Fig 2: Aposome nanoparticles made using nanofluidic synthesis method show higher apoB-100 enrichment of nanoparticles compared to TLE method – (A) Size, polydispersity index and surface charge for nanoparticles (NPs) made using a nanofluidic method and thin layer evaporation using two different protein sources for LDL derived from two different plasma fractions (B) Flow cytometry analysis of surface apoB-100 show that NPs made using a microfluidic method show higher LDL enrichment than similar particles synthesized using thin layer evaporation, based on these results it was determined that particles made with FPLC fraction 13 and using a microfluidic method was most effective for LDL enrichment. From this point onward Aposome refers to these particles unless specified otherwise (C) Cryo-TEM imaging of liposomes and Aposomes, Aposomes show increased membrane thickness of the lipid bilayer assessed using Fiji software.

Fig 3: Separation of extracellular vesicles (EVs), lipoproteins, and free proteins from plasma using density gradient centrifugation.Levels of CD9, CD63, CD81, albumin, and ApoB-100 were measured by Simoa in individual 1 ml fractions (collected from the top) after density gradient centrifugation of plasma using an iodixanol gradient. Error bars represent the standard deviation of two replicates of each measurement. Figure 3—source data 1.Simoa data (protein concentrations) for different density gradient centrifugation fractions.

Fig 4: Comparison of novel columns for extracellular vesicle (EV) isolation from plasma using electron microscopy and Simoa.(A) Schematic of the columns being compared: size exclusion chromatography (SEC) column comprised of 10 ml Sepharose CL-6B, dual-mode chromatography (DMC) columns comprised of 10 ml Sepharose CL-6B SEC resin atop 2 ml Fractogel cation exchange resin, Tri-Mode Chromatography (TMC) columns comprised of 10 ml Sepharose CL-6B SEC resin atop 2 ml 2:1 ratio of 2 ml Fractogel cation exchange resin to Capto Core 700 multimodal chromatography resin. (B) Electron microscopy of EVs isolated from plasma using SEC (left), DMC (middle), or TMC (right) columns. EVs indicated with red arrows (among background of lipoproteins). (C) EV recovery is calculated for EV isolation from plasma for SEC (fractions 7–10), DMC (fractions 9–12), or TMC (fractions 9–12). Simoa measurements in the designated fractions for CD9, CD63, and CD81 are taken as a ratio relative to measurements of these proteins from diluted plasma and these three ratios are then averaged to calculate recovery. (D) Purity of EVs with respect to free proteins is determined by dividing relative EV yield (the average of the ratios of CD9, CD63, and CD81) by relative levels of albumin in each condition. (E) Purity of EVs with respect to lipoproteins is determined by dividing relative EV yield (the average of the ratios of CD9, CD63, and CD81) by relative levels of ApoB-100 in each condition. Error bars represent the standard deviation of four column measured on different days with two technical replicates each. Figure 4—source data 1.Simoa data (protein concentrations) for TMC columns with different ratios of resins in the bottom layer. Figure 4—source data 2.Simoa data (protein concentrations) for different fractions of SEC and DMC columns. Figure 4—source data 3.Simoa data (protein concentrations) comparing SEC (fractions 7-10), DMC and TMC columns (fractions 9-12).

Fig 5: The EV-CATCHER assay releases functional small-EVs. a. Six convalescent serum samples (Second set of covid-19 serum samples) distributed in two groups based on presence of immunoglobulins (IgG) targeting the Receptor Binding Domain (RBD) of SARS-CoV-2 spike protein as antigen. Three high anti-spike IgG titer (left) and three below level of quantification (BLQ) anti-spike IgG titer serum samples were quantified by ELISA (Dr. Perlin's Laboratory). High anti-spike IgG titers were estimated with activity remaining at a 10,000x (fold) dilution, while below level of quantification (BLQ) IgG serum samples did not contain detectable IgG against the spike protein RBA region of SARS-CoV-2 at the same dilution. b. Western blot analyses of CD63+ small-EVs purified with anti-CD63 EV-CATCHER from sera of high anti-spike (RBD region) IgG (lanes 1–3) and BLQ IgG (lanes 4–6) serum samples using anti-ApoB, -Alix, -CD63, -Albumin, -ApoA1, -CD9, and -CD81 antibodies. c. Representative Transmission electron microscopy (TEM) images of small-EVs isolated from high anti-spike IgG (CDI -001, Figure 6b - Lane 1) and BLQ anti-spike IgG (CDI-004, Figure 6b - Lane 6) convalescent sera using the anti-CD63 EV-CATCHER assay. Direct magnification was 20,000x and scale bars are for 200 nm. d. Nanoparticle size distribution characterized using the Spectradyne nCS1 instrument with a TS400 microfluidic cartridges of CD63+ small-EVs purified from all six serum donors using the anti-CD63 EV-CATCHER assay. e. Schematic representation of the experimental procedure used to obtain high-purity small-EVs from convalescent sera by ultracentrifugation. f. In vitro assessment of SARS-CoV-2 infection using Vero E6 cells treated with whole sera or small-EVs purified from the different sera. Healthy Vero E6 cells Control (bar 1, no virus) were subjected to mNG SARS-CoV-2 (bar 2, virus), small-EVs ultracentrifuged (EV control) from serum with high anti-spike IgG (bar 3) and serum with BLQ anti-spike IgG (bar 4), mNG SARS-CoV-2 with whole sera with high anti-spike IgG (bars 5, 7 and 9) or BLQ anti-spike IgG (bars 11, 13 and 15), mNG SARS-CoV-2 after Vero E6 cells were treated with CD63+ small-EVs purified with anti-CD63 EV-CATCHER from high anti-spike IgG sera (bars 6, 8, and 10), or with CD63+ small-EVs purified with anti-CD63 EV-CATCHER from BLQ anti-spike IgG sera (bars 12, 14, and 16), and finally UNG digest buffer (bar 17) and UNG digest buffer with mNG SARS-CoV-2 (bar 18) for 72 h. Statistical analyses were performed using one-way ANOVA with Bonferroni post hoc correction. All results are presented as mean ± SEM (n = 3), and **** = P < 0.0001 vs. virus. g. Fluorescent imaging of Vero E6 cells infected with the mNeonGreen SARS-CoV-2 reporter virus. Hoechst 33342 and mNeonGreen was visualized using fluorescent Celigo Cell Imaging. Representative fluorescent images display healthy Vero E6 cells (no virus control), infected with mNG SARS-CoV-2 and treated with high anti-spike IgG whole serum (high anti-spike IgG serum), infected with mNG SARS-CoV-2 and treated with CD63+ small-EVs purified with the anti-CD63 EV-CATCHER assay from high anti-spike IgG serum (High anti-spike IgG patient# CDI-001), infected with mNG SARS-CoV-2 (virus control), infected with mNG SARS-CoV-2 with BLQ anti-spike IgG whole serum (BLQ IgG serum; Patient # CDI-004), infected with mNG SARS-CoV-2 and treated with CD63+ small-EVs purified with the anti-CD63 EV-CATCHER assay from BLQ anti-spike IgG serum (small-EVs (BLQ serum)). Scale bars on images was at 500 µM