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Antibody-displaying extracellular vesicles for targeted cancer therapy – Nature Biomedical Engineering

Cell culture

FreeStyle 293F cells were cultured in FreeStyle medium (FreeStyle 293; Gibco Life Technology) supplemented with 1% Antibiotic Antimycotic (Gibco Life Technology) and 4.5 g l−1 pyruvate (Invitrogen). HEK293T, SKBR-3 and B16F10 cells were cultured in DMEM high glucose medium (DMEM; Gibco Life Technology), supplemented with 1% Antibiotic Antimycotic (Invitrogen) and 10% fetal bovine serum (FBS) (Gibco). Mycoplasma testing was routinely performed, and all cell lines were maintained at 37 °C with 5% CO2 in a humidified atmosphere.

Production of engineered EVs

Plasmid constructs and cloning

For the different Fc-binding constructs, the amino acid sequence for all nine EV-sorting domains (Supplementary Fig. 12), nine Fc binders (listed in Supplementary Fig. 13), mNG (Supplementary Fig. 13) and nLuc (Supplementary Fig. 13) were confirmed by Uniprot. They were subsequently synthesized (Integrated DNA Technologies) as gene blocks and cloned into a lentiviral p2CL9IPwo5 backbone downstream of the SFFV promoter using NotI and EcoRI and upstream of an internal ribosomal entry site-puromycin resistance complementary DNA cassette. To create the plasmid-expressing mNG fused to the C- or N-terminus of the EV-sorting domains, mNG was subcloned into the p2CL9IPw5-EV (plasmid was kindly given by H. Hanenberg, University Hospital Essen, Germany) sorting domain construct using NotI and SphI or AgeI and EcoRI, respectively. The nLuc was subcloned into the p2CL9IPw5-EV-sorting domain construct using NotI and SphI. The Fc binders were then cloned into the p2CL9IPw5-EV-sorting domains using Kpn2I and Bsp119I. Before using the constructs, all plasmid constructs were confirmed by Sanger sequencing (Eurofins Genomics).

Virus production and transduction

Lentiviral supernatants were produced as described previously16. In brief, HEK-293T cells were co-transfected with the p2CL9IPw5 construct, including CD63-Fc fused either to mNG or nLuc proteins, the human foamy virus envelope construct pcoPE and the helper construct pCD/NL-BH using the transfection reagent JetPEI (Polyplus). Eighteen hours post transfection, gene expression from the human cytomegalovirus immediate-early gene enhancer/promoter was induced with 10 mM sodium butyrate (Sigma-Aldrich) for 6 to 7 h before fresh media was added to the cells, and the supernatant was collected after 22 h. Viral particles were then pelleted at 25,000 g for 90 min at 4 °C. The pellet was resuspended in 1 ml of Iscove’s modified Dulbecco’s media supplemented with 20% FBS and 1% antibiotic antimycotic after the supernatant had been removed. Until use, the viruses were kept at 80 °C. The FreeStyle 293F cells were then transduced overnight with the virus and passaged a minimum of five times under puromycin selection to make stable cell lines.

EV isolation

EVs were isolated, as described in a previous publication13. In brief, once a concentration of 2 × 106 ± 2 × 105 FreeStyle 293F cells per ml was obtained, the condition media was collected and centrifuged at 700 RCF for 5 min to remove cells. The pellet was discarded, and the condition media was centrifuged for an additional 10 min at 2,000 g to remove cell debris and additional larger contaminants. Subsequent purification by 0.22 µm filtration was performed (Nalgene Rapid-Flow; Thermo Fisher Scientific), and the EVs were concentrated by tangential flow filtration (KrosFlo Research 2i TFF system; Spectrum labs) at a flow rate of 100 ml min−1 and a transmembrane pressure around 20,684 kPa using a 300 kDa polyether sulfone hollow fibre filter (MidiKros, 370 cm2 surface area, SpectrumLabs). The sample was diafiltered with twice the original condition media volume using 0.22 µm filtered (Nalgene Rapid-Flow; Thermo Fisher Scientific) 0.1 M PBS and collected at a final volume of 20–35 ml. The concentrate was purified by 0.22 µm suction filtration (Nalgene Rapid-Flow; Thermo Fisher Scientific), and the filtrate was further concentrated by ultrafiltration at 4,000 RCF in 10 kDa spin columns (Amicon Ultra-15; Millipore) to a final volume of 400–600 µl. The EVs were stored in PBS-HAT (HEPES, Albumin, Trehalose) buffer at −80 °C until further use, as described in ref. 47.

Nanoparticle tracking analysis

NTA was performed with the NS500 nanoparticle analyser (NanoSight) to measure the size distribution and concentration of EVs as described in ref. 6. The samples were diluted in 0.1 M PBS to achieve a particle count of between 2 × 108 and 2 × 109 per ml. The camera focus was adjusted to make the particles appear as sharp dots. Using the script control function, five 30 s videos for each sample were recorded, incorporating a sample advance and a 5 s delay between each recording. The analysis was performed with the screen gate set at 10 and the detection threshold at 7, with all remaining settings set at automatic.

Transmission electron microscopy

Purified Fc-EVs or ctrl-EVs were incubated with 1 μl of 1% BSA diluted in PBS for 5 min. Rabbit anti-goat 10 nm antibody conjugated with gold nanoparticles (BBI Solutions) was added and incubated for 45 min. Finally, 5 μl of labelled EVs were added onto glow-discharged formvar-carbon-type-B-coated electron microscopy grids (Ted Pella) for 3 min. The grid was dried with filter paper, washed twice with distilled water and blotted dry with filter paper. After the wash, the grid was stained with 2% uranyl acetate in double-distilled H2O (Sigma-Aldrich) for 10 s and filter paper dried. The grid was air dried and visualized on a transmission electron microscope (Tencai 10).

Protein detection by western blot

The presence of the EV-associated tetraspanin CD63 fused to the Nanoluc (Nluc) protein as well as the intravesical proteins Alix and TSG101 was examined by western blot. About 2 × 106 HEK293 cells from the production culture were pelleted at 300 RCF for 5 min, washed with cold PBS and pelleted once more. The cell pellet and 1 × 1010 EVs were lysed separately using 100 µl of radioimmunoprecipitation buffer (BioRad). Both samples were incubated on ice for 30 min and vortexed for 10 s each fifth minute. Lipids and additional large contaminants were removed from the cell lysis by centrifugation at 12,000 RCF for 12 min at 4 °C. About 24 µl of the supernatant was transferred to a new tube on ice. The collected supernatant and 24 µl EVs were mixed with 8 µl loading buffer (10% glycerol, 8% sodium dodecyl sulfate, 0.5 M dithiothreitol and 0.4 M sodium carbonate). The protein was further denatured to its primary structure by incubation at 65 °C for 5 min before being loaded onto the NuPAGE+ (Invitrogen, Novex 412% Bis–Tris gel) and run at 120 V for 2 h. The proteins were transferred by iBlot system (iBlot 2 Dry Blotting System; Invitrogen) for 7 min to an iBlot membrane (iBlot 2 Transfer Stacks; Invitrogen). The membrane was treated by blocking buffer (Odyssey Blocking Buffer; LI-COR Biosciences) at room temperature for 60 min to avoid unspecific binding and later incubated overnight at 4 °C with newly prepared primary antibodies (anti-CD63 (ab134045, Abcam) diluted 1:1,000, anti-Alix (MA1-83977, Thermofisher) diluted 1:1,000, anti-nLuc (N7000, Promega) diluted 1:1,000 and anti-Tsg101 (ab30871, Abcam) diluted 1:1,000. The membranes were washed 4 times with tris-buffered saline with 0.1% tween for 5 min each on a shaker and then incubated with secondary antibody (goat anti-mouse (C00322) diluted 1:10,000, goat anti-rabbit (C90827-25) diluted 1:10,000) at room temperature for 1 h. The washing was repeated with one additional PBS wash, and the results were visualized by both 700 and 800 nm channels of an infrared imaging system (LI-COR Odyssey CLx).

Stability of antibody display of Fc-EVs

To test the stability of antibody-functionalized EVs, Fc-EVs (with intravesicular mNG reporter) were first decorated with human IgG1-APC antibody (Miltenyi, catalogue number 130-11-434). Specifically, 1 × 1011 Fc-EVs were mixed with 25 µg antibody in a total volume of 0.2 ml, followed by 2 h incubation at 37 °C. After dilution with 0.2 ml Dulbecco’s phosphate-buffered saline (DPBS), EVs were purified using qEV size exclusion chromatography columns (Izon; SP1) according to manufacturer’s instructions. To examine the stability of engineered EVs in plasma, 1 × 108 APC-labelled EVs (in 20 µl) were incubated with 10 µl plasma (from healthy human or mice) in 96-well V-bottom plates at 37 °C. After 1, 10 or 30 min, the samples were diluted with DPBS by 1,500-fold and analysed using CellStream flow cytometer as described below. Stability in the presence of other antibodies was similarly tested by incubating 1 × 108 APC-labelled EVs with 25, 50 or 250 ng phycoerythrin (PE)-conjugated antibody for 30 min. The percentage of APC+mNG+ (double positive) among total mNG+ EVs was calculated. The following PE-conjugated antibodies were included: hIgG1-PE (Miltenyi, catalogue number 130-113-438), hIgG4-PE (BioLegend, catalogue number 403704), mIgG1-PE (Miltenyi, catalogue number 130-113-200) and mIgG2-PE (Miltenyi, catalogue number 130-092-215).

Binding affinity of Fc-EVs with antibody

To characterize the affinity of Fc-EVs with antibodies, 1 × 108 EVs (with intravesicular mNG reporter, in 20 µl) were incubated with different amount of PE-conjugated antibody (0.0125–10 µg ml−1; in 10 µl) in 96-well V-bottom plates at 37 °C. After 2 h, the samples were diluted with DPBS by 1,500-fold and analysed using CellStream as described below. The percentage of APC+mNG+ (double positive) among total mNG+ EVs was calculated and fitted against antibody concentration using the one-site total binding model from GraphPad. The predicted equilibrium dissociation constant Kd was presented for each type of antibody if available. The following PE-conjugated antibodies were included: hIgG1-PE (Miltenyi, catalogue number 130-113-438), hIgG4-PE (BioLegend, catalogue number 403704), mIgG1-PE (Miltenyi, catalogue number 130-113-200) and mIgG2-PE (Miltenyi, catalogue number 130-092-215).

Antibody quantification per EVs

To determine the number of antibodies that bind to Fc-EVs, the Fc-EVs (with intravesicular mNG reporter) were first decorated with human IgG1-APC antibody (Miltenyi, catalogue number 130-11-434) or mouse IgG2b (Miltenyi Biotec 130-092-215) and ctrl-EVs (with intravesicular mNG reporter) decorated with human IgG1-APC antibody (Miltenyi, catalogue number 130-11-434) or mouse IgG2b (Miltenyi Biotec 130-092-215). Specifically, 1 × 1011 Fc-EVs or ctrl-EVs were mixed with 25 µg antibody in a total volume of 200 µl, followed by 2 h incubation at 37 °C. After dilution with 200 µl DPBS, Fc-EVs were purified using qEV columns (Izon; SP1) according to manufacturer’s instructions. For quantification of antibodies per EVs, the SPP was performed24. Briefly, fluorescence intensity (Supplementary Fig. 14a) fluctuations were recorded by observing free diffusion of fluorescently labelled EVs through the observation volume. The histogram of fluorescent brightness of antibodies per single EV was converted into the histogram in terms of antibody number per particle (Supplementary Fig. 14b) using the fluorescence brightness of free antibody, which was quantified by fluorescence correlation spectroscopy (Supplementary Fig. 14c–e). Curves were fitted with the following three-dimensional diffusion, and molecular brightness was quantified:

$$G(tau )=frac{1}{langle Nrangle }{left(1+frac{tau }{{tau }_{rm{D}}}right)}^{-1}{left(1+frac{tau }{A{R}^{2}times {tau }_{rm{D}}}right)}^{1/2}$$

where G (τ) is a correlation function, τ is delay time, τD is diffusion time, AR is for aspect ration of the observation volume and N is an average number of molecules within the observation volume. Molecular brightness is quantified as counts per second per particle. SPP was performed using the set-up for fluorescence correlation spectroscopy on a Zeiss LSM 780 microscope. A 488 nm argon ion laser was used for mNG and a 633 nm He–Ne laser was used for APC detection. A 40 × 1.2 NA water immersion objective was used to focus the light. The laser power was set to 0.1–0.5% of the total laser power, corresponding to 2–10 μW. The emission detection windows were set to 490–560 for mNG and 650–700 for APC.

Flow cytometry

Validation of purified EVs was performed by flow cytometry using two different techniques described in the following sections.

Multiplex bead-based flow cytometry analysis

Multiplex bead-based flow cytometry analysis (MACSPlex Exosome Kit, human, Miltenyi Biotec) was implemented to characterize the general surface protein composition of Fc-EVs and to assess the binding of specific antibodies to the Fc receptor expressed on the EVs. Assays were performed on the basis of a previously described protocol16. In brief, EVs were used at an input dose of 1 × 109 NTA-based particles per assay, diluted with MACSPlex buffer to a total volume of 120 μl and incubated with 15 μl of MACSPlex exosome capture beads overnight in wells of a pre-wet and drained MACSPlex 96-well 0.22 μm filter plate on an orbital shaker at 450 rpm at room temperature. The beads were washed with 200 μl MACSPlex buffer, and the liquid was removed by applying a vacuum (Sigma-Aldrich, Supelco PlatePrep; −100 mBar). For counterstaining of captured EVs, either a mixture of APC-conjugated anti-CD9, anti-CD63 and anti-CD81 detection antibodies (supplied in the MACSPlex kit, 5 μl each) or AlexaFluor647-conjugated human IgG Fc fragments (The Jackson Laboratory, catalogue number 009-600-008, 100 ng) was added to each well in a total volume of 135 μl, and the plates were incubated on an orbital shaker at 450 rpm for 1 h at room temperature. Next, the samples were washed twice, resuspended in MACSPlex buffer and analysed by flow cytometry with a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec). FlowJo (v.10.6.2, FlowJo) was used to analyse flow cytometry data.

Single-vesicle imaging flow cytometry

Single-vesicle imaging flow cytometry was performed as described previously11,47 by using either an Amnis ImageStream X Mk II or an Amnis Cellstream instrument (Amnis/Luminex). In brief, 2.5 × 108 EVs (NTA-based particles) were stained (unless indicated otherwise) in a total volume of 25 µl with 8 nM of fluorescent antibodies against the tetraspanins CD9, CD63 and CD81 or recombinant antibodies (isotypes control) (REA(S))-APC human IgG isotype control antibodies (Miltenyi Biotec). EVs were incubated overnight at room temperature in the dark with a subsequent dilution to a concentration of 1 × 107 EVs per ml in a final volume of 100 µl before data acquisition. Unstained samples and non-EV-containing samples incubated with antibodies were included as controls, respectively. PBS-HAT buffer was used as diluent for all steps47. The results were analysed with FlowJo software (v. 10.7.2; FlowJo LLC).

Analysis of tissue, blood and tumour single-cell suspensions by flow cytometry

To remove cell aggregates, appropriate dilutions of tissue and tumour cell suspensions (1:10 in PBS/1% FBS for tumour, liver and spleen cell suspensions; no dilution for peripheral blood mononuclear cell (PBMC) suspensions and lymph node cell suspensions) were prepared and filtered through a 40 µm cell strainer cap directly into a 5 ml round bottom tube (Corning) for flow cytometry to remove aggregates. All samples were subjected to four different antibody staining protocols to detect PD-L1 simultaneously with T cells (CD3, CD8), B cells (CD45R/B220), monocytes (CD11b) or macrophages (F4/80), respectively. For all stainings, 25 µl of cell suspension was added to each well of a 96-well V-bottom microplate (Sarstedt) and 25 µl of PBS/1% FBS was added containing a mix of the respective antibody combinations. Cells were stained for 30 min at 4 °C in the dark. To wash off unbound antibodies, wells were filled up with PBS/1% FBS, and the plate was centrifuged at 900 g for 5 min. Cell pellets were resuspended in 90 µl PBS/1% FBS and kept in the dark at 4 °C until flow cytometric measurement on a MACSQuant Analyzer 10 instrument equipped with three lasers (405 nm, 488 nm, 635 nm, Miltenyi Biotec) using the 96-well plate experiment layout. For live/dead cell discrimination 10 µl of a 10× DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich) stock solution was added to each well. The data were analysed using the FlowJo Analysis Software (v. 10.8, FlowJo LLC, BD Biosciences). Antibodies (fluorophore, final dilution, clone and manufacturer) were rb-α-ms PD-L1 (APC, 1:100, 485, Sino Biological), rt-α-ms CD3 (PerCP/Cy5.5, 1:100, 17A2, BioLegend), rt-α-ms CD8a (BV510, 1:100, 53-6.7, BioLegend), rt-α-ms CD45R/B220 (BV510, 1:100, RA3.6B2, BioLegend), rt-α-ms CD11b (BV510, 1:100, M1/70, BD Biosciences) and rt-α-ms F4/80 (BV510, 1:100, T45-2342, BD Biosciences).

Size exclusion chromatography

To confirm the binding affinity of Fc-EVs with antibodies, 1 × 1011 mNG-Fc-EV or control mNG-EV was incubated for 2 h at 37 °C in 400 µl of 0.22 µm filtered PBS with 2.5 µg REA(S)-APC human IgG isotype control antibodies (Miltenyi Biotec). Following this, the samples were fractionated using 70 nm, 500 µl qEV columns (qEV original; IZON Science LTD), yielding 25 fractions of 1 ml per fraction. The fractions were analysed by fluorometer (SpectraMax i3x; Molecular Devices LLC).

Cell-uptake experiment

For Huh-7 cells uptake of human IgG Fc fragment bound to Fc-EVs, 100,000 Huh-7 cells were seeded overnight in a 24-well plate before Fc-EV treatment. About 1 × 1011 mNG+ ctrl-EV or Fc-EVs were incubated with 2 µg of human IgG Fc fragment (AF647 conjugated) overnight at 4 °C with rotation. The next day, Huh-7 cells were treated with the Fc fragment, with or without ctrl-EV or Fc-EVs and incubated at 37 °C with 5% CO2 for 2 h. The Huh-7 cells were then analysed in a motorized Olympus IX-81 inverted-fluorescence microscope equipped with an XM10-monochrome camera and narrow band-filter cube for UV (DAPI), green (GFP) and red (APC) excitation.

For B16F10, stimulated with or without 40 ng ml−1 of IFNγ (PMC4031, Gibco Lifetechnology) and SKBR-3 cell lines, the cells were seeded in a 96-well plate format at 1 × 104 and 2.5 × 104 cells per well, respectively. The cells were incubated and maintained at 37 °C with 5% CO2 in a humidified atmosphere for 24 h before treatment. After that, the cells were treated for 2 h with either mNG-Fc-EVs + anti-PD-L1 (atezolizumab) or anti-HER2 (trastuzumab) antibodies or control mNG-EVs + anti-PD-L1 (atezolizumab) or anti-HER2 (trastuzumab) antibodies. The cells were then washed with PBS, trypsinized and resuspended in fresh DMEM medium for flow cytometry using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec). FlowJo (v. 10.6.2, FlowJo) was used to analyse flow cytometry data.

To assess the effect on EV uptake by pretreatment of HER2-Ab, 25 × 103 SKBR-3 cells were seeded in a 96-well plate format per well for 24 h in DMEM high-glucose medium (DMEM; Gibco Life Technology), supplemented with 1% antibiotic antimycotic (Invitrogen) and 10% FBS (Gibco). The plate was maintained at 37 °C with 5% CO2 in a humidified atmosphere. The cells were then treated with 2.5 µg of anti-HER2 (trastuzumab) or left untreated for 2 h. mNG-Fc-EVs + 2.5 µg of anti-HER2 (trastuzumab) or Fc-EVs alone or mNG-Fc-EVs + 2.5 µg of human IgG isotype control antibody (catalogue number 12000C, Thermo Fisher) were subsequently added to the cells for another 2 h. The cells were subsequently washed with PBS, trypsinized and resuspended in fresh DMEM medium for flow cytometry using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec). FlowJo (v. 10.6.2, FlowJo) was used to analyse flow cytometry data.

Kidney and liver function tests

Tests for creatinine (Abcam: ab65340) and urea (Abcam: a234052) for kidney function were performed based on the manufacturer’s instructions. Briefly, serum samples were separated by 10 kDa and spin-filtered at 10,000 g for 10 min at 4 °C. After collecting the filtrate, 2–10 µl of samples was added to a 96-well plate; the final volume was adjusted to 50 µl with distilled H2O, and absorbance was measured by fluorometer (SpectraMax i3x; Molecular Devices LLC) at 570 nm and 505 nm, respectively. Aspartate aminotransferase (Abcam: ab105135) and alanine transaminase (Abcam: ab105134) for liver function tests were performed based on the manufacturer’s instructions. In brief, serum samples were directly diluted in the assay buffer. Samples were prepared at up to 50 μl per well with assay buffer in a 96-well plate. Absorbance was measured by fluorometer (SpectraMax i3x; Molecular Devices LLC) at 450 nm and 570 nm, respectively.

Fluorescence microscopy

B16F10 cells were seeded at 20,000 cells per well in 8-well glass-bottom Nunc Lab-Tek II Chamber slide (Thermo Scientific). Cells were allowed to adhere for 24 h and then stimulated with or without 40 ng ml−1 of IFNγ (PMC4031, Gibco Lifetechnology). Cells were treated for 2 h with labelled antibody (anti-PD-L1-APC, Sinobiological, catalogue number 50010-R485-A) or mNG+ Fc-EVs alone, or mNG+ Fc-EVs with PD-L1-Ab (atezolizumab or anti-PD-L1-APC) as described above. After the treatment period, cells were washed once with PBS and then fixed with 4% paraformaldehyde (Fisher Scientific) for 10 min at room temperature. Both PBS and fixation solutions were warmed up to 37 °C, and the pipetting was conducted swiftly to avoid sample drying. Following fixation, nuclei were stained with DAPI (Fisher Scientific) for 15 min. Stained cells were then washed three times with PBS. Images were acquired using a confocal microscope (A1R confocal, Nikon) and analysed by the NIS-Elements software (v. 5.21.00, Nikon).

Nanoimager microscopy

For the antibody to Fc-EVs conjugation, 5 μg of antibody was mixed with 1 × 1011 Fc-EVs in PBS 0.2% human albumin serum in a final volume of 100 μl, overnight on a thermoblock plate at 25 °C with a 300 r.p.m. rotation and protected from light. For sample purification, a qEV column was initially equilibrated with 2 ml volume of PBS. Following reaction completion, the samples were purified by adding the full 100 μl of the reaction mixture onto a qEV column (Izon qEVsingle Single-use Columns, Fisher Scientific, catalogue number 15928090). Fraction collection was performed by adding 3 ml of PBS to the column and eluting by counting eight drops per fraction, on a total of 21 fractions. For imaging, each well of a µ-Slide 18 Well Glass Bottom slide (IbiDi, catalogue number 81817) was washed with distilled water twice followed by a wash with potassium hydroxide 1 M and a final wash with distilled water for surface cleaning. For surface coating, 100 μl of poly-l-lysine solution (Sigma, catalogue number 25988-63-0) was added and incubated at 37 °C for 2 h. Upon incubation period completion, poly-l-lysine solution was carefully removed with a pipette, and the sample of antibody-Fc-EVs was added (50 μl); the plate was left overnight at 4 °C. Before imaging, the plate was allowed to reach room temperature. The high-resolution experiment was performed using a Nanoimager S Mark II microscope from ONI (Oxford Nanoimaging) equipped with a ×100, 1.4 NA oil immersion objective, an XYZ closed-loop piezo 736 stage and triple emission channels split at 488, 555 and 640 nm. For the evaluation of antibody-Fc-EV conjugation the manufacturer’s protocol was used.

Loading Fc-EVs with Dox

Electroporation was conducted based on our recently published protocol39. Dox (doxorubicin hydrochloride, Sigma-Aldrich; Merck) was diluted to a concentration of 10 mM with ultrapure water, to avoid aggregation at later stages. The Dox was mixed with EVs diluted with 0.22 µm filtered (Nalgene Rapid-Flow; Thermo Fisher Scientific) 0.1 M PBS and incubated at 4 °C for 30 min. Prepared electroporation buffer (400 mM sucrose in PBS) was added to the EVs in a 1:1 ratio, and 400 µl of the sample was electroporated in 0.4 cm cuvettes by exponential pulse using an electroporation system (GenePulser Xcell; BioRad). The sample was then incubated at 37 °C for 30 min, and the EVs were subsequently isolated by SEC, using 70 nm 500 µl qEV columns (qEVoriginal; IZON Science LTD), collecting the 4th and 5th millilitres of elute. The elute was then concentrated to a final volume of 200–300 µl, using 10 kDa spin filters (Amicon Ultra; Millipore) spun at 4,000 RCF. Dox was analysed by fluorometer (SpectraMax i3x; Molecular Devices LLC), and the concentrations were determined in relation to a standard curve starting at 100 µM halving 12 times to 40 mM and blanks of ultrapure water and PBS, using the dedicated software (SoftMax Pro v.7; Molecular Devices LLC). The samples were portioned at 90 µl, excited at 488 nm and read at 530 nm. Effective loading was calculated as a function of the concentration of Dox (mM per billion EVs).

Stability of encapsulated Dox in plasma

Dox was loaded into Fc-EVs by electroporation, as described above. The electroporated Fc-EVs were purified by SEC to remove unloaded Dox. After SEC, the sample was concentrated by a 2 ml 10 kDa spin filter. The concentrated samples were mixed with 1 ml of mouse plasma and incubated at three time points (1, 15 and 30 min) at 37 °C. The samples were subsequently purified by SEC and concentrated using a 2 ml 10 Kd spin filter. Encapsulated Dox was analysed by fluorometer (SpectraMax i3x; Molecular Devices LLC), and the concentrations were determined in relation to a standard curve starting at 100 µM halving 12 times to 40 mM and blanks of ultrapure water and PBS, using the dedicated software (SoftMax Pro v.7; Molecular Devices LLC). The samples were portioned at 90 µl, excited at 488 nm and read at 530 nm.

Viability assay

The viability of the cells after treatment was assessed by 80 µl per well Cell Titre Glo (CellTiterGlo; Promega Biotech) following the provided protocol. About 1 × 104 B16F10 cells were seeded in a 96-well plate 24 h before treatment, and the plate was read 48 h post treatment, with a luminometer (GloMax 96 Microplate Luminometer: Promega Biotech) using the predesigned protocol provided by the manufacturer. The plate was read each third minute until the signal stabilized.

Animal experiments

All of the animal experiments were performed in accordance with ethical permissions approved by the Swedish Local Board for Laboratory Animals and designed to minimize the suffering and pain of the animals. The animals were supplied by Scanbur or Janvier Labs.

Malignant melanoma model

The tumours were established as previously described48. Briefly, female C57BL/6 (20 ± 2 g) mice were subcutaneously implanted with 5–7 × 105 B16F10 cells on day 0. They were monitored daily and developed palpable tumours within 7 days. The mice were killed at pre-decided time points, if the tumour size exceeded 1,500 mm3 or if the mice exceeded the scoring of pre-set humane end points. For distribution experiments, the mice (n = 7–10) were injected intravenously (if not otherwise indicated) when the tumours had reached 1,000 mm3 (on day 14 ± 2) with 1 × 1011 Fc-EVs alone or following 4 °C incubation over-night with 2.5 µg anti-PD-L1 (atezolizumab) or 2.5 µg REA(S)-APC human IgG isotype control antibody (Miltenyi Biotec). After the injection the mice were killed for tissue and blood collection at different time points (15 min, 30 min, 1 h, 3 h, 24 h, 48 h or 72 h). For luminescent reading, nLuc positive Fc-EVs were used, whereas mNG+ Fc-EV were used for flow cytometry applications (30 min time point only). The tissues were processed as described in ref. 29, with lysed tissue and nLuc+ EV input being analysed for nanoluc luminescence or for single-cell suspension for flow cytometry, as described below. For tumour treatment experiments with repetitive injections, mice were treated by intraperitoneal injections every third day starting from 8 days after inoculation of mice with palpable tumours. In the short-term experiment all mice were killed no later than day 20. The treatment groups, n = 10–20, were as follows:

  1. 1.

    PBS

  2. 2.

    1 × 1011 Fc-EVs alone

  3. 3.

    25 μg anti-PD-L1 (atezolizumab)

  4. 4.

    25 μg IgG human isotype control (catalogue number 12000C, Thermo Fisher),

  5. 5.

    50 μg Dox (Sigma-Aldrich)

  6. 6.

    Fc-EVs + PD-L1-Ab (11 × 1011 Fc-EVs which had been incubated for 2 h at 37 °C in 0.22 µm filtered PBS with 25 µg anti-PD-L1 (atezolizumab))

  7. 7.

    Fc-EVs + Dox (1 × 1011 Dox-loaded Fc-EVs alone (prepared as described above)

  8. 8.

    Fc-EVs + Dox + PD-L1-Ab (11 × 1011 Dox-loaded Fc-EVs which had been incubated for 2 h at 37 °C in 0.22 µm filtered PBS with 25 µg anti-PD-L1 (atezolizumab))

  9. 9.

    Fc-EVs + Dox + PD-L1-Ab (11 × 1011 Dox-loaded Fc-EVs which had been incubated for 2 h at 37 °C in 0.22 µm filtered PBS with 25 µg anti-PD-L1 (atezolizumab))

In the long-term experiment, the mice were killed no later than day 35 and were given treatment, n = 10, using groups 1, 8 and 9 as described above. The mice were observed daily, and tumour size was measured every third day using a calliper. Blood was collected when possible before killing the animals.

Preparation of single-cell suspensions from tissue

Mice were killed, and organs (liver, spleen, lymph nodes) were surgically resected and kept on ice submerged in appropriate amounts of PBS supplemented with 1% FBS until further processing. Next, tissues were homogenized by mashing the cells through a 70 μm Falcon cell strainer (Thermo Fisher Scientific) with a syringe plunger. The cell strainer was washed several times with ice-cold PBS/1% FBS buffer to elute all remaining cells into a 50 ml conical tube. Cell suspensions were filled up to 25 ml with ice-cold PBS/1% FBS. Here, 10% of liver suspensions were transferred into a new tube with sufficient amounts of PBS/1% FBS for further processing. Next, suspensions were centrifuged at 500 g for 5 min to collect the cells, and the supernatants were carefully decanted. Liver cells and spleen cells were resuspended in 500 µl 1× BD Pharm Lyse lysing solution (BD Biosciences) and incubated for 10 min at room temperature in the dark to lyse remaining red blood cells (RBCs). To stop the lysis, suspensions were filled up to 30 ml with ice-cold PBS/1% FBS and centrifuged at 500 g for 5 min. The supernatants were removed carefully by pipetting. Pellets from lymph node cell suspensions were not subjected to RBC lysis but processed in parallel in all subsequent steps. Next, cell pellets were washed by resuspension in 10 ml PBS/1% FBS, transferred to fresh 15 ml conical tubes and centrifuged at 500 g for 5 min. Supernatants were carefully removed, and pellets were resuspended in 500 µl PBS/1% FBS. The final cell suspensions were kept at 4 °C until further processing for flow cytometry.

Preparation of PBMCs from blood

To obtain suspensions of erythrocyte-depleted PBMCs, mice were killed, and 200 µl blood was collected into BD Microtainer tubes (BD Biosciences) containing EDTA by heart puncture. About 180 µl EDTA–blood was transferred to a 1.5 ml tube and centrifuged at 500 g for 7 min at 4 °C. The supernatant was removed, cells were gently resuspended in 200 µl FBS/1% PBS, transferred to 15 ml conical tubes containing 500 µl 1× BD Pharm Lyse lysing solution (BD Biosciences) and incubated for 10 min at room temperature in the dark to lyse RBCs. To stop the lysis, suspensions were filled up to 10 ml with ice-cold PBS/1% FBS and centrifuged at 500 g for 5 min. After supernatants were removed carefully, the RBC lysis step was repeated for 8 min, followed by lysis stop and centrifugation as before. The final cell pellet was resuspended in 500 µl PBS/1% FBS and kept at 4 °C until further processing for flow cytometry.

Preparation of single-cell suspension from B16F10 cell-engrafted tumours

After mice were killed, tumours were surgically resected, placed into a 60 mm dish, and submerged in room temperature PBS. The tissue was minced with a scalpel to obtain small pieces (1–3 mm3), and pieces were transferred into a 50 ml conical tube containing 5 ml pre-warmed B16F10 cell culture medium. The tissue was centrifuged at 100 g for 5 min at room temperature, and the supernatant was removed carefully. Next, tissue pieces were submerged in 4.7 ml pre-warmed B16F10 cell culture medium and 250 µl 20× collagenase II (2,500 collagen digestion unit ml−1 (20 mg ml−1, Sigma-Aldrich) and 50 µl 100× DNase I (10,000 Kunitz per ml, Sigma-Aldrich) were added to final concentrations of collagenase II and DNase I at 1 mg ml−1 and 100 Kunitz per ml, respectively. The tubes were fixed on a rocking platform and incubated at 37 °C for 60 min. After digestion, the cell suspensions were carefully pipetted for at least 25 times with a 10 ml serological pipette until the suspensions appeared homogenous without apparent tissue pieces. Next, the suspensions were filtered through a 70 μm Falcon cell strainer (Thermo Fisher Scientific) into a fresh 50 ml conical tube, followed by a subsequent filtration through a 40 μm Falcon cell strainer (Thermo Fisher Scientific) into another fresh 50 ml conical tube. The cell strainer was washed slowly with 10 ml of pre-warmed B16F10 cell culture medium to collect remaining cells. Cell suspensions were centrifuged at 500 g for 10 min to pellet the cells, and the supernatant was removed carefully. The cell pellets were resuspended in 500 µl cold PBS/1% FBS and kept at 4 °C until further processing for flow cytometry.

Human breast cancer HER2 positive model

Biodistribution studies were performed in SKBR-3-bearing Swiss nude mice as previously described35. Briefly, 35 six-week-old female Swiss nude mice (Charles River) were orthotopically grafted (mammary fat pad) with 5 × 106 SKBR-3 cells in 60% matrigel (CLS354234-1EA, Sigma-Aldrich). They were monitored daily and developed palpable tumours within 2 months. The mice were killed at pre-decided time points if the tumour size exceeded 1,500 mm3 or if the mice exceeded the scoring of pre-set humane end points. The mice (n = 5–9) were injected intravenously on day 60 with 1 × 1011 Fc-EVs, which had been incubated for 2 h at 37 °C in 0.22 µm filtered PBS with 25 µg anti-HER2 (trastuzumab) or 25 µg human IgG isotype control antibody (catalogue number 12000C, Thermo Fisher). Thirty minutes after the injection, the mice were killed for tissue and blood collection. The tissues were processed as described in ref. 34, with lysed tissue and nLuc+ EV input being analysed for nanoluc luminescence or for single-cell suspension for flow cytometry, as described above, but with the HER2 Ab (R&D Systems, FAB9896V-100UG) used for flow cytometry applications.

Data analysis

Statistical analyses of the data were performed using Prism 8.0 (GraphPad) by one- or two-way analysis of variance (ANOVA) for all P values. All results are expressed as mean ± s.d. with upper and lower limits. Graphs were made in Prism 8.0 (GraphPad).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.