Cholesterol-Anchored Ac-SDKP Modified Platelet sEVs Effectively Deliver Anti-Inflammatory Peptides

סיכום

A protocol for preparing platelet-derived small extracellular vesicles (sEVs) and functionalizing them with the anti-inflammatory tetrapeptide Ac-SDKP through cholesterol-mediated membrane insertion is described. The engineered vesicles exhibit efficient peptide incorporation, minimal cytotoxicity, preserved hemostatic function, and enhanced suppression of endothelial inflammatory responses compared with free peptide.

תקציר

Small extracellular vesicles (sEVs) released from thrombin-activated platelets carry adhesion proteins inherited from the parent cell membrane. Whether these vesicles can function as carriers for short anti-inflammatory peptides depends on a methodical evaluation of loading performance, biological compatibility, and intrinsic inflammatory potential. This protocol describes the isolation of platelet sEVs by thrombin-triggered vesiculation and differential ultracentrifugation, followed by surface decoration with the anti-inflammatory tetrapeptide Ac-SDKP via a cholesterol-polyhistidine anchor. The isolated vesicles were 90–300 nm in diameter, spherical by transmission electron microscopy, and positive for the platelet markers CD41 and CD62P. Cholesterol-directed membrane insertion gave loading efficiencies of 93.7% to 97.0% across four peptide-to-vesicle feeding ratios. The drug payload rose from 9.75% to 37.5% (w/w) as peptide input increased, without measurable change in vesicle diameter or zeta potential. Incubation with human coronary artery endothelial cells or human umbilical vein endothelial cells for 24 h produced no loss of metabolic activity at the concentrations tested. A single intravenous dose of unmodified sEVs at 20 mg/kg in mice did not prolong tail bleeding time or alter serum ALT, AST, BUN, or creatinine over a 7-day observation window. Histological examination of the lung, liver, spleen, and kidney showed no evidence of injury. Thrombin stimulation triggered substantial release of TNF-alpha, IL-6, and IL-1beta from intact platelets; the sEV fraction released negligible amounts of these cytokines, irrespective of thrombin exposure. In lipopolysaccharide-treated endothelial monolayers, Ac-SDKP-decorated sEVs suppressed cytokine secretion more effectively than an equal concentration of free Ac-SDKP. These results describe a practical method for generating peptide-functionalized platelet sEVs and provide an initial dataset on their pharmaceutical properties, supporting further investigation of this platform for anti-inflammatory delivery in vascular disorders.

מבוא

Kawasaki disease (KD) is an acute febrile illness of early childhood. The inflammatory process targets medium-sized arteries, and the coronary circulation bears the greatest burden. Complications include dilatation, aneurysm formation, thrombosis, and stenosis^1,2,3. High-dose intravenous immunoglobulin combined with acetylsalicylic acid remains the standard first-line treatment. This regimen lowers aneurysm rates substantially, but 10%–20% of affected children do not respond. Those with IVIG resistance carry a higher risk of developing coronary artery lesions^4,5,6. Current management relies on broad immunosuppression and platelet inhibition. Neither strategy achieves site-specific anti-inflammatory activity at the coronary wall, and both carry liabilities. Systemic immunosuppression increases infection risk. Platelet inhibition raises the hazard of bleeding^{7,8,9,10,11,12}.

The endogenous tetrapeptide Ac-SDKP (N-acetyl-Ser-Asp-Lys-Pro) is produced through sequential cleavage of thymosin-beta4 by meprin-alpha and prolyl oligopeptidase¹³. Anti-fibrotic and inflammation-resolving effects have been documented in models of angiotensin II-induced hypertension, myocardial infarction, silica-induced pulmonary fibrosis, and diabetic nephropathy^14,15. Translation of these findings is limited by pharmacokinetics. Angiotensin-converting enzyme (ACE) cleaves the Asp-Lys bond with high catalytic efficiency, and the plasma half-life of the free peptide is measured in minutes¹⁶. The molecule is small, highly hydrophilic, and lacks endogenous homing domains. These properties restrict the duration and local concentration achievable with bolus parenteral administration. Incorporating Ac-SDKP into a biogenic nanoparticulate carrier could offer a path around these obstacles. Shielding from circulating peptidases, extended residence time, and preferential accumulation at sites of vascular inflammation are all feasible with an appropriately designed carrier.

Vesicles shed from the plasma membrane of activated platelets, designated here as platelet-derived small extracellular vesicles (platelet sEVs), are lipid bilayer particles with diameters typically between 100–400 nm^17,18,19. Their surface retains a subset of platelet transmembrane and membrane-associated proteins, notably integrin alphaIIb-beta3 (CD41/CD61) and P-selectin (CD62P). The latter engages PSGL-1 on leukocytes and on activated endothelium. This interaction provides a molecular basis for sEV adhesion to inflamed vascular segments^20,21. Platelet sEVs differ from their parent cells in an important respect. They lack nuclei, mitochondria, dense tubular systems, secretory granules, and translational machinery. Consequently, they are incapable of mounting a sustained, agonist-driven secretory response^22,23. The retention of platelet-derived surface addressins, combined with the loss of granule-based secretory capacity, has motivated investigation of platelet sEVs as biomimetic vectors for small-molecule and peptide drugs^24,25,26.

This protocol proceeds through five stages: (1) preparation of platelet sEVs from washed mouse platelets by controlled thrombin activation and differential ultracentrifugation; (2) surface functionalization with Ac-SDKP using a synthetic cholesterol-hexahistidine conjugate; (3) physicochemical characterization by electron microscopy, dynamic light scattering, electrophoretic light scattering, and immunoblotting; (4) evaluation of in vitro metabolic activity and in vivo hemostatic function, serum chemistry, and organ histology; and (5) assessment of cytokine release from the sEV carrier and of the anti-inflammatory potency of Ac-SDKP-decorated sEVs in lipopolysaccharide-stimulated endothelial monolayers.

פרוטוקול

All procedures involving animals were reviewed and approved by the Animal Ethics Committee of Soochow University (approval no. SUDA20241104A02) and were performed in compliance with the 3R principles (Replacement, Reduction, Refinement). The reagents and the equipment used are listed in the Table of Materials.

1. Animals, cell lines, and reagents
Male C57BL/6J mice (specific-pathogen-free grade, 4 weeks of age, 11–13 g body weight) were obtained from a commercial supplier (see Table of Materials) and acclimatized for at least 5 d before use. Animals were group-housed under a 12 h/12 h light/dark cycle at 22 °C ± 2 °C with free access to standard chow and autoclaved water. Human coronary artery endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) were propagated in endothelial growth medium-2 (EGM-2) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin solution. Cultures were maintained in a humidified incubator at 37 °C under 5% CO₂. Only cells between passages 3 and 6 were used experimentally. Detailed supplier information, catalog identifiers, and antibody dilutions for all reagents are compiled in the Table of Materials.

2. Preparation and purification of platelet-derived small extracellular vesicles
Whole blood (approximately 0.8–1.0 mL per mouse) was drawn by cardiac puncture under deep isoflurane anesthesia (5% in oxygen for induction, 2%–3% for maintenance, delivered via a precision vaporizer). The collecting syringes were pre-loaded with acid-citrate-dextrose (ACD) solution at a 1:9 (v/v) ratio of ACD to anticipated blood volume. Prostaglandin E1 (PGE1, 1 uM final concentration) was added immediately to limit platelet preactivation during handling. Blood from 6–8 mice was combined for each independent preparation.

The anticoagulated blood pool was centrifuged at 200 x g for 10 min at 22–25 °C to generate platelet-rich plasma (PRP). The upper PRP layer was transferred to a fresh polypropylene tube, taking care not to disturb the buffy coat, and PGE1 was replenished (1 uM). Platelets were pelleted by centrifugation at 800 x g for 15 min at room temperature. The supernatant platelet-poor plasma was discarded. The platelet pellet was gently resuspended in modified Tyrode buffer (composition: 137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl₂, 1.8 mM CaCl₂, 0.4 mM Na₂HPO₄, 5.5 mM D-glucose, 5.0 mM HEPES; titrated to pH 7.4) containing 1 µM PGE1. The suspension was centrifuged at 800 x g for 10 min; this wash step was performed twice. Following the final wash, platelets were suspended in PGE1-free Tyrode buffer at 1 x 10⁹ platelets per mL and rested at 37 °C for 30 min to recover responsiveness.

Vesiculation was triggered by adding bovine thrombin to a final activity of 1 U/mL and incubating the platelet suspension at 37 °C for 30 min with orbital agitation (approximately 60 rpm). The reaction was quenched with hirudin (2 U/mL final). The activated mixture was centrifuged at 2,000 x g for 20 min at 4 °C to sediment platelet remnants and large fragments. The supernatant was collected and subjected to a second clarification spin at 10,000 x g for 30 min at 4 °C. The clarified supernatant was ultracentrifuged at 100,000 x g for 90 min at 4 °C (Type 70 Ti rotor, k-factor approximately 44). The resulting sEV-enriched pellet was resuspended in sterile-filtered Dulbecco phosphate-buffered saline (D-PBS, pH 7.4, Ca2+ and Mg2+-free) and washed by a second identical ultracentrifugation step. The final washed pellet was gently resuspended in a minimal volume of D-PBS, and total protein content was quantified by the bicinchoninic acid (BCA) assay using bovine serum albumin as the calibration standard. Aliquots were used immediately or stored at -80 °C. Throughout this protocol, vesicle doses and concentrations are expressed as total protein mass unless noted otherwise.

Per MISEV2023 recommendations, and because the 100,000 x g pellet contains particles of heterogeneous biogenic origin, the isolated material is designated as small extracellular vesicles (sEVs). The term platelet sEVs refers specifically to sEVs derived from thrombin-activated mouse platelets.

3. Physicochemical characterization of platelet sEVs
For transmission electron microscopy (TEM), a 10 µL droplet of vesicle suspension (approximately 0.1 mg protein per mL) was deposited onto a glow-discharged, carbon-coated 300-mesh copper grid. After 2 min of adsorption, excess fluid was wicked away with filter paper, and the grid was floated on a drop of 2% (w/v) aqueous uranyl acetate for 60 s for negative staining. Grids were air-dried and examined at 80 kV accelerating voltage. Digital micrographs were captured at instrument magnifications of 20,000x to 50,000x.

Hydrodynamic diameter and polydispersity index (PDI) were determined by dynamic light scattering (DLS). Vesicle samples were diluted in 0.22 µm-filtered D-PBS to a protein concentration of approximately 0.05 mg/mL and placed in disposable polystyrene cuvettes. Measurements were acquired at a fixed scattering angle of 90° with the sample chamber equilibrated at 25 °C. Each reported size distribution represents the intensity-weighted mean of three consecutive runs of 10–15 acquisitions each. Resting and thrombin-activated platelet suspensions were analyzed at equivalent particle counts. For storage stability, aliquots of freshly prepared vesicles were stored at minus -80 °C, and size measurements were repeated at 0, 1, 3, and 7 days.

Electrophoretic mobility was measured by laser Doppler micro-electrophoresis. Samples were diluted in 10 mM NaCl (pH 7.4) and injected into folded capillary cells. Zeta potential was calculated from electrophoretic mobility using the Smoluchowski approximation. All measurements were performed in triplicate at 25 °C.

For immunoblotting, platelet and vesicle lysates (20 µg total protein per lane) were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels under reducing conditions and electrotransferred to 0.22 µm polyvinylidene difluoride membranes. Membranes were blocked in 5% (w/v) non-fat dry milk dissolved in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) for 1 h at 22–25 °C and then incubated overnight at 4 °C with primary antibodies recognizing CD41 (1:1,000), CD62P (1:1,000), or Na+/K+-ATPase (1:2,000, loading control). After three 10-min washes in TBST, blots were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000) for 1 h at room temperature. A chemiluminescent signal was developed with an enhanced chemiluminescence substrate and captured with a digital imaging system. Band intensities were quantified with ImageJ.

4. Construction of peptide-functionalized sEVs and determination of loading performance
A synthetic Ac-SDKP variant modified at the C-terminus with a hexahistidine spacer and a cholesterol moiety (Ac-SDKP-His6-CHOL) was obtained by solid-phase peptide synthesis. The cholesterol group was coupled to the epsilon-amino group of a C-terminal lysine through an amide linkage; the His6 segment was placed between the peptide sequence and the lipid anchor to improve aqueous solubility and to provide additional interfacial contacts with the phospholipid headgroup region.

For membrane incorporation, platelet sEVs (200 µg total protein) were combined with Ac-SDKP-His6-CHOL at four mass ratios: 200:20, 200:40, 200:60, and 200:80 (µg vesicle protein to ug peptide), in a final volume of 200 µL D-PBS. These ratios were selected to span a range of surface-density conditions, from low coverage (approximately 10% peptide-to-carrier, w/w) to near-saturation, based on pilot experiments. Mixtures were incubated at 37 °C for 2 h with gentle agitation (300 rpm on a thermomixer). Unincorporated peptide was removed by ultracentrifugation at 100,000 x g for 90 min at 4 °C. The supernatant was collected for the determination of free peptide, and the Ac-SDKP-sEV pellet was resuspended in D-PBS.

Free Ac-SDKP was measured with a competitive ELISA kit. Loading efficiency (LE) and drug-loading content (DLC, w/w) were computed as: LE (%) = (B / T) x 100; DLC (%) = [B / (m_PEV + B)] x 100, where T is the total mass of peptide added and B is the bound mass (T minus free peptide in the supernatant). All determinations were carried out in triplicate.

5. In vitro biocompatibility assessment
HCAECs and HUVECs were plated in 96-well plates at 5,000 cells per well in 100 µL complete EGM-2 and allowed to adhere overnight. Culture supernatants were replaced with fresh medium containing unmodified sEVs at final total protein concentrations of 1.25, 2.5, 5, 10, 20, and 40 µg/mL, or Ac-SDKP-sEVs at Ac-SDKP-equivalent concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 µg/mL. After 24 h of co-incubation at 37 °C, the treatment medium was aspirated, cells were rinsed once with D-PBS, and 100 µL of fresh medium containing 10% (v/v) CCK-8 reagent was added per well. The plate was incubated for a further 2 h, after which absorbance at 450 nm was recorded on a microplate spectrophotometer. Cell viability was expressed as a percentage of the absorbance obtained from untreated control wells on the same plate. For each concentration, three technical replicates were included in each of three independent biological experiments.

6. In vivo safety evaluation
Tail bleeding time was determined in C57BL/6J mice (n = 6 per treatment group) under isoflurane anesthesia (3% for induction, 1.5%–2% for maintenance in oxygen). The distal 3 mm of the tail was transected with a number 10 scalpel blade, and the tail was immediately submerged in sterile 0.9% (w/v) NaCl pre-equilibrated to 37 °C. The interval from transection to the first 30-s period without visible bleeding was recorded. Mice received a single injection via the lateral tail vein (PBS vehicle, freshly prepared washed mouse platelets at 20 mg/kg total protein, or platelet sEVs at 20 mg/kg total protein) 30 min before the bleeding time measurement.

For evaluation of systemic toxicity, C57BL/6J mice (n = 5 per group) received a single intravenous bolus of platelet sEVs at 20 mg/kg total protein or an equivalent volume of sterile PBS. On days 1 and 7 post-injection, animals were anesthetized with isoflurane (3% induction, 1.5%–2% maintenance), and approximately 200 µL of blood was drawn from the retro-orbital venous plexus. Serum was obtained by centrifugation at 3,000 x g for 10 min at 4 °C and analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) with an automated clinical chemistry analyzer. On day 7, immediately after blood collection, mice were euthanized by cervical dislocation while still under deep anesthesia. Lung, liver, spleen, and kidney were excised, immersion-fixed in 4% (w/v) paraformaldehyde in PBS (pH 7.4) for 24 h at 4 °C, dehydrated in graded ethanol, cleared with xylene, and embedded in paraffin. Sections of 5 µm thickness were cut on a rotary microtome and stained with hematoxylin and eosin (HE). Slides were examined on a bright-field microscope at total magnifications of 100x and 400x by an observer blinded to treatment assignment.

7. Evaluation of the cytokine release profile of platelet sEVs
Freshly isolated mouse platelets (1 x 10⁸ per mL) and platelet sEVs (100 µg protein per mL) were suspended in Tyrode buffer. Samples were incubated at 37 °C for 30 min in the presence or absence of thrombin (1 U/mL). Following incubation, samples were centrifuged at 13,000 x g for 5 min at 4 °C, and the cell- and vesicle-free supernatants were harvested. Murine TNF-alpha, IL-6, and IL-1beta concentrations were measured with commercial sandwich ELISA kits; absorbance at 450 nm was read on the microplate spectrophotometer described above. Cytokine concentrations were interpolated from four-parameter logistic standard curves. All determinations were performed in triplicate across three independent biological replicates.

8. In vitro anti-inflammatory activity of Ac-SDKP-functionalized sEVs
HCAECs and HUVECs were seeded in 24-well plates at 50,000 cells per well and cultured to approximately 90% confluence. Endothelial inflammation was induced by exchanging the growth medium for fresh EGM-2 containing 1 µg per mL lipopolysaccharide (LPS, from Escherichia coli O111:B4). Concurrently with LPS stimulation, wells received one of the following: (1) D-PBS (LPS-only reference), (2) Ac-SDKP-sEVs at an Ac-SDKP-equivalent concentration of 1 µg/mL, (3) free Ac-SDKP at 1 µg per mL, or (4) unmodified sEVs at a protein concentration matched to the Ac-SDKP-sEV condition. Untreated, unstimulated wells served as the baseline control. After 8 h of incubation at 37 °C, conditioned medium was collected and cleared by centrifugation at 13,000 x g for 5 min. Human TNF-alpha, IL-6, and IL-1beta in the supernatants were quantified by ELISA, as described above. Each treatment condition was assayed in triplicate within three independent biological experiments.

9. Statistical analysis
Summary data are reported as mean ± standard deviation (SD). Comparisons between two groups were performed with the unpaired, two-tailed Student t-test. For comparisons among three or more groups, one-way analysis of variance (ANOVA) was applied, and pairwise differences were evaluated with the Tukey honestly significant difference (HSD) post hoc procedure. The threshold for statistical significance was set at p < 0.05. Computations were carried out in statistical or graphing software. Exact sample sizes (n) and definitions of replicates (biological versus technical) are stated in the respective figure legends.

תוצאות

Isolation and characterization of platelet-derived small extracellular vesicles
The sEV preparation workflow is outlined in Figure 1A. Negative-stain TEM showed spherical, membrane-delimited particles (Figure 1B). DLS of resting platelets gave a single broad peak centered near 1,990 nm (Figure 1C). After thrombin treatment, a second population appeared in the 100–300 nm range (Figure 1D). The purified sEV fraction was distributed between 90–300 nm, with the main intensity peak at approximately 141.8 nm (Figure 1E). Zeta potential values were negative for both platelets and sEVs and did not differ measurably between the two (Figure 1F). The mean hydrodynamic diameter of sEVs stored at −80 °C remained stable over the 7-day monitoring period, with measurements performed on days 0, 3, 5, and 7. (Figure 1G). Immunoblots confirmed the presence of CD41 and CD62P in the sEV preparation. The relative abundance of CD62P was comparable to that of unstimulated platelets (Figure 1H–J).

Cholesterol-anchored incorporation of Ac-SDKP
Figure 2A,B show the chemical structure of Ac-SDKP-His6-CHOL and a schematic of the loading strategy. Loading efficiencies exceeded 93% across the four feeding ratios: 97.01% ± 0.18% (200:20), 96.37% ± 0.13% (200:40), 96.11% ± 0.10% (200:60), and 93.69% ± 0.38% (200:80) (Figure 2C). Drug-loading content increased with peptide input, from 9.70% ± 0.02% to 37.48% ± 0.15% (Figure 2D). Neither median hydrodynamic diameter nor zeta potential changed measurably after peptide decoration (Figure 2E,F; p > 0.05).

Cytocompatibility and preliminary in vivo tolerability
Unmodified sEVs at concentrations of 1.25–40 µg/mL and Ac-SDKP-sEVs at Ac-SDKP-equivalent concentrations of 0.05–1.6 µg/mL did not significantly reduce metabolic activity in either HCAEC or HUVEC monolayers after 24 h (Figure 3A,B). Tail bleeding times were 166.3 s ± 23.2 s (PBS), 161.0 s ± 17.7 s (platelets, 20 mg/kg), and 164.7 s ± 19.8 s (sEVs, 20 mg/kg). No intergroup differences were detected (p > 0.05; Figure 3C). Serum ALT, AST, BUN, and CREA concentrations remained at control levels on days 1 and 7 post-injection (Figure 3D). HE sections of lung, liver, spleen, and kidney showed no inflammatory infiltrates, necrosis, or architectural disruption on day 7 (Supplementary Figure 1).

Cytokine release by sEVs and anti-inflammatory potency of Ac-SDKP-sEVs
Under basal conditions, intact platelets and sEVs both released small quantities of TNF-alpha, IL-6, and IL-1beta. Thrombin stimulation evoked a clear, statistically significant increase in all three cytokines from platelets but did not increase cytokine concentrations in sEV-conditioned supernatants (Figure 4A–C).

LPS treatment raised TNF-alpha, IL-6, and IL-1beta secretion by HCAECs and HUVECs well above baseline (Figure 4D–F). Free Ac-SDKP (1 µg per mL) produced a modest attenuation. At the same peptide-equivalent concentration, Ac-SDKP-sEVs suppressed all three cytokines to a greater extent than free Ac-SDKP (p < 0.05 for each comparison). Blank sEVs, applied at the matched protein concentration, did not alter the LPS-induced cytokine profile.

DATA AVAILABILITY:
Raw data supporting all figures and the corresponding statistical analyses have been deposited in a public repository (ScienceDB; https://www.scidb.cn) and are freely accessible.

Figure 1: Preparation, physicochemical characterization, and verification of the platelet origin of platelet-derived small extracellular vesicles. (A) Schematic workflow for the preparation of small extracellular vesicles (sEVs) from mouse platelets. (B) Representative transmission electron microscopy (TEM) image of purified sEVs following uranyl acetate negative staining. Scale bar = 200 nm. (C) Dynamic light scattering (DLS) intensity-weighted size distribution of resting platelets. (D) Size distribution of thrombin-activated platelets, showing the emergence of a 100–300 nm particle population. (E) Size distribution of purified sEVs, with a principal peak at approximately 141.8 nm. (F) Zeta potential of resting platelets and sEVs (n = 3). (G) Hydrodynamic diameter of sEVs during storage at −80 °C for 7 days, measured on days 0, 3, 5, and 7 (n = 3). (H) Immunoblot analysis of CD41, CD62P, and Na+/K+-ATPase in lysates of resting platelets and sEVs. (I,J) Densitometric quantification of CD41 and CD62P normalized to Na+/K+-ATPase (n = 3). Data are presented as the mean ± standard deviation (SD). Please click here to view a larger version of this figure.

Figure 2: Preparation and loading efficiency of Ac-SDKP-functionalized small extracellular vesicles. (A) Chemical structure of Ac-SDKP. (B) Schematic illustration of Ac-SDKP-His6-CHOL incorporation into the sEV lipid bilayer through cholesterol-mediated membrane anchoring. (C) Loading efficiency at platelet extracellular vesicle (PEV)-to-peptide feeding mass ratios of 200:20, 200:40, 200:60, and 200:80 (n = 3). (D) Drug-loading content (% w/w) at the corresponding feeding ratios (n = 3). (E) Median hydrodynamic diameter of sEVs before and after peptide incorporation (n = 3). (F) Zeta potential of sEVs before and after peptide incorporation (n = 3). Data are presented as the mean ± SD. Please click here to view a larger version of this figure.

Figure 3: Biocompatibility and preliminary in vivo safety evaluation of platelet-derived sEVs and Ac-SDKP-sEVs. (A) Cell metabolic activity of HCAECs and HUVECs following 24 h incubation with unmodified sEVs at 1.25, 2.5, 5, 10, 20, and 40 µg/mL. (B) Cell metabolic activity of HCAECs and HUVECs following 24 h incubation with Ac-SDKP-sEVs at Ac-SDKP-equivalent concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 µg/mL. (C) Tail bleeding time in mice measured 30 min after intravenous administration of phosphate-buffered saline (PBS; control), washed platelets (20 mg/kg), or sEVs (20 mg/kg) (n = 6 per group). (D) Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) levels measured on days 0, 1, and 7 after a single intravenous dose of sEVs (20 mg/kg) (n = 5 per group). Data are presented as the mean ± SD. Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CREA, creatinine. Please click here to view a larger version of this figure.

Figure 4: Cytokine release profile of platelet-derived sEVs and in vitro anti-inflammatory activity of Ac-SDKP-sEVs. (A–C) Tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β concentrations in the supernatants of intact platelets and platelet-derived sEVs under basal (unstimulated) and thrombin-stimulated (1 U/mL) conditions (n = 3). (D–F) Effects of PBS (LPS control), Ac-SDKP-sEVs (1 µg/mL Ac-SDKP equivalent), free Ac-SDKP (1 µg/mL), and blank sEVs (protein concentration matched) on TNF-α, IL-6, and IL-1β secretion by lipopolysaccharide (LPS; 1 µg/mL)-stimulated HCAEC and HUVEC monolayers (n = 3). Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 versus the indicated comparison groups. Abbreviation: ns, not significant. Please click here to view a larger version of this figure.

Supplementary Figure 1: Histopathological evaluation of major organs following administration of platelet-derived small extracellular vesicles. Representative hematoxylin and eosin (HE)-stained sections of the lung, liver, spleen, and kidney collected 7 days after a single tail-vein injection of PBS (control) or platelet-derived sEVs (20 mg/kg). Upper panels: scale bar = 100 µm. Lower panels: scale bar = 50 µm. Please click here to download this file.

דיון

This protocol describes the preparation of platelet sEVs, their surface engineering with a cholesterol-tagged anti-inflammatory peptide, and an initial set of pharmaceutical and functional characterizations. Four main findings emerge. First, thrombin-initiated vesiculation combined with stepwise centrifugation yields platelet sEVs that display the expected nanoscale morphology, express platelet-characteristic membrane proteins, and maintain colloidal stability for at least 7 days at -80 °C. Second, a cholesterol-polyhistidine Ac-SDKP conjugate incorporates into the sEV membrane with high efficiency. The payload is tunable across a wide range, and the loading process does not perturb vesicle diameter or surface charge. Third, both unmodified and peptide-decorated sEVs are well tolerated by cultured endothelial cells at the concentrations tested. A single intravenous dose of 20 mg/kg does not impair hemostatic plug formation or produce biochemical or histological evidence of organ injury in mice over a 7-day observation window. Fourth, platelet sEVs release negligible amounts of TNF-alpha, IL-6, and IL-1beta in response to thrombin, in contrast to the brisk secretory response of intact platelets. Ac-SDKP-decorated sEVs suppress LPS-induced endothelial cytokine secretion more effectively than an equal concentration of free peptide.

The cholesterol-anchoring approach exploits the native phospholipid bilayer as a passive insertion matrix. No covalent modification of surface proteins is required, which reduces the risk of altering bioactivity or immunogenicity^27,28,29. The hexahistidine spacer between the peptide and the cholesterol moiety positions the tetrapeptide at the membrane-water interface, where it remains sterically accessible. This design element probably contributes to the high loading efficiencies observed. The payload could be adjusted from approximately 10% to nearly 40% (w/w) simply by changing the peptide-to-vesicle ratio. At the highest feeding ratio tested (200:80), loading efficiency declined modestly, from above 96% to approximately 94%. This decline is consistent with the approach of a saturation limit, at which further cholesterol conjugate insertion begins to perturb bilayer packing. The tunability of the system, together with this apparent ceiling, provides practical guidance for selecting dosing ratios in future efficacy experiments.

The cytokine release data require careful interpretation. Platelet sEVs are not biologically inert. They carry P-selectin, integrins, and other surface molecules capable of engaging leukocytes and endothelium³⁰. The data demonstrate a narrower point: under the specific conditions tested, thrombin does not trigger cytokine output from sEVs the way it does from intact platelets. This dissociation is mechanistically plausible³¹. The sEVs have shed the dense granules, alpha-granules, mitochondria, and signal transduction infrastructure that support agonist-driven secretion in the parent cell. Several limitations of the cytokine assessment should be noted. The panel was restricted to three analytes (TNF-alpha, IL-6, IL-1beta) and did not include CXCL4, soluble CD40L, CCL5, TGF-beta1, or other platelet-derived mediators. Only one agonist (thrombin) was tested. Experiments were conducted in a simplified buffer system rather than whole blood or tissue. Primary macrophages and monocytes were not examined³². Stimulation conditions beyond rest versus thrombin, including spontaneous release, collagen, ADP, and calcium ionophore, were not evaluated. The observation that platelet sEVs show negligible thrombin-dependent cytokine output is therefore a defined experimental result, not a general claim of immunological safety. Broader inflammatory profiling, including alternative agonists, additional cell types, and in vivo models of vascular inflammation, is necessary to establish the full immunological profile of this carrier^33,34,35.

The basis for the enhanced anti-inflammatory activity of Ac-SDKP-sEVs relative to free peptide remains to be determined. At least three mechanisms could contribute. Membrane anchoring may shield the tetrapeptide from ACE-mediated proteolysis in the culture medium, effectively extending its functional half-life. Multivalent display of Ac-SDKP on the vesicle surface could increase binding avidity for the molecular target. P-selectin on the sEV surface may mediate adhesion to activated endothelium via PSGL-1, concentrating the peptide payload at the target cell surface. Distinguishing among these possibilities will require peptide degradation kinetics, receptor-blocking antibodies, and quantitative fluorescence imaging of vesicle-endothelial interactions.

Several limitations of the protocol should be noted. First, the characterization does not fully meet MISEV2023 standards³³. Generic EV markers (CD9, CD63, CD81, TSG101, Alix) and negative markers (calnexin, GM130) were not evaluated. Particle concentration per microgram of protein was not measured by nanoparticle tracking analysis or tunable resistive pulse sensing. Second, the loading assay quantifies total peptide associated with the sEV pellet and cannot resolve membrane-inserted peptide from superficially adsorbed peptide. Protease protection and density-gradient co-migration experiments are required to confirm bilayer incorporation. Third, the ELISA used for free Ac-SDKP was validated for the unmodified tetrapeptide. Cross-reactivity with the cholesterol-His6-modified derivative has not been rigorously established, and the reported loading values should be considered provisional pending orthogonal validation by HPLC or LC-MS/MS. Fourth, the storage stability assessment tracked only particle diameter over 7 days. Particle concentration, aggregation state, and retention of bioactivity over longer periods remain to be characterized. Fifth, the in vivo safety data were obtained at a single dose level (20 mg/kg) with five animals per group and a 7-day observation window. Multi-dose regimens, recovery groups, extended observation, and immunogenicity screening are prerequisites for translational development. Sixth, the LPS-stimulated endothelial monolayer model does not reproduce the coronary inflammatory microenvironment of KD. Validation in disease-relevant models, such as Candida albicans water-soluble fraction (CAWS)- or Lactobacillus casei cell wall extract (LCWE)-induced coronary arteritis in mice, is essential. Seventh, group sizes of three to six per condition provide limited statistical power for detecting small-to-moderate effect sizes, and formal a priori power calculations were not conducted.

The protocol presented here provides a systematic framework for isolating platelet sEVs, decorating them with a cholesterol-anchored anti-inflammatory peptide, and performing an initial set of pharmaceutical and functional characterizations. The combination of efficient loading, preserved vesicle integrity, acceptable acute tolerability, and enhanced bioactivity relative to free peptide indicates that the platform merits further investigation. Priority areas for subsequent work include: full MISEV2023-compliant characterization; orthogonal validation of peptide incorporation; biodistribution and pharmacokinetic profiling in KD-relevant animal models; efficacy testing in CAWS- or LCWE-induced vasculitis; development of cleavable linker chemistries for stimuli-responsive peptide release; and evaluation of the platform with additional anti-inflammatory payloads, alone and in combination, building on recent advances in extracellular vesicle surface engineering, lipid-anchor functionalization, hybrid vesicle systems, cargo-free therapeutic vesicles, and inflammation-targeted nanoplatforms^{36,37,38,39,40}.

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רשימת החומרים שנעשה בהם שימוש במאמר זה

שם	חברה	מספר קטלוג	הערות
Ac-SDKP ELISA kit	Cusabio Technology LLC (Houston, TX, USA)	CSB-EQ027552HU	Competitive ELISA; validated for unmodified Ac-SDKP; cross-reactivity with Ac-SDKP-His6-CHOL pending verification
Acid-citrate-dextrose (ACD) solution, Formula A	Sigma-Aldrich (St. Louis, MO, USA)	C3821	Anticoagulant; 1:9 (v/v) ratio with whole blood
Ac-SDKP-His6-CHOL peptide	GL Biochem (Shanghai) Ltd. (Shanghai, China)	Custom synthesis	C-terminal His6- and cholesterol-modified Ac-SDKP; purity >95% by HPLC; characterized by MS
Ammonium formate (LC-MS grade)	Sigma-Aldrich (St. Louis, MO, USA)	70221	For mobile phase preparation
Automated biochemistry analyzer	Shenzhen Mindray Bio-Medical Electronics Co., Ltd. (Shenzhen, China)	BS-240VET	For serum ALT, AST, BUN, CREA measurement
BCA protein assay kit	Thermo Fisher Scientific (Waltham, MA, USA)	23225	Pierce BCA Protein Assay; BSA standard included
Bovine serum albumin (BSA)	Sigma-Aldrich (St. Louis, MO, USA)	A7030	Fraction V; ≥98% purity; used as standard for BCA assay
C57BL/6J mice (male, ~4 weeks, 11-13 g)	Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China)	N/A	SPF grade; acclimatized ≥5 d before use
Carbon-coated copper grids (300 mesh)	Electron Microscopy Sciences (Hatfield, PA, USA)	CF300-Cu	Glow-discharged before use for TEM
CCK-8 assay kit	Dojindo Molecular Technologies, Inc. (Kumamoto, Japan)	CK04	WST-8 based; used at 10% (v/v) in medium, 2 h incubation
CD41 antibody (anti-integrin αIIb)	Abcam plc (Cambridge, UK)	ab134131	Rabbit monoclonal; WB 1:1,000
CD62P antibody (anti-P-selectin)	Abcam plc (Cambridge, UK)	ab255822	Rabbit monoclonal; WB 1:1,000
Chemiluminescence imaging system	Bio-Rad Laboratories, Inc. (Hercules, CA, USA)	ChemiDoc MP Imaging System	For ECL-based western blot imaging
D-Glucose	Sigma-Aldrich (St. Louis, MO, USA)	G8270	≥99.5%; for modified Tyrode's buffer
Disposable polystyrene cuvettes for DLS	Malvern Panalytical Ltd. (Malvern, UK)	DTS0012	For size measurement at 90° detection angle
DLS / Zeta potential analyzer	Malvern Panalytical Ltd. (Malvern, UK)	Zetasizer Nano ZS	He-Ne laser (633 nm); 90° detection angle; Smoluchowski model for zeta
DMEM/F12 medium	Gibco / Thermo Fisher Scientific (Waltham, MA, USA)	11320033	For HL-1 cell culture
EGM-2 endothelial growth medium	Lonza Group AG (Basel, Switzerland)	CC-3162	BulletKit; contains 2% FBS, growth factors; for HCAEC and HUVEC
Enhanced chemiluminescence (ECL) substrate	Thermo Fisher Scientific (Waltham, MA, USA)	34580	SuperSignal West Pico PLUS Chemiluminescent Substrate
Fetal bovine serum (FBS), heat-inactivated	Gibco / Thermo Fisher Scientific (Waltham, MA, USA)	10082147	Origin: Australia; heat-inactivated at 56 °C for 30 min
Folded capillary cells for zeta potential	Malvern Panalytical Ltd. (Malvern, UK)	DTS1070	For electrophoretic mobility / zeta potential measurement
Formic acid (LC-MS grade)	Sigma-Aldrich (St. Louis, MO, USA)	F0507	For mobile phase preparation
GraphPad Prism (version 9.0)	GraphPad Software, Inc. (San Diego, CA, USA)	N/A	Statistical analysis and graphing software
HCAEC (human coronary artery endothelial cells)	ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA)	6000	Primary cells; passages 3–6 used
HEPES (≥99.5%)	Sigma-Aldrich (St. Louis, MO, USA)	H3375	For modified Tyrode's buffer, pH 7.4
Hirudin (recombinant)	Sigma-Aldrich (St. Louis, MO, USA)	H0393	Thrombin inhibitor; 2 U/mL final to quench thrombin
HRP-conjugated anti-rabbit IgG	Cell Signaling Technology, Inc. (Danvers, MA, USA)	7074S	Goat anti-rabbit; WB 1:5,000
Human IL-1β ELISA kit	R&D Systems, Inc. (Minneapolis, MN, USA)	DLB50	Quantikine ELISA; solid-phase sandwich format
Human IL-6 ELISA kit	R&D Systems, Inc. (Minneapolis, MN, USA)	D6050	Quantikine ELISA; solid-phase sandwich format
Human TNF-α ELISA kit	R&D Systems, Inc. (Minneapolis, MN, USA)	DTA00C	Quantikine ELISA; solid-phase sandwich format
HUVEC (human umbilical vein endothelial cells)	ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA)	8000	Primary cells; passages 3–6 used
ImageJ (version 1.53)	National Institutes of Health (Bethesda, MD, USA)	N/A	Public domain; for WB densitometry; https://imagej.net
Isoflurane (USP)	RWD Life Science Co., Ltd. (Shenzhen, China)	R510-22	Inhalation anesthetic; 3-5% induction, 1.5-2% maintenance in O2
Lipopolysaccharide (LPS) from E. coli O111:B4	Sigma-Aldrich (St. Louis, MO, USA)	L4391	Ultrapure; used at 1 µg/mL for inflammatory induction
Magnesium chloride (MgCl2, ≥98%)	Sigma-Aldrich (St. Louis, MO, USA)	M8266	Anhydrous; for modified Tyrode's buffer
Methanol (LC-MS grade)	Merck Millipore (Darmstadt, Germany)	1.06035	For metabolite extraction (when applicable)
Microplate reader (absorbance)	BioTek Instruments, Inc. (Winooski, VT, USA)	Synergy H1	For ELISA and CCK-8 absorbance at 450 nm
Mouse IL-1β ELISA kit	R&D Systems, Inc. (Minneapolis, MN, USA)	MLB00C	Quantikine ELISA; solid-phase sandwich format
Mouse IL-6 ELISA kit	R&D Systems, Inc. (Minneapolis, MN, USA)	M6000B	Quantikine ELISA; solid-phase sandwich format
Mouse TNF-α ELISA kit	R&D Systems, Inc. (Minneapolis, MN, USA)	MTA00B	Quantikine ELISA; solid-phase sandwich format
Na+/K+-ATPase antibody	Abcam plc (Cambridge, UK)	ab76020	Rabbit monoclonal; WB 1:2,000; loading control
Non-fat dry milk powder	Sigma-Aldrich (St. Louis, MO, USA)	M7409	For WB blocking buffer; 5% (w/v) in TBST
Paraformaldehyde, 16% (w/v) aqueous solution	Electron Microscopy Sciences (Hatfield, PA, USA)	15710	Diluted to 4% in PBS for tissue fixation
Penicillin-streptomycin (100×)	Gibco / Thermo Fisher Scientific (Waltham, MA, USA)	15140122	10,000 U/mL penicillin; 10,000 µg/mL streptomycin
Phosphate-buffered saline (D-PBS), pH 7.4	Gibco / Thermo Fisher Scientific (Waltham, MA, USA)	10010023	Ca2+- and Mg2+-free; sterile-filtered
Potassium chloride (KCl, ≥99%)	Sigma-Aldrich (St. Louis, MO, USA)	P9333	For modified Tyrode's buffer
Prostaglandin E1 (PGE1)	Cayman Chemical Company (Ann Arbor, MI, USA)	13010	Platelet activation inhibitor; 1 µM final concentration
Protease inhibitor cocktail	Sigma-Aldrich (St. Louis, MO, USA)	P8340	EDTA-free; for RIPA lysis buffer
PVDF membrane (0.22 µm)	Merck Millipore (Darmstadt, Germany)	ISEQ00010	Immobilon-PSQ; for western blot protein transfer
RIPA lysis buffer	Thermo Fisher Scientific (Waltham, MA, USA)	89900	For protein extraction from sEVs and platelets
SDS-PAGE gels (10% precast)	Bio-Rad Laboratories, Inc. (Hercules, CA, USA)	4561034	Mini-PROTEAN TGX Precast Protein Gels; 10-well, 30 µL
Sodium chloride (NaCl, ≥99.5%)	Sigma-Aldrich (St. Louis, MO, USA)	S7653	For Tyrode's buffer and diluent preparation
Sodium phosphate dibasic (Na2HPO4, ≥99%)	Sigma-Aldrich (St. Louis, MO, USA)	S0876	For modified Tyrode's buffer
SPSS Statistics (version 26.0)	IBM Corporation (Armonk, NY, USA)	N/A	Statistical analysis software
Thrombin (from bovine plasma)	Sigma-Aldrich (St. Louis, MO, USA)	T4648	Lyophilized powder; reconstituted in PBS; 1 U/mL final
Transmission electron microscope	Hitachi High-Technologies Corporation (Tokyo, Japan)	HT-7700	80 kV accelerating voltage; for negative-stain TEM
Tris-buffered saline with 0.1% Tween-20 (TBST, 10×)	Cell Signaling Technology, Inc. (Danvers, MA, USA)	9997S	Diluted to 1× with deionized water before use
Ultracentrifuge	Beckman Coulter, Inc. (Brea, CA, USA)	Optima XPN-100	Type 70 Ti rotor; k-factor ~44 at max speed
Ultracentrifuge tubes (polycarbonate)	Beckman Coulter, Inc. (Brea, CA, USA)	355618	26.3 mL capacity; for 100,000 × g operations
Uranyl acetate dihydrate	Electron Microscopy Sciences (Hatfield, PA, USA)	22400	2% (w/v) in distilled water; negative stain for TEM
Water (LC-MS grade)	Merck Millipore (Darmstadt, Germany)	1.15333	For LC-MS mobile phase preparation