10 mg/mL, respectively) (Fig. 3A). Considering the smaller particle sizes obtained for PLGA 502 H (carboxylic acid-terminated) and the feasibility for tuning particle size in the lower range of concentrations, it became the polymer of choice. Afterwards, the behaviour of PLGA 502 H for forming PCs was tested with acetonitrile as the organic solvent [46,47]. PCs elaborated with acetone as the organic solvent were smaller than those prepared with acetonitrile for all three concentrations (144.73 ±4.08 vs 194.17 ± 12.14 nm; 120.16 ±1.05 vs. 143.40 ±1.28 nm and 124.57 ±1.52 vs. 158.01 ±5.44 nm for acetone vs. acetonitrile at 1, 5 and 10 mg/mL, respectively), though no statistical significance was found for 5 mg/mL (Fig. 3B). Thus, acetone was selected as the organic solvent to work with. Furthermore, the intermediate polymer concentration (5 mg/mL) was chosen as it was not too low to produce excessively big PCs, nor too high to compromise PCs coating by extrusion. All PCs were filtered through 0, 22 µm membranes right after elaboration to remove possible aggregates that could end up in clogging the extrusion membrane and hindering the coating process. As a next step, a GapmeR was encapsulated into the selected PCs (PLGA 502 H at 5 mg/mL in acetone) to obtain PCs-G and their properties were compared to blank PCs. As shown in Fig. 3C, blank PCs happened to be bigger than PCs-G (120.94 ±3.36 vs. 101.81 ±3.35 nm, respectively) and were more negatively charged (-33.25 ±1.32 vs. −25.64 ±0.68 mV) (Fig. 3C). To assess the encapsulation efficiency of the GapmeR, the amount of non-encapsulated FAM-labelled GapmeR was measured. The encapsulation efficiency of GapmeR in PCs-G was 85.8 ±10.8 %. Therefore, these PLGA-protamine PCs seem to be suitable nanocarriers for GapmeRs, concerning the encapsulation yield. 3.3. Biomimetic nanoparticles with a core-shell structure BMNPs were elaborated by the extrusion method, forcing PCs and EVs together through a 0.2 µm pore size membrane, aiming to coat the PCs with EVs. As seen in Fig. 4A, there is no statistical difference in size between PCs and BMNPs (120.94 ±3.36 and 123.04 ±7.15 nm, respectively, p>0.05). This is expected, considering the small thickness of the EVs membrane and the intrinsic variability within PCs size measured by DLS. However, both PCs and BMNPs were statistically smaller than EVs (p<0.05), that showed an average size of 249.41 ± 48.21 nm (Fig. 4A). Moreover, there was statistical difference in ZP between PCs and BMNPs (-33.25 ±1.32 vs −23.21 ±2.56 mV, p<0.05), and both systems with EVs (-10.65 ±1.45 mV). Differences in ZP were also expected and an increased value might prove a successful PCs coating as is closer to the EVs ZP. BMNPs-G were also elaborated by extruding Gapmer loaded PCs with EVs membranes. Results showed that, as happened with PCs and PCs-G, BMNPs-G were statistically smaller than BMNPs (98.72 ±3.56 nm vs. 123.04 ±7.15, respectively, p<0.05), but no statistical difference in ZP was observed (-32.39 ±2.96 mV vs. −23.21 ±2.56, p<0.05), contrary to the effect of the GapmeR incorporation in plain PCs (Fig. 4B). This might be due to the coating process as the outer layer is coincident for both kinds of nanoparticles. Moreover, the fluorescent signal of the released GapmeR after PCs coating was undetectable, indicating no GapmeR was lost throughout the coating process. The stability data of PCs and BMNPs in PBS at 37 ⁰C (Fig. 4C) showed no significant changes in size for neither of the formulations during the initial 6 h of study. However, BMNPs incubated for 24 h at 37⁰C depicted a significant increase in size. On the other hand, when incubated in the presence of 10 % FBS at the initial points of study (0–6 h) BMNPs showed lower size increments compared to BMNPs at time 0 (Fig. 4D). Moreover, PCs depicted highly variable mean diameters between batches ranging from Fig. 4. Hydrodynamic diameter (bars) and ZP (diamonds) measured by DLS and ELS, respectively, of (A) PCs, BMNPs and EVs and (B) BMNPs and BMNPs-G. Stability of PCs and BMNPs in DPBS (C) and 10 % FBS (D) at 37 ⁰C. * indicates differences in size compared to fresh BMNPs in DBPS (B-C) or 10 % FBS (D), # indicates differences in ZP, & indicates differences both in size and ZP. E. Briffault et al. Colloids and Surfaces B: Biointerfaces 239 (2024) 113951 7 479.1 ±202.5 nm to 840 ±207.3 nm just after 2 h of incubation with the FBS solution. This increase in size, reached just after the first timepoint, was kept constant for the rest of the study. Conversely, BMNPs behaved more similarly to previously observed in DPBS with more homogenous mean diameters between batches and an increase in the mean hydrodynamic diameter over time that ended being statistically significant after 24 h of incubation. This behavior could be indicative to a modification of the lipid shell composition and integrity. This shell initially confers stability to the BMNPs and can be partially lost over time when incubated in saline solutions at 37⁰C containing or not proteins. Despite previous studies have reported no significant changes in the biomimetic nanoparticles size when incubated in PBS and/or 10 % FBS the experiments were not performed at the physiological temperature (37 ⁰C) [48,49]. Temperature is a crucial factor controlling diffusion and could explain the different behavior observed in our experiment. EVs, PCs and BMNPs were also analysed by NTA confirming the results obtained by DLS for PCs and BMNPs (Fig. 5A-D). PCs presented an average size of 110.1 ±0.5 nm, while for BMNPs this value is 129.63 ± 25.88 nm. However, differences between DLS and NTA determinations Fig. 5. A-D) NTA size distributions of PCs (A), EVs (B) and BMNPs (C) along with mean size and SD (D). E-J) Transmission electron microscopy images of PCs (E, F), EVs (G, H) and BMNPs (I, J), negatively stained with 1 % uranyl acetate. E. Briffault et al. Colloids and Surfaces B: Biointerfaces 239 (2024) 113951 8 were observed for EVs, with a lower mean size (110.9 ±0.4 nm) than the one observed by DLS. Measurements from NTA, however, show no difference in size between PCs and EVs. TEM images are shown in (Fig. 5 E-J). PCs can be seen as clearly delimited round-shaped particles, while EVs show their typical and characteristic cup-shape, as expected based on previous reports [50]. Thus, EVs look like hollow structures, unlike PLGA PCs, which look consistent inside. TEM images confirm a successful coating of PCs with EVs, as clear structures can be seen surrounding the PCs in BMNPs, which are assumed to be EV-membranes, forming a clear core-shell structure. FCCS provides information about the interaction between two fluorescently labelled components obtaining fluorescence fluctuations in two different channels associated with the different labelled components. The diffusion of these components will result in correlated signals (Fig. 6) from both fluorophores (DiD and ATTO 488) (Fig. 6A and Fig. 6B, respectively) and for the cross-correlation between them (Fig. 6C) [43,45]. When labelled components are attached, they are expected to pass through the confocal or detection volume at the same time obtaining a positive cross-correlation. As shown in Fig. 6C, a positive crosscorrelation was obtained confirming the expected polymeric coremembrane shell composition previously observed in the TEM images. 3.4. Cell interactions with the developed systems A cellular uptake assay was performed for BMNPs and PCs in MSCs, fibroblasts and macrophages to determine whether the EVs membranes coating increases the cell uptake compared to naked PCs. BMNPs showed higher uptake rates than PCs (p<0.05) in all the three cell lines to a similar extend. The internalisation values were 43.1 ±3.2 % for BMNPs and 31.2 ±1.7 % for PCs for macrophages; 35.7 ±2.5 % and 27.0 ±2.2 % for fibroblasts and 47.4 ±3.7 % and 37.5 ±2.2 % MSCs (Fig. 7). BMNPs were, as expected, more easily internalised by all three cell types, which further confirms the biomimetic nature of these nanoparticles. This coating, however, unspecifically enhanced cellular uptake versus non-coated nanoparticles. The increased uptake rate for BMNPs was similar for all three cell types, not observing an uptake rate enhancement for the source cells (MSCs) nor an uptake reduction in other cell lines; not even in immune cells (macrophages). The intracellular distribution of the nanoparticles was analysed in MSCs, fibroblasts and macrophages. As shown in Fig. 8 BMNPs were located on the cell cytoplasm more clearly observed in the orthogonal images (Fig. 8C). Moreover, lower amount of internalized nanoparticles were observed for PCs when compared to BMNPs where the Fig. 6. Fluorescent cross-correlation curves obtained for PCs and membrane components. (A) represents the correlation curve for PCs (DiD), (B) represents the correlation curve for DOPE-Atto488, and (C) represents the obtained cross-correlation. Fig. 7. Cell uptake rates of PCs and BMNPs in MSCs, fibroblasts and macrophages. * indicates differences vs. PCs in each cell line. E. Briffault et al. Colloids and Surfaces B: Biointerfaces 239 (2024) 113951 9 nanoparticles are non-associated to cells and/or attached to the cell membrane (Fig. 8B) showing the same tendency as previously observed in the cell uptake studies. The internalization behaviour was similar in the other two cell lines studied (Supplementary Figures S2 and S3). 4. Discussion The development of nanocarriers able to faithfully mimic biological membranes and successfully interact with the desired cells or tissues is constantly advancing. Both cell membranes and EVs have been previously used as coatings for PCs to build up BMNPs, with the latter being a more novel strategy [21,51–55]. The use of EVs as a source of biological membranes allows for simpler protocols without needing so many membrane purification steps and taking advantage of EVs membrane proteins and enhanced capability for being uptaken by cells. However, the isolation and recovery of EVs need to be tuned beforehand. In our case, we tested several methods and finally isolated EVs by ultracentrifugation. This agrees with the trend collected in MISEV18 guidelines [56] regarding EVs isolation. Regarding the PCs preparation, our findings contrast with previous results published in the literature, according to which particle size should increase along with the polymer concentration [57], which we also confirmed using PLGA 502 (Supplementary Figure 1, S1). These differences are probably owed to the presence of protamine, a highly positively charged molecule. This positive charge might be the responsible of the bigger sizes observed for the 1 mg/mL concentration, as it may interact with the negative charges of the polymer. Thus, when polymer concentration decreases too much while keeping the protamine concentration constant, the polymer tends to nucleate forming bigger particles. PLGA 502 is alkyl-ester functionalised but includes few unfunctionalized residuals (carboxy-terminated groups). This may lead to much bigger PCs as more polymer molecules would be able to interact Fig. 8. Confocal microscopy images of mesenchymal stem cells, non-treated, treated with PCs or with BMNPs, for 2 h (A). Representative orthogonal sections at additional 3X magnification of the cells treated with PCs (B) and BMNPs (C) showing the intracellular distribution of the particles. Cell cytoplasm is stained in green (Phalloidin-Alexa Fluor 488), cell nuclei in blue (DAPI) and nanoparticles in red (DiD). Arrows indicate observed BMNPs. E. Briffault et al.