Modification of Extracellular Vesicles by Fusion with Liposomes for the

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Modification of Extracellular Vesicles by Fusion with Liposomes for the Design of Personalized Biogenic Drug Delivery Systems Max Piffoux, Amanda K. A. Silva, Claire Wilhelm, Florence Gazeau, and David Tareste ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b02053 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 5, 2018

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Modification of Extracellular Vesicles by Fusion with Liposomes for the Design of Personalized Biogenic Drug Delivery Systems

Max Piffoux,1 Amanda K. A. Silva,1 Claire Wilhelm,1 Florence Gazeau,1 David Tareste2,3,4

1

Université Paris Diderot, Sorbonne Paris Cité, Laboratoire Matière et Systèmes Complexes,

CNRS UMR 7057, F-75013 Paris, France. 2

Université Paris Diderot, Sorbonne Paris Cité, Institut Jacques Monod, CNRS UMR 7592, F-

75013 Paris, France. 3

Université Paris Descartes, Sorbonne Paris Cité, Centre de Psychiatrie et Neurosciences,

INSERM UMR 894, F-75014 Paris, France. 4

Université Paris Descartes, Sorbonne Paris Cité, Membrane Traffic in Health & Disease,

INSERM ERL U950, F-75014 Paris, France.

Corresponding authors: Florence Gazeau ([email protected]) and David Tareste ([email protected])

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Abstract Extracellular vesicles (EVs) are recognized as the nature's own carriers to transport macromolecules throughout the body. Hijacking this endogenous communication system represents an attractive strategy for advanced drug delivery. However, efficient and reproducible loading of EVs with therapeutic or imaging agents still represents a bottleneck for their use as a drug delivery system. Here, we developed a method for modifying cellderived EVs through their fusion with liposomes containing both membrane and soluble cargoes. The fusion of EVs with functionalized liposomes was triggered by polyethylene glycol (PEG) to create smart biosynthetic hybrid vectors. This versatile method proved to be efficient to enrich EVs with exogenous lipophilic or hydrophilic compounds, while preserving their intrinsic content and biological properties. Hybrid EVs improved cellular delivery efficiency of a chemotherapeutic compound by a factor of 3-4, as compared to the free drug or the drug-loaded liposome precursor. On one side, this method allows the bio-camouflage of liposomes by enriching their lipid bilayer and inner compartment with biogenic molecules. On the other side, the proposed fusion strategy enables efficient EV loading, and the pharmaceutical development of EVs with adaptable activity and drug delivery property.

Keywords: extracellular vesicles, liposomes, fusion, polyethylene glycol, drug delivery


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In the search for more efficient, biocompatible and personalized drug delivery systems (DDS), bio-inspired strategies hold promises to overcome the physical and biological barriers that limit the access of DDS to target tissues. The need for personalized vectors adapted to the pathology of each patient motivates the use of cellular components, such as the cell membrane, that can be derived from the patient’s own cells. In this regard, the future generation of bio-camouflaged vectors with long circulation time, enhanced capacity to cross biological barriers, low immunogenicity and natural targeting properties is in its infancy.1, 2, 3 Among technical limitations, the methodologies to engineer cell-derived products and transform them into versatile DDS are still limited.4 To date, synthetic nanocarriers such as liposomes are far beyond biogenic DDS in terms of versatility, production yield, drug loading efficiency, surface modification and standardization. Indeed, liposomes are well-established DDS supported by five decades of research, which led to the clinical approval and marketing authorization of more than a dozen of formulations (Doxil, Myocet, Ambisome, Depocyt, to name a few).5 Coupling liposomes ease of production and modification with the intrinsic functionality of biogenic delivery systems (biocompatibility, targeting and uptake properties) would result in bioengineered DDS merging assets from both synthetic and biological objects. A promising bio-inspired strategy is to harness the communication and delivery system mediated by extracellular vesicles (EVs). Since their first description in plasma as platelet-derived particles with a procoagulant activity.6 EVs were discovered in every biological fluids, regulating communication between cells,7 and tissue homeostasis,8 and accomplishing various roles in coagulation,9,

10

immunity,11 pregnancy,12 infections13 and

cancer progression.14, 15 Mesenchymal stem cells-derived EVs display regenerative properties in various models such as cardiac failures,16, 17 nerve remyelination18 or liver injury.19 EVs can also play an immunomodulatory role, either limiting or promoting immune responses,


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depending on their cellular origin.20 The intrinsic biological activity of EVs thus raised therapeutic opportunities, ranging from regenerative medicine to vaccination either in oncology or infectious disease field.2, 21, 22 EVs are described as membrane-bound compartments containing transmembrane proteins in their lipid bilayer and cell cytosol components – including proteins, mRNA and miRNA23 – in their lumen.24, 25 Recent findings suggest that EVs are enriched in membrane rafts26 and contain specific nucleic acids sequences and proteins at higher concentrations than in the parental cell cytosol.27 First evidences of horizontal information transfer through EVs were described with the transport of mRNA from embryonic stem cells to bone marrowderived mast cells,8,

23

as well as between endothelial cells28 or between cancer cells.7 The

concept of EVs as delivery vectors was launched with the transfer of exogenous GFP mRNA in vitro through transwell co-cultures.23 Recently, exosomes engineered to carry RNA specific to oncogenic KrasG12D, a common mutation in pancreatic cancer, were shown to target oncogenic KRAS and to display enhanced therapeutic efficacy compared to liposomes, due to natural phagocytosis-inhibition factors contained in exosomes.29 Thus, the assets of EVs together with their biological tolerability and natural targeting properties are now regarded as an opportunity to deliver condensed packages of biologically active macromolecules, while protecting them from enzymes circulating in body fluids.30,

31, 32

Nevertheless, a major

challenge is to endow EVs with active exogenous compounds such as imaging agents, therapeutic molecules, nanoparticles or biological agents such as siRNA, proteins, genes, or even viral vectors. Thus far, two major types of loading techniques have been described, based on either modifications of the parental cells before EV generation or modifications of EVs after their production. To modify parental cells, spontaneous loading or transfection techniques have been investigated.33, 34 The most commonly used method for post-production loading of EVs


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is the electroporation approach. It was used to load various compounds into EVs such as siRNA,35, 36, 37, 38 DNA39 and doxorubicin.32 However, electroporation induced the formation of siRNA aggregates that were co-purified with exosomes, thus leading to an overestimation of the loading efficiency.40 Methods involving destruction/reformation of EVs such as sonication, osmotic shock, extrusion41 or freeze/thaw in the presence of a drug to be loaded have also been used, but these approaches may affect EV membrane stability and integrity.34, 42, 43

Achieving reproducible, efficient and quantifiable loading of costly cargoes into EVs,

while preserving EV biological integrity and identity, is thus a critical issue for the pharmaceutical development of EV-based DDS. In this regard, we propose herein to modify EVs by fusing them with liposomes. Membrane fusion is a naturally occurring process allowing the mixing of cellular compartments without leakage or loss of lipid bilayer integrity. Our method relies on polyethylene glycol (PEG)-induced fusion between liposomes of defined composition and cell-derived EVs to design hybrid EVs with tunable composition and properties. From another standpoint, this approach is expected to provide a strategy to translate liposomes, in particular clinically approved ones, into bio-friendly personalized DDS.

Results and Discussion

Polyethylene Glycol Induces Efficient Fusion between Extracellular Vesicles of Various Cellular Origins and Liposomes of Various Compositions Protein-free membrane fusion can be triggered in vitro using chemical agents with the capacity to mediate close-contact and dehydration of lipid bilayer structures.44 Polyethylene glycol (PEG) has been largely employed to mediate cell-cell fusion and to gain insight into the molecular mechanisms of membrane fusion.45 Here, we used PEG to mediate the fusion 5 ACS Paragon Plus Environment

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between EVs and liposomes of defined size and composition. We first sought to determine the experimental conditions allowing optimal PEG-mediated fusion between EVs and liposomes. EVs were isolated from Human Umbilical Vein Endothelial Cells (HUVEC) and liposomes were produced by extrusion. Liposomes were composed of phosphatidylcholine and phosphatidylethanolamine (the main lipids found in natural membranes) in order to avoid important changes in the EV lipid composition after fusion. The fusion process was monitored using a previously described FRET-based lipid mixing assay.46 Briefly, donor liposomes containing FRET pairs of fluorescent NBD and Rhodamine lipids dilute their lipids in the membrane of non-fluorescent acceptor EVs during fusion, which increases the distance between NBD and Rhodamine dyes and thus decreases FRET efficiency. Fusion is therefore measured as the increase of NBD fluorescence over time (Figure 1a). We first investigated the effect of PEG size on the fusion process. EVs were incubated with liposomes (1/1 EV/liposome molar ratio) in the presence of 10% (w/v) PEG of different molecular weights (3000, 6000 and 8000 g/mol). NBD fluorescence after 2 hours was twofold higher with PEG 8000 compared to PEG molecules of lower molecular weights, indicating more efficient fusion with PEG 8000 (Figure S1a). EVs were next incubated with liposomes in the presence of various concentrations of PEG 8000, ranging from 0 to 30% (w/v) (Figure 1a). The fusion extent was clearly dependent on PEG concentration, reaching up to 36% NBD fluorescence after 2 hours in the presence of 30% (v/w) PEG 8000. To give a more quantitative insight on the fusion efficiency, we have converted the percentage of NBD fluorescence into rounds of fusion as previously described (Figure S1b).47 As an example, 10% NBD fluorescence corresponds to 0.42 rounds of fusion per liposome, meaning that if there is one liposome per EV, around 40% of EVs will fuse with a liposome. We also compared the efficiency of fusion using EVs from another cell type, i.e. murine Mesenchymal Stem Cells (MSC), and liposomes of different compositions, containing


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either PEGylated or cationic lipids. Fusion was slightly less efficient with MSC-derived EVs compared to HUVEC-derived EVs (e.g. 27% vs. 36% NBD fluorescence after 2 hours in the presence of 30% (w/v) PEG 8000) but again clearly increased with PEG 8000 concentration (Figures 1b and 1c). PEGylated liposomes reduced the extent of fusion compared to nonPEGylated liposomes (e.g. 12 % vs. 23% NBD fluorescence for MSC-derived EVs after 2 hours in the presence of 20% (w/v) PEG 8000) but still allowed very efficient fusion (Figure 1c). Interestingly, microvesicles (MVs) or exosomes (Exos) further purified from EVs fused with liposomes with the same efficiency as the whole population of EVs, suggesting that there is no sub-population of EVs displaying higher or lower fusion activity (Figure S1d). Finally, the incorporation of positively charged lipids into the liposome membrane increased PEGmediated fusion with HUVEC-derived EVs, most likely because of their electrostatic interactions with the negatively charged EV membrane surface (Figure S1e). We nevertheless decided to avoid the use of such cationic lipids due to their non-physiological nature and their previously reported toxic effect.48 Overall, PEG 8000 proved to be a potent trigger of fusion between liposomes and EVs, MVs or Exos. PEGylated liposomes fused also efficiently with EVs, which will allow surface modification of EVs with PEGylated lipids to increase their circulation time.

Imaging Flow Cytometry Confirms EV-Liposome Interaction at the Single Vesicle Level The transfer of lipids from liposomes to EVs was also confirmed by Imaging flow cytometry (ImageStreamX) which allows high throughput imaging and analysis of single vesicles.49 EVs exhibiting phosphatidylserine on their outer monolayer were labeled with Annexin V-FITC while the liposome membrane contained fluorescent Rhodamine lipids (Figure 2a). After fusion in the presence of PEG, the population of double positive objects featuring hybrid EVs contained most of the Rhodamine lipid fraction (69%) (Figure 2c).


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Moreover, the Rhodamine fluorescence intensity of these double positive objects was much higher than in the absence of PEG (Figure 2d). A size analysis of the vesicle population (detected through their fluorescence) was performed based on their bright field image. Although ImageStreamX only detects the largest population of vesicles, it showed that PEGmediated fusion of EVs with liposomes produced a higher percentage of large particles in the range 0.5-1 µm (Figure 2e).

PEG Mediates Full Fusion between EVs and Liposomes Membrane fusion usually includes an intermediate hemifused state, where the outer lipid monolayers have mixed while the inner lipid monolayers remain separated. Hemifusion then resolves into full fusion, i.e. the merging of inner lipid monolayers and the transfer of soluble contents initially enclosed by the two membranes. In some instances, hemifusion does not transit to full fusion and constitutes a dead-end state preventing content mixing.50, 51, 52 To assess whether lipid mixing measured between EVs and liposomes was due to hemifusion and/or full fusion, we incubated fluorescent liposomes with sodium dithionite which selectively quenches the NBD fluorescence signal in the outer monolayer of liposomes. Any fluorescence dequenching measured by the FRET-based lipid mixing assay with dithionitetreated liposomes would therefore be the result of inner monolayer fusion, and the signature of full fusion between liposomes. The fusion curves obtained between EVs and liposomes treated or not with sodium dithionite were very similar (Figure S1f), indicating that EVs and liposomes essentially undergo full fusion. We also used cryo-Transmission Electron Microscopy (cryo-TEM) to investigate the fusion between EVs and liposomes. Liposomes contained biotinylated lipids, allowing their detection with streptavidin gold nanoparticles, whereas EVs were identified using Annexin V gold nanoparticles, which bind to phosphatidylserine lipids present in the EV membrane.


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Following incubation with PEG, various double stained vesicles (hybrid EVs) were identified, confirming that full fusion had occurred between EVs and liposomes (Figure S2). One should note that homotypic fusion events between liposomes or between EVs may also take place in addition to the reported heterotypic EV-liposome fusion. Cryo-TEM analysis in fact showed an increase in size of the vesicles obtained following homotypic or heterotypic PEG-mediated fusion reactions involving EVs and liposomes (Figures S3a-d). The extent of homotypic fusion between liposomes was quantified with the FRET-based lipid mixing assay as a function of PEG concentration (Figure S3e). We also checked whether PEG-induced EV/liposome aggregation was reversible after PEG dilution by comparing the size distribution of hybrid EVs measured by cryo-TEM (where vesicles were analyzed one by one) with that measured by NTA (which cannot distinguish single large objects from aggregates). There was only a slight increase of the average vesicle size measured by NTA versus cryo-TEM (Figure S3c, dashed red curve versus grey curve), which we attributed to the limited ability of NTA to detect small biological objects, confirming that PEG dilution induced cluster disaggregation. FRET-based experiments and cryo-TEM analysis thus converge to confirm that PEGmediated fusion between EVs and liposomes can be used to generate semi-synthetic bioengineered EVs.

Control and Optimization of Hybrid EVs Loading We next investigated ways to control the fraction of total hybrid EV surface containing material coming from liposomes. EVs fusing with an excess of liposomes are expected to contain more lipids originating from liposomes. So we first checked the effect of varying the EV/liposome molar ratio using the FRET-based lipid mixing assay (Figure 3a). An EV/liposome ratio of 9/1 led to the fusion of much more liposomes with EVs than the 1/1 and 1/9 ratios (20% NBD fluorescence / 0.85 round of fusion vs. 5.5% NBD fluorescence /


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0.25 rounds of fusion and 3.5% NBD fluorescence / 0.14 round of fusion, respectively). However, it is important to note that the 1/9 EV/liposome ratio, even if it leads to a lower FRET efficiency, creates a population of hybrid EVs whose total surface is composed on average of 56% of lipids coming from liposomes (1.26 liposomes fusing per EV), compared to 19% at a 1/1 ratio (0.25 liposomes fusing per EV) and 9% at a 9/1 ratio (0.09 liposomes fusing per EV), assuming a similar diameter for both EVs and liposomes. Detection of fusion in the FRET-based lipid mixing assay is limited at low EV/liposome molar ratios, mostly because the fluorescence signal is dominated by the contribution from unfused liposomes. To overcome this issue, we designed another way to investigate the outcome of fusion quantitatively. We separated EVs from liposomes using a density gradient, and we quantified the fluorescence in the different zones of the gradient to evaluate the transfer of fluorescent molecules from liposomes to EVs. This approach presents the additional advantage of separating hybrid EVs from the unfused liposomes, which are the dominant species at low EV/liposome ratios. As a proof of concept, we first compared the fluorescence distribution of a solution of liposomes containing Rhodamine lipids (Rho-PE) with that of PKH26-labeled EVs in a discontinuous density gradient of Nycodenz constituted by 3 layers, as illustrated in Figure 3b. The fluorescence of liposomes with a density of about 153 was retrieved in the first fractions within the 0-1.5 mL zone (94 % in the 0-0.75 mL zone). By contrast, PKH26-labeled EVs distributed in the 0.5-3 mL zone, with a peak between the fractions 1.5 and 2 mL. This is consistent with the previously reported higher density of EVs (1.15) compared to liposomes.54 According to these distributions, we have therefore defined three zones: the first one (0-0.75 mL) that essentially contains liposomes, the second one (1-3 mL) to retrieve EVs, and the third one (3.25-5 mL) defined as the leaking zone, containing soluble compounds with higher densities.


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Using the EV-liposome density gradient separation method described above, we verified the efficiency of fluorescent lipid transfer from liposomes to non-fluorescent MSCderived EVs by their fusion in the presence of PEG, using an EV/liposome ratio of 1/9, 1/1, or 9/1 (Figures 3c-e). No fluorescence was detected in the leaking zone (3.25-5 mL) for all samples, which is in agreement with the fact that fluorescent lipids stay associated with the liposome or EV membrane. Fluorescence of the fused samples was located within a higher density zone than that of the unfused samples (without PEG), or the liposomes alone (with or without PEG) (Figures 3b-e), and interestingly this fluorescence varied depending on the EV/liposome ratio. A “liposome-like” hybrid EVs population (less dense and closer to the liposome zone) was obtained at a 1/9 ratio (Figure 3c), whereas an “EV-like” hybrid EVs population (denser and clearly in the EV zone) was observed when using a 9/1 ratio (Figure 3e). The amount of fluorescence in the fused samples (in the presence of PEG) which was above the fluorescence level of the unfused samples (without PEG) was quantified and considered as the amount of fused liposomes; we obtained 62%, 49% and 26% for 9/1, 1/1 and 1/9 EV/liposome ratios, respectively. The percentage of total hybrid EV surface composed of lipids coming from liposomes was then calculated in each condition using equation 3, leading to 6% at the 9/1 ratio (0.07 liposome fusing per EV), 33% at the 1/1 ratio (0.49 liposome fusing per EV), and 69% at the 1/9 ratio (2.34 liposomes fusing per EV). These results are consistent with those obtained with the FRET-based lipid mixing assay, thus validating the quantitative analysis of fusion by two independent methods. The density gradient approach also confirmed fusion of EVs with PEGylated liposomes (Figure S4a-b), as well as fusion of MVs and Exos subpopulations with liposomes (Figure S4c). The density gradient separation method is therefore an efficient way to isolate hybrid EVs from unfused liposomes and tune their composition for drug delivery application. The


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EV/liposome ratio turned out to be an important control parameter allowing the modulation of membrane composition and thus surface properties of hybrid EVs.

EV can be Loaded with Lipophilic and Hydrophilic Molecules Without Losing their Intrinsic Cargo Fusion is a process that can be leaky and thus lead to the loss of internal cargoes in the surrounding medium.52, 55 We took advantage of our fusion test using flotation in a density gradient to investigate the leakiness of the fusion process induced by different concentrations of PEG. We compared the fusion of MSC-derived EVs with liposomes loaded either with a lipophilic fluorescent probe (Rho-PE) that should not leak, or a hydrophilic Rhodamine probe (Rho) encapsulated within the liposome lumen. A 1/1 molar ratio was chosen in order to limit the concentration of unfused liposomes or unfused EVs. In the case of the lipidic probe, as expected, no fluorescence was found in the leaking zone (Figure 4a). Step-wise increase of PEG concentration (0, 10, 20 and 30%) led respectively to the transfer of 6%, 35%, 75% and 93% of the Rho-PE fluorescence from the liposome zone to the EV zone. In contrast, the hydrophilic probe was transferred from the liposome zone to distribute between the EV zone and the leaking zone (Figure 4b). Fusion in the presence of 0, 10, 20 and 30% PEG led respectively to the transfer of 8%, 27%, 39% and 43% of the Rho fluorescence from the liposome zone to the EV zone. Rho leakage also tended to increase with PEG concentration (3%, 9%, 24% and 43% of the total dose in the presence of 0, 10, 20 and 30% PEG, respectively). To investigate whether EV content was preserved during fusion with liposomes, we used EVs generated from MDCK cells expressing cytosolic GFP. We first confirmed that such EVs were encapsulating GFP, and we tested GFP leaking during PEG-mediated fusion with non-fluorescent liposomes (Figure 4c). Interestingly, GFP was mostly found in the EV


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zone of the gradient, whether EVs had previously fused or not with liposomes, whereas purified soluble GFP used as a positive control was found in the leaking zone. This suggests that there is no significant leakage of compounds residing in the inner volume of EVs during PEG-mediated fusion with liposomes, at least in the case of molecules with similar size as GFP. A second series of experiments was conducted to demonstrate simultaneously the transfer of molecules from liposomes to EVs and the conservation of EV cargo during fusion. Magnetic nanoparticles-loaded EVs were produced from cells having previously internalized 9 nm magnetic iron oxide nanoparticles as described before.49,

56, 57

The magnetic

nanoparticles-loaded magnetic EVs were then fused with liposomes labeled with Rho-PE lipids or containing hydrophilic Rho in their lumen. The fusion mix was subjected to a high gradient magnetic field (150 T/m) in order to separate magnetic EVs or magnetic hybrid EVs from the mix by pelleting them on a magnetic tip (Figure S5a). The magnetic pellet was then imaged to quantify the amount of fluorescent material transferred from liposomes to magnetic EVs.58 Magnetic EVs were clearly loaded with fluorescent compounds, either Rho-PE or hydrophilic Rho, as a result of their fusion with liposomes (Figures S5b and S5c). It thus confirms that EVs have acquired lipophilic or hydrophilic molecules from donor liposomes while keeping their internal cargo (herein magnetic nanoparticles). Taken together, this series of experiments shows that PEG-mediated fusion of EVs with liposomes preserves the intrinsic cargo of EVs, thus likely maintaining their physiological properties.

Interaction of EVs with Macrophages can be Tuned by their Fusion with PEGylated Liposomes


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The advantage of using EVs for drug delivery is to benefit from their intrinsic ability to interact with cells due to their specific membrane protein and lipid composition. However, the half-life in the blood stream of different EV types was estimated to be between 2 and 60 minutes following intravenous injection, due to their capture by the reticulo-endothelial system.56,

59, 60

PEGylated liposomes were shown to limit opsonization and to have less

interaction with cells, leading to a prolonged half-life in the blood stream.61 The insertion of PEGylated-lipids in platelet and neuronal cell-derived EVs by a post-insertion method based on mixing with micelles was also shown to prolong their circulation time.59 Thus, we thought of fusing EVs with PEGylated liposomes in order to modify their membrane properties and thus lower their interaction with macrophages. Hybrid EVs were generated by PEG-mediated fusion between MSC-derived EVs and PEGylated or non-PEGylated liposomes functionalized with the DiR membrane probe. Liposomes or hybrid EVs were then incubated with THP1-derived macrophages to monitor their cellular uptake (Figures 5a and 5b). As expected, PEGylated hybrid EVs were less internalized compared to non-PEGylated ones (about 8-fold decrease of DiR fluorescence) and the liposomes followed the same trend (about 4-fold decrease of DiR fluorescence for PEGylated versus non-PEGylated liposomes) with an overall lower uptake than hybrid EVs. The obtained data therefore support that cellular uptake of EVs can be modulated by their fusion with PEG-functionalized liposomes.

Photosensitizer Delivery in Cancer Cells is Enhanced by Hybrid EVs in Comparison to Commercial Therapeutic Liposomes We next investigated whether it was possible to deliver drugs using hybrid EVs. As a proof of concept, we chose mTHPC, a fluorescent clinically-approved anti-tumor photosensitizer, also available encapsulated in a liposomal form (Foslip) used for advanced


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photodynamic therapy (PDT). mTHPC is activated only in the presence of light, allowing the production of radical oxygen species that lead to cell death. It represents a convenient model to (i) test the production of therapeutic hybrid EVs from their fusion with commercial liposomes; (ii) compare the transfer of the drug to cancer cells by detecting its fluorescence; (iii) test if hybrid EVs keep their physiological properties inherited from MSC parental cells before activation by the laser; and (iv) investigate the PDT effect while activated with a laser. To generate mTHPC-containing hybrid EVs, Foslip liposomes were fused with EVs in the presence of PEG. The drug encapsulation performance of the obtained hybrid EVs was compared to that of EVs generated by cells pre-loaded with mTHPC as reported previously49. The drug encapsulation efficiency was calculated as the percentage of the total drug added to the EV parental cells or contained into the precursor liposomes, and found to be close to 90% for hybrid EVs compared to only 3% for EVs generated by mTHPC-loaded parental cells (Figure S6a). This clearly shows the advantage of the fusion approach compared to the spontaneous loading strategy of parental cells in terms of drug encapsulation yield. The density gradient method confirmed the transfer of mTHPC fluorescence from the liposome zone to the EV zone, showing that 65% of the total hybrid EVs volume was made of Foslip mTHPC content (Figure 6a). We next compared the delivery of mTHPC drug to mouse colon carcinoma CT26 cells in 2D culture using hybrid EVs, liposomes (Foslip) and free mTHPC at three different mTHPC doses (2.5, 0.5 and 0.1 µM). After 4 hours of incubation, the mTHPC fluorescence in cells increased with the amount of incubated drug, and was higher with hybrid EVs compared to Foslip or free mTHPC (transfer of 60-70% of the incubated dose with hybrid EVs versus 10-20% with Foslip or free mTHPC) (Figure 6b). This demonstrates that hybrid EVs are bestperforming carriers for mTHPC delivery in CT26 cancer cells in comparison to liposomes. Internalization of hybrid EVs labeled with Rho-PE lipids was also tested on HUVEC and


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compared with internalization by CT26 cells (Figures S7a and S7b), confirming that hybrid EVs internalization was possible by many cell types. Considering the expected cell proliferative activity of MSC-derived EVs,62 we next assessed the metabolic activity of CT26 cells exposed, in the absence of irradiation, to drugfree MSC-derived EVs and mTHPC-loaded hybrid EVs in comparison to Foslip or free mTHPC. Drug-free MSC-derived EVs had a cell proliferative effect that depended on their concentration: the metabolic activity raised up to 137% of the control value for high EVs concentration (preparation of drug-free EVs at high, medium and low concentrations corresponded in terms of EV numbers to hybrid EVs containing 2.5, 0.5 and 0.1 µM of mTHPC, respectively) (Figure 6c). This proliferative effect was conserved for hybrid EVs resulting from PEG-mediated fusion of EVs with Foslip. The metabolic activity notably raised up to 133% of the control value for hybrid EVs containing 2.5 µM of mTHPC, similarly to drug-free EVs. On the contrary, Foslip displayed a slight toxicity (down to 11% decrease compared to the control value). Thus, the intrinsic proliferative effect of MSC-derived EVs was preserved after their fusion with liposomes. In order to compare the cytotoxic activity of drug-loaded hybrid EVs and liposomes, we next investigated their light-induced PDT effect with an interval between cell exposure and light irradiation of 24 hours. The metabolic activity of cells was assessed 24 hours after 650 nm laser irradiation at 10 J/cm² (Figure 6c). After irradiation, every conditions tested displayed nearly 100% toxicity, except the lowest dose (0.1 µM) of free mTHPC (43%). In complement, fluorescence microscopy was used for the 0.5 µM dose to assess mTHPC delivery and cell morphology, as well as cell death by apoptosis (Annexin V) or necrosis (Propidium iodide) (Figure 6d). We observed that mTHPC fluorescence was 3-4 fold higher when using hybrid EVs compared to Foslip or free mTHPC. Once activated by the laser, mTHPC induced cell morphological changes in all cases. Interestingly, free mTHPC induced


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a much higher level of intracellular Annexin V and Propidium iodide compared to the other conditions. The drug delivery performance of EVs, Foslip and hybrid EVs was also assessed in a 3D model of colon carcinoma using CT26 cells. Tumor spheroids were incubated at equivalent mTHPC concentrations with free mTHPC, Foslip, hybrid EVs and EVs generated from drug-loaded cells (denoted as mTHPC EVs). Spheroids were then analyzed in terms of drug penetration depth by confocal microscopy and total drug uptake by spectrophotometry. mTHPC EVs, Foslip and hybrid EVs displayed an equivalent penetration potential (Figures S6b-d), which was lower than that of the free drug (most likely because of its much smaller size). Laser-irradiated spheroids previously incubated with free mTHPC, mTHPC EVs, Foslip or hybrid EVs did not adhere or proliferate in cell culture wells (Figure S6b). This indicated photodynamic-induced cell death in contrast to laser irradiated control spheroids, which remained viable, flattening and proliferating (Figure S6b). Importantly, drug uptake into the whole tumor spheroid was better when using hybrid EVs compared to the free drug, mTHPC EVs or Foslip (Figure S6e). It is worth noting that, although the ligands of the EV membrane were diluted after fusion with liposomes, it did not affect their interaction with tumor cell receptors since hybrid EVs internalization was efficient (Figure S6e). Together, these results provide evidence that (i) generating hybrid EVs by fusion with liposomes allows highly efficient drug loading, and (ii) hybrid EVs display a drug delivery performance that is slightly better than EVs. Besides, hybrid EVs display a unique biological signature making them better bio-mimetic carriers than liposomes.

Conclusion In this study, we have shown that PEG can induce the fusion between EVs of different cellular origins and liposomes of various biocompatible compositions. Using a FRET-based


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lipid mixing assay, we have identified critical parameters to optimize fusion efficiency, such as the liposome-to-EV ratio, the liposome bilayer composition, and the PEG molecule size and concentration. We have demonstrated that PEG-mediated fusion was complete, without any detectable hemifusion, and that the obtained hybrid EVs could be visualized and characterized by cryo-TEM and imaging flow cytometry. Importantly, hybrid EVs could be efficiently separated from unfused liposomes in a Nycodenz density gradient and their lipophilic and hydrophilic contents coming from liposomes could be accurately quantified. In optimized conditions, more than 60% of membrane and soluble contents could be transferred from liposomes to EVs, demonstrating efficient functionalization by the fusion process accompanied by limited leakage of small molecules from the inner core of liposomes. Similarly, leakage assay on GFP-loaded EVs and magnetic assay on nanoparticle-loaded EVs confirmed that EVs kept their cargo upon fusion. The composition of hybrid EVs could be designed by varying the liposome-to-EV ratio and the composition of precursor liposomes. The interest of the method was demonstrated by the fusion of MSC-derived EVs with PEGylated liposomes, enabling lower internalization by macrophages in situ. In addition, MSC-derived hybrid EVs generated upon fusion with commercial therapeutic liposomes displayed a 3-4 fold increased ability to deliver anti-tumor drugs to tumor cells compared to the precursor liposomes. Interestingly, even after fusion, MSC-derived hybrid EVs kept their proliferative effect, indicating that their intrinsic properties were maintained by the fusion process. The PEG-mediated fusion method therefore enables the engineering of hybrid EVs with tunable properties, while preserving their inner cargo and biological activity. This method can be applied to load EVs of any cellular origin with potentially any compound associated to synthetic liposomes: from hydrophilic compounds encapsulated in their inner core (siRNA, miRNA, chemotherapeutic agents, proteins, nanoparticles, imaging contrast


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agents, etc.) to lipophilic compounds residing at their bilayer surface (membrane proteins, targeting agents, PEGylated lipids, etc.). We anticipate for example that PEGylation of EVs will increase their circulation time, resulting in an improved targeting and enhancedpermeability-and-retention (EPR) effect, and that their intrinsic biocompatibility will be an advantage to avoid vector-mediated toxicity. This fusion method also enables the addition of biogenic functionalities to liposomes such as enhanced cellular uptake. This is a promising perspective for upgrading the properties of several liposomal formulations, in particular those that have already received market authorization5, as it will allow the design of personalized cell-friendly DDS. This study therefore erases the frontiers between synthetic liposomes and endogenous EVs, combining the assets of both towards the generation of DDS at the crossroad of bioengineered liposomes and semi-synthetic EVs.

Materials and Methods Chemicals 1,2-dioleoyl-sn-glycero-3-phosphocholine

(DOPC),

1-palmitoyl-2-oleoyl-sn-glycero-3-

phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2dioleoyl-sn-glycero-3-phospho-L-serine

(DOPS),

1,2-dioleoyl-sn-glycero-3-

ethylphosphocholine (EPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N-(7-nitro-2-1,3benzoxadiazol-4-yl)

(NBD-PS),

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-

(lissamine rhodamine B sulfonyl) (Rho-PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamineN-(cap

biotinyl)

(Biotin-PE)

and

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-

[methoxy(polyethylene glycol)-2000] (PEG 2000-PE) were purchased from Avanti Polar lipids as chloroform solutions. n-Dodecyl β-D-maltoside (DDM, ULTROL grade) and sodium dithionite (grade for analysis) were purchased from Merck. PEG 8000 (BioUltra grade), Sulforhodamine B (laser grade), Streptavidin-Gold from Streptomyces avidinii (14 nm


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particle size), DiR and PKH26 were purchased from Molecular Probes/Sigma-Aldrich. Nycodenz was purchased from Proteogenix. Citrate-coated magnetic iron oxide (γFe2O3) nanoparticles (NPs) of 9 nm diameter were produced by alkaline co-precipitation of FeCl2 and FeCl3 salts as previously described.63 Their molarity was measured by atomic emission spectroscopy. Annexin5 gold NPs of 11 nm diameter were synthesized as previously described.64 GFP carrying an N-terminal His6-tag was expressed in the BL21(DE3) Escherichia coli bacterial strain and purified by nickel affinity chromatography as described previously.65 mTHPC [3,3’,3”,3”’-(2,3-dihydroporphyrin-5,10,15,20-tetrayl)tetraphenol] and mTHPC liposomal formulation (Foslip®) were provided by Biolitec research GmbH (Jena, Germany).

Preparation of Liposomes Liposomes were prepared using the extrusion method. 1 µmol of the appropriate lipid mixture (in chloroform) was dried in a glass tube for 10 min under a gentle stream of argon, and evaporation was continued for 2 hours under vacuum to remove all trace of organic solvent. The dried lipid film was hydrated by adding 1 mL of phosphate-buffered saline (PBS, including 1-2 µM of cargoes for cargo-loaded liposomes) to obtain a final lipid concentration of 1 mM, and the solution was vortexed vigorously for 1 hour at room temperature. Multilamellar vesicles were frozen in liquid nitrogen (~30 sec) and then thawed in a 40°C water bath (~5 min); the sequence was repeated 10 times. Liposomes were homogenized by extrusion through a 0.1 µm polycarbonate membrane using the Avanti Mini-Extruder; at least 19 passages were performed. Cargo-loaded liposomes were purified from non-encapsulated cargoes by flotation in a Nycodenz density gradient (as described below).

Cell Culture and Isolation of EVs


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Human umbilical vein endothelial cells (HUVEC), murine mesenchymal stem cell line C3H (MSC) and free GFP expressing Mardin-Darby Canine Kidney (MDCK) were cultured at 37°C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL of penicillin and streptomycin. To trigger EV release, cells were cultured in serum-free DMEM for 3 days. EVs were isolated from the conditioned culture medium with a differential ultracentrifugation method as previously described.66 First, cell debris were eliminated by 2,000 g centrifugation for 10 min. From the supernatant, microvesicles (MVs) were isolated after a 10,000 g ultracentrifugation step for 20 min and exosomes (Exos) were further isolated after a final 100,000 g step for 1 hour. Alternatively, the total population of extracellular vesicles (EVs, containing both MVs and Exos) were isolated with a simple 100,000 g step for 1 hour without the 10,000 g step. Vesicles were then resuspended in PBS. To produce magnetic EVs loaded with iron oxide nanoparticles, HUVEC cells were first incubated with 9 nm iron oxide nanoparticles (5 mM iron in serum-free RPMI medium supplemented with 5 mM sodium citrate) during 2 hours at 37 °C, as previously described.56 To generate mTHPC EVs from drug-loaded cells, HUVEC cells were incubated with 10 µM mTHPC drug for 12 hours at 37°C in DMEM supplemented with 10% fetal bovine serum as described in 49. Incubation was followed by 2 washing steps in serum-free RPMI medium and 2 hours chase in serum-supplemented DMEM medium in order to allow nanoparticle internalization by cells. The cells were further cultured in serum-free culture medium to trigger EV release as described above.

FRET-based Lipid-Mixing Assay Liposomes and EVs were mixed in Nunc F96 MicroWell White plates maintained at 40°C in the SpectraMax M5 plate reader (Molecular Devices). The total volume of the reaction


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mixtures was 40-200 µL. EVs and liposomes were mixed at a liposome/EV ratio of 1/1 (2 x 1010 objects), 1/9 (2 x 1010 objects) or 9/1 (2 x 1011 objects) in PBS. Objects were counted with Nanoparticle Tracking Analysis, as described below. PEG was added at a final concentration of 5-30% (w/v). Lipid mixing during EV-liposome fusion was monitored by Fluorescence Resonance Energy Transfer (FRET) as described previously.46 Briefly, donor fluorescent liposomes were prepared containing equal molar ratio (1.5%) of the fluorescent lipids NBD-PS and Rho-PE. Upon excitation of NBD, energy is transferred to Rhodamine in a FRET process, which is strongly dependent on the distance between the two fluorophores. Therefore, dilution of the NBD-PS and Rho-PE lipids within the membrane of acceptor non fluorescent EVs (when donor liposomes and acceptor EVs fuse) results in an increase of NBD fluorescence. Lipid mixing was monitored by following the NBD fluorescence intensity (excitation at 460 nm, emission at 535 nm, cut-off at 530 nm) over time at 40°C. After 2 hours, the fusion reaction was stopped by the addition of 10 µL of DDM at 2.5% (w/v) to solubilize all liposomes and thus measure the NBD fluorescence intensity at infinite dilution: Max (NBD). Fusion curves were normalized using the following equation: NBD Fluorescence Increase (%) = [NBD – Min(NBD)] / [Max(NBD) – Min(NBD)]

(eq 1),

where Min (NBD) is the lowest NBD fluorescence value from all time points. The NBD fluorescence increase was related to the average number of fusion events per donor liposome (rounds of fusion) as previously described

47

and confirmed in our setup (Figure

S1b): Rounds of fusion = (0.49666*exp(0.036031*X)) – (0.50597*exp( – 0.053946*X))

(eq

2), where X is the percentage of NBD fluorescence increase at any time point of the kinetic measurement. The same method was applied to assess homotypic fusion between donor


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liposomes containing the FRET pair of fluorescent lipids NBD-PS and Rho-PE and unlabeled acceptor liposomes.

Dithionite Assay to Probe for Hemifusion vs. Full Fusion To assess the possibility of hemifusion between liposomes and EVs, fluorescent donor liposomes (0.75 mM lipids) were preincubated with 5 mM sodium dithionite for 10 min at 37°C and then transferred to 4°C before mixing with acceptor EVs. The efficiency of NBD reduction to non-fluorescent N-(7-amino-2,1,3-benzoxadiazole- 4-yl) was estimated to be 5060% of the total NBD-PS content. In addition, dithionite was shown to completely loose its activity after 10 min at 37°C, ensuring that NBD-PS from the inner monolayers are not quenched following DDM addition.

PKH26 Labeling of EVs EVs were incubated with 1 µM of fluorescent lipophilic tracer PKH26 with the provided buffer at room temperature prior to density gradient ultracentrifugation (see below). Fluorescence was quantified using the SpectraMax M5 plate reader (excitation at 560 nm, emission at 595 nm, cut-off at 590 nm).

Purification of Hybrid EVs by Density Gradient Ultracentrifugation Hybrid EVs were generated by incubating EVs and liposomes for 2 hours at 40°C in a Thermomixer (Eppendorf) using the same experimental conditions as in the FRET-based lipid-mixing assay, and purified in a three-step density gradient consisting of 0%, 15% and 40% (w/v) Nycodenz solutions in PBS. The sample to purify was mixed with 80% Nycodenz solution in a 1/1 (v/v) ratio, to obtain a 40% mixed solution. 3 mL of this 40% mixture, 1.5 mL of 15% Nycodenz in PBS, and 0.5 mL of pure PBS solutions were put on top of each


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other in a 6 mL open top Ultra-clear centrifuge tube (Beckman Coulter) and centrifuged in a SW 55 Ti rotor (Beckman Coulter) at 246,000 g for 4 hours at 4°C. Fractions of 0.25 mL were collected from the top of the gradient, solubilized with DDM and the fluorescence was quantified for each of the fractions using the SpectraMax M5 plate reader (excitation at 560 nm, emission at 595 nm, cut-off at 590 nm for Rhodamine; excitation at 395 nm, emission at 504 nm, cut-off at 495 nm for GFP).

Characterization of Magnetic Hybrid EVs To investigate the magnetic content (i.e. magnetic nanoparticles loading) of magnetic hybrid EVs, 30 µL of hybrid EVs samples were inserted in a glass slide/coverslip chamber featuring an integrated nickel microrod.67,

68

When the microrod was magnetized by a permanent

magnet, the local magnetic field generated at its extremity attracted magnetic objects toward the micromagnet, covering the tip. The nickel microrod was submitted to a 150 mT uniform magnetic field from a rectangular magnet positioned aside. The chambers were observed by means of an Olympus JX81/BX61 Device/Yokogawa CSU Device spinning disk microscope (Andor Technology plc, Belfast, Northern Ireland) equipped with a 60X Plan-ApoN oil objective lens (60X/1.42 oil, Olympus). The excitation wavelength for Rhodamine was 405 nm and a 685 nm filter was used for collecting fluorescence emission. Image J software was used to follow fluorescence on the tip by selecting the tip zone on the bright field picture, and quantifying the fluorescence on the corresponding zone of the fluorescence picture. Each sample was analysed with 3-4 tips, and the mean value was calculated.

Nanoparticle Tracking Analysis EV and liposome size distribution and concentration were determined with Nanoparticle Tracking Analysis (NTA) using a Nanosight LM10-HS (NanoSight, UK) with a 532 nm laser.


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Before measurements, EVs or liposomes were diluted to an appropriate concentration (from 3 x 108 to 2 x 109 particles/mL) with sterile PBS (confirmed to be particle-free). For each sample, 10 movies of 12 sec were recorded using camera level 16. Fluorescent EVs among all EVs were quantified using a filter to eliminate the light from non-fluorescent objects. Data was analyzed with NTA Analytical Software suite. For counting Foslip liposomes, one movie of 60 sec was recorded using camera level 16. It has to be taken into account that the number of EVs seen by NTA is highly depending on the purity of the sample. In our case, EVs were produced in serum-free culture medium, therefore limiting protein aggregates contamination. With this EV generation method, the purity ratio in particle/µg of proteins is 2 x 1010 particles/µg, corresponding to a pure sample.69

Calculation of the Total Surface and Volume of Hybrid EVs coming from Liposomes The fraction of total hybrid EV surface coming from liposomes’ membrane is calculated using the following formula: FSLipo = (NLipo x [percentage of fused liposomes] x SLipo) / (NLipo x [percentage of fused liposomes] x SLipo + NEVs x SEVs)

(eq 3)

The fraction of total hybrid EV volume coming from liposomes’ lumen is calculated using the following formula: FVLipo = (NLipo x [percentage of fused liposomes] x [% of content transfer] VLipo) / (NLipo x [percentage of fused liposomes] x [% of content transfer] x VLipo + NEVs x VEVs)

(eq 4)

NLipo is the number of liposomes, NEVs is the number of EVs, SLipo and VLipo are respectively the surface and the volume of a liposome (radius ~ 50 nm, measured by NTA), SEVs and VEVs are respectively the surface and the volume of EVs (radius ~ 50 nm, measured by cryo TEM). The percentage of fused liposomes is either determined by the fusion assay calibration curve


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(eq 2) or by flotation in the Nycodenz density gradient. Percentage of leaking is determined by flotation in the Nycodenz density gradient.

Cryo-Transmission Electron Microscopy (cryo-TEM) To visualize the size and shape distribution of HUVEC-derived EVs, 1 µL of 12 nm Annexin V gold NP solution at 1–3 x 1016 NPs/L and 1 µL of 100 mM CaCl2 solution were mixed with 8 µL of the sample of interest, incubated for 15 min at room temperature, and then processed for cryo-TEM as previously described.64 For hybrid EVs made from the fusion with biotinylated liposomes in the presence of 10% (w/v) PEG 8000, the fusion mix was diluted 10 times in 10 mM HEPES, 140 mM NaCl and 7 µL of the mix were added to 1 µL of Annexin V gold NP solution (1–3 x 1016 NPs/L), 1 µL of 100 mM CaCl2 solution and 1 µL of 14 nm Streptavidin gold NPs from Streptomyces avidinii (Sigma), incubated for 15 min at room temperature, and then processed for cryo-TEM. For this specific sample, it has to be noted that EVs sometimes displayed an aggregated phenotype, forming clumps of EVs. These clumps are probably due to the fact that the observation was made in the presence of 1% (w/v) PEG 8000, and confirmed that PEG induced liposomes and EVs aggregation prior to fusion. Briefly, 4 µL sample aliquot was deposited onto an EM grid coated with a perforated carbon film (Ted Pella, Redding, CA, USA), the excess liquid was blotted off with a filter paper, and the grid was then quickly plunged into liquid ethane using a Leica EMCPC cryo-chamber. EM grids were stored in cryo-boxes under liquid nitrogen until use, then mounted in a Gatan 626 cryo-holder and transferred in a Tecnai F20 microscope operated at 200 kV. Images were recorded with an USC1000-SSCCD Gatan camera. Lamellarity was quantified by counting the number of bilayers of each object with the Fiji software. Size was quantified with Fiji, by deducing EVs radius from their projected area.


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Flow Cytometry EV images were acquired with the ImageStreamX multispectral imaging flow cytometer (Amnis Corporation, Seattle) as previously described.49,

70

About 10,000 events per sample

were collected at 60X magnification. Liposomes were labeled with Rho-PE, and EVs were stained with Annexin V-FITC assay kit (Annexin-V-Fluos kit, Roche) according to the manufacturer’s instructions. The excitation/emission wavelengths were 488 nm / [470–560] nm for Rhodamine and 488 nm / [470–560] nm for FITC. Dark field images were acquired using a 785 nm laser. Single stained controls were used for compensating bleed-through of fluorescence between channels. Non-fluorescent speed beads of 1 µm (calibration SpeedBeads, Amnis, Seattle) were continuously imaged during the operation of the ImageStreamX. Multifluo 0,5 µm calibration beads were used for size measurement. Images were analyzed using IDEAS® image-analysis software (Amnis, Seattle). Regions of interest were determined using Annexin V-labeled EVs and Rhodamine-labeled liposomes (Figures S8a and S8b).

Internalization Assay into Macrophages THP-1 monocyte cell line (ATCC) were cultured in suspension at 37°C and 5% CO2 in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and streptomycin. The THP-1 cells were maintained between 105 and 106 cells/mL. In the exponential phase of growth, cells were seeded onto sterile homemade serum coated 1.5 glass cover slips of 1 cm² at 100,000 cells per well for 48 hours in 5 ng/ml phorbol myristate acetate (PMA) to induce differentiation into macrophages and adherence to the plate. Macrophages were then incubated for 4 hours with DiR-labeled hybrid EVs or liposomes containing the same dose of DiR (1 mol %, quantified by spectroscopy at excitation/emission 750/780 nm using the EnSpire plate reader from Perkin Elmer), and then washed with PBS


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before observation of the transferred fluorescence. Observation was done with Olympus JX81/BX61 Device/Yokogawa CSU Device spinning disk microscope (Andor Technology plc, Belfast, Northern Ireland). For each sample, fluorescence was quantified using the mean value of n > 3 pictures.

In vitro Photodynamic Therapy For experiments in 2D culture, human colon carcinoma CT26 cells were seeded in 24-well plates and incubated overnight at 37°C in a humidified 5% CO2 atmosphere in DMEM medium supplemented with 10% FBS. After PBS rinsing, cells were incubated for 4 hours in the dark at 37°C in a humidified 5% CO2 atmosphere with 400 µL of free mTHPC, mTHPCloaded commercial liposomes (Foslip) or mTHPC-loaded hybrid EVs at equivalent mTHPC concentrations in DMEM media without serum. For experiments in 3D culture, 500 human colon carcinoma CT26 cells were resuspended in 30 µL DMEM medium supplemented with 10% FBS on the inverted lid of a petri dish containing PBS for 3 days to allow the formation of spheroids. Spheroids were then transferred to petri dishes and incubated for 24 hours in the dark at 37°C in a humidified 5% CO2 atmosphere with free mTHPC, mTHPC-loaded commercial liposomes (Foslip), EVs produced by parental mTHPC-loaded cells (mTHPC EVs) or mTHPC-loaded hybrid EVs at equivalent mTHPC concentrations in DMEM medium without serum. In 2D and 3D experiments, supernatant containing mTHPC was then removed and replaced by complete DMEM medium without phenol red. Some wells were used to detect mTHPC fluorescence quantified using the EnSpire plate reader (excitation 405 nm, emission 650 nm) after 24 hours. Triton X-100 was used at 0.3% (w/v) final concentration in order to lyse membranes. Quantification was carried out using a calibration curve of free mTHPC fluorescence in 0.3% (w/v) Triton X-100.


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In 2D and 3D experiments, 24 hours after the beginning of mTHPC uptake, cells were irradiated at a light fluency of 10 J/cm² (100 mW/cm² for 100 sec) by means of a 650 nm laser diode featuring a fiber delivery system (BWT, China). Cells were incubated for another 24 hours before cytotoxicity assessment by Alamar Blue test (Invitrogen) for 2D experiments, according to the supplier’s instructions. Cells were incubated with 10% Alamar Blue in complete medium for 2 hours. After incubation, the medium was transferred to a 96-well plate for analysis using the EnSpire plate reader (excitation at 550 nm, emission at 590 nm). Cytotoxicity for 3D experiments was assessed by observing the ability of the spheroids to adhere to the surface once transferred to 24-well plates. Confocal experiments to investigate mTHPC

penetration

in

spheroids

were

conducted

with

Olympus

JX81/BX61

Device/Yokogawa CSU Device spinning disk microscope (Andor Technology plc, Belfast, Northern Ireland), and image analysis was performed with Fiji.

Statistical Data Analysis Statistics are presented as standard deviation from the mean (n ≥ 3). Student t-test was carried out using Prism 3.0 version of GraphPad software (USA) to determine whether a difference between two groups was significant. A minimum of 95% confidence level was considered significant (*** indicates P < 0.0001; ** indicates P < 0.01; * indicates P < 0.05 and NS means not statistically significant).

Acknowledgments This work has been supported by the ITMO-Inserm Plan Cancer 2014-2019. MP received a PhD fellowship from Servier Laboratories, is supported by “Ecole de l’INSERM-Lilliane Bettencourt”, and funds by the PhD Program “Frontières du Vivant (FdV) – Cursus Bettencourt”. DT is funded by the “Association Française contre les Myopathies” (AFM


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Research grant 20123), the “Fondation pour la Recherche Médicale” (FRM), and the Labex “Who am I?”. The authors thank Biolitec research GmbH (Jena, Germany) for kindly providing mTHPC and Foslip products. We are also grateful to the team of A. Brisson (University of Bordeaux, CNRS UMR 5248) and C. Ménager (Phenix, University Pierre and Marie Curie, CNRS UMR 8234) for kindly providing Annexin V gold nanoparticles and citrate-coated magnetic iron oxide nanoparticles, respectively.

Supporting Information Supporting information available online contains data about: calibration and optimization of the liposome fusion assay (Figure S1); characterization of fusion by cryo-TEM (Figures S2 and S3); fusion with microvesicles, exosomes or PEGylated liposomes (Figure S4); micromagnetophoresis on magnetic hybrids (Figure S5); delivery of mTHPC to 3D tumor spheroids (Figure S6); internalization of hybrids by HUVEC and CT26 cells (Figure S7); calibration of flow cytometry (Figure S8).


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Figure 1

Figure 1: PEG mediates robust lipid mixing between EVs and liposomes. (a) Representative set of fusion experiments between non-fluorescent HUVEC-derived EVs and fluorescent liposomes composed of 67% POPC, 30% DOPE, 1.5% NBD-PS and 1.5% Rho-PE (1/1 EV/liposome molar ratio) in the presence of 0-30% (w/v) PEG 8000. The kinetics and extent of fusion clearly increased with PEG 8000 concentration (see also pannel b). (b) Average extent of fusion after 2 hours of reaction (performed as in pannel a) between liposomes and HUVEC-derived EVs. (c) Average extent of fusion after 2 hours of reaction between MSC-derived EVs and liposomes with the same lipid composition as in panels a and b (filled bars) or PEGylated liposomes composed of 62% POPC, 30% DOPE, 1.5% NBD-PS, 1.5% Rho-PE and 5% PEG 2000-PE (empty bars) in the presence of 0-30% (w/v) PEG 8000. Fusion was slightly less efficient when the liposome membrane contained PEGylated lipids.


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Results are expressed as mean ± SD with n ≥ 3 independent EV preparations. (d) FRET-based lipid mixing assay used to monitor the fusion between EVs and liposomes.


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Figure 2

Figure 2: Imaging flow cytometry confirms EV-liposome interaction at the single particle level. HUVEC-derived EVs and fluorescent liposomes consisting of 67% POPC, 30% DOPE, 3% Rho-PE were incubated for 2 hours at 40°C (1/1 EV/liposome molar ratio) in the absence or presence of 10 % (w/v) PEG 8000. EVs were detected with Annexin V-FITC fluorescence and liposomes with Rhodamine fluorescence. (a) Representative images of EVs (Annexin V positive), liposomes (Rhodamine positive) and hybrid EVs (double positive) after fusion in the absence or presence of PEG. (b) Biparametric dot plot of Rhodamine versus Annexin VFITC intensity in the mixture of EVs and liposomes following their incubation in the absence or presence of PEG. (c) Fraction of total Rhodamine fluorescence contained in the hybrid EVs (double positive) region after fusion with or without PEG. (d) Rhodamine intensity in hybrid EVs (double positive events) after fusion with or without PEG. (e) Size distribution of EVs 33 ACS Paragon Plus Environment

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before and after their incubation with liposomes in the absence or presence of PEG. Objects size was determined on bright field pictures by comparison with calibrated beads. Results in c and d are expressed as mean ± SD with n ≥ 3 independent EV preparations.


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Figure 3

Figure 3: Tuning the composition of hybrid EVs: quantitative analysis of fusion and purification of hybrid EVs by flotation in a density gradient. (a) NBD fluorescence in MSC-derived EVs after their fusion with liposomes composed of 67% POPC, 30% DOPE, 1.5% NBD-PS and 1.5% Rho-PE using different EV/liposome ratios in the presence (filled bars) or absence (empty bars) of 5% (w/v) PEG 8000. Varying the EV/liposome ratio leads to a total membrane surface of hybrid EVs composed of different fractions of liposome-originating surface (red) and EV-originating surface (green) deduced using equations 2 & 3. (b) Flotation profiles after ultracentrifugation in a Nycodenz density gradient of (i) MSC-derived EVs labeled with the PKH26 membrane dye (green curve), or (ii) liposomes composed of 67% POPC, 30% DOPE, 1.5% NBD-PS and 1.5% Rho-PE before (red curve) and after (grey curve) incubation in the presence of PEG (without EVs). Three zones were distinguished: 0-1 mL encompassing mostly liposomes, 1.25-3 mL corresponding to EVs (fused or not with liposomes) with higher density than liposomes, and 3.25-5 mL corresponding to the leaking zone containing soluble compounds of higher densities or unable to move along the gradient due to their small size. (c-e) Rhodamine fluorescence along the Nycodenz density gradient after PEG-mediated fusion of MSC-derived EVs with liposomes 35 ACS Paragon Plus Environment

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composed of 67% POPC, 30% DOPE, 1.5% NBD-PS and 1.5% Rho-PE using various EV/liposome ratios (c: 1/9, d: 1/1 or e: 9/1). Fusion leads to Rhodamine fluorescence transfer from the low density zone (liposomes) toward the higher density zone (EVs). The percentage of fused liposomes is defined as the percentage of fluorescence that shifted toward larger densities (hatched zone). The fraction of total hybrid EVs surface coming from liposomes (red) is calculated using equation 3. All fusion reactions in this figure were conducted with 5% (w/v) PEG 8000. Results are expressed as mean ± SD with n ≥ 3 independent EV preparations.


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Figure 4

Figure 4: PEG-mediated fusion allows loading of membrane and soluble/hydrophilic compounds without losing EVs intrinsic cargo. (a) MSC-derived EVs were fused with liposomes labeled with Rho-PE lipids (liposomes composed of 67% POPC, 30% DOPE, 1.5% NBD-PS and 1.5% Rho-PE) using 0-30% (w/v) PEG 8000 (1/1 EV/liposome ratio). Each sample was then purified in a Nycodenz density gradient and fluorescence from Rho-PE lipids was quantified along this gradient and categorized as unfused liposomes, hybrids or leaking using zones defined in Figure 2b. (b) MSC-derived EVs were fused with liposomes (consisting of 70% POPC and 30% DOPE) loaded with 2 µM hydrophilic Rhodamine (Rho) using 0-30 % (w/v) PEG (1/1 EV/liposome ratio). Each sample was then purified in a Nycodenz density gradient and fluorescence from Rho was quantified along this gradient and categorized as unfused liposomes, hybrids or leaking. (c) EVs derived from MDCK cells expressing cytosolic GFP were fused with nonfluorescent liposomes (consisting of 70% POPC and 30% DOPE) using 20% (w/v) PEG 8000 (1/2 EV/liposome ratio). The sample was then purified in a Nycodenz density gradient, and GFP fluorescence was quantified along this gradient and compared to the flotation profile of a recombinant GFP (green curve). Results in a-c are expressed as mean ± SD with n ≥ 3 independent EV preparations.


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Figure 5

Figure 5: PEGylation of hybrid EVs reduces their internalization by THP1-derived macrophages. MSC-derived EVs and PEGylated or non-PEGylated liposomes, consisting respectively of 69% POPC, 30% DOPE and 1% DiR or 64% POPC, 30% DOPE, 1% DiR and 5% PEG 2000-PE, were fused using 30% (w/v) PEG 8000 (1/1 EV/liposome ratio). Hybrid EVs or liposomes alone were then added to THP1-derived macrophages. Representative pictures (a) and quantification (b) of DiR fluorescence in THP1-derived macrophages in each condition. Results in b are expressed as mean ± SD with n ≥ 4 independent EV preparations.


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Figure 6

Figure 6: Hybrid EVs deliver mTHPC in cancer cells more efficiently than liposomes. (a) MSC-derived EVs and mTHPC-loaded Foslip liposomes were fused in the presence of 20% (w/v) PEG 8000 (1/2 EV/liposome ratio). Hybrid EVs and unfused liposomes were separated in a Nycodenz density gradient and mTHPC fluorescence transfer was quantified (~90%), confirming that nearly all Foslip liposomes had fused with EVs. The total volume of hybrid EVs was constituted of 64% mTHPC Foslip content (measured using equation 4). (b) Hybrid EVs, Foslip or free mTHPC were added to CT26 colon cancer cells and their internalization efficiency was measured as the ratio between the fluorescence of intracellular mTHPC and the fluorescence of the total incubated dose. (c) Cells were submitted to 650 nm laser irradiation in order to activate mTHPC (light ON, filled bars) and evaluate its lightinduced toxicity using measurement of the cell metabolic activity by the Alamar blue test. Controls without light irradiation (light OFF, empty bars) allowed us to compare the intrinsic dark toxicity of each vector and to show the proliferation effect of MSC-derived EVs and hybrid EVs (100% corresponds to toxicity when cells are irradiated in the presence of PBS). 39 ACS Paragon Plus Environment

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(d) Representative pictures of cells in the case of 0.5 µM mTHPC added from solution (free form) or encapsulated within Foslip liposomes or hybrid EVs. Cells are clearly showing morphological changes and are labeled with Annexin V (apoptosis marker) and Propidium iodide (necrosis marker) after irradiation. mTHPC uptake is larger when using hybrid EVs. Results in a-c are expressed as mean ± SD with n ≥ 3 independent EV preparations.


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Graphical Table of Content


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Modification of Extracellular Vesicles by Fusion with Liposomes for the

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