Fig 2: Conjugation of RBCEVs with anti‐CD33 antibody enables targeted delivery of RNA cargo to leukaemia cells (A) Outline of the streptavidin‐mediated approach for antibody conjugation following enzymatic ligation of a biotinylated peptide. (B) Western blot analysis of anti‐CD33 antibody conjugated to RBCEVs compared to a serial dilution of biotinylated anti‐human CD33 monoclonal antibody. The blot was probed with anti‐mouse IgG heavy and light chain (H + L). (C) Single‐EV flow cytometric analysis (NanoFCM) of monoclonal antibody conjugation on RBCEVs using the streptavidin‐mediated conjugation method. AF488‐conjugated anti‐mouse secondary antibody was used to detect the anti‐CD33 and IgG isotype control monoclonal antibody, CD33‐EV: anti‐CD33 antibody‐conjugated RBCEVs, IgG‐EV: isotype monoclonal antibody‐conjugated RBCEVs. (D) Flow cytometry analysis of CD33 expression on AML MOLM13 cells and leukaemic lymphoid CEM cells. (E, F) Flow cytometric analysis of CFSE indicating the uptake of CFSE‐labelled RBCEVs conjugated with CD33 mAb by CD33‐positive MOLM13 cells and CD33‐negative CEM cells, presented as the percentage and mean fluorescent intensity of CFSE. (G) Workflow for antibody conjugation of RBCEVs and subsequent ASO loading. (H) Flow cytometry analysis of FAM fluorescence reflecting the delivery of a FAM‐conjugated ASO (FAM ASO) to MOLM13 cells by CD33‐targeting or non‐targeting RBCEVs. The graphs present the mean ± SEM. Student's one‐tailed t‐test: ns, not significant, ***p < .001, ****p < .0001

Fig 3: Conjugation of RBCEVs with anti‐CD33 antibody enhances the delivery of miR‐125b ASO loaded RBCEVs and its effect in suppressing leukaemia progression in patient derived AML xenografts. (A) Knockdown of miR‐125b in MOLM13 cells using NC ASO or miR‐125b ASO‐loaded RBCEVs, with or without CD33 targeting, presented as average log10 fold change relative to the untreated control, determined using Taqman RT‐qPCR, normalized to U6b RNA. (B) Viability of MOLM13 cells following treatment with miR‐125b ASO‐loaded RBCEVs with or without CD33 targeting, determined using CCK8 assay. (C) Experimental schema of AML PDX model. (D) Phenotype of PDX mice at late stage of AML, detecting the accumulation of human CD45+CD33+ leukaemia cells in the bone marrow, liver, spleen, and peripheral blood of PDX mice using flow cytometry analysis. (E) Outline of the treatment scheme with miR‐125b ASO loaded RBCEVs with or without CD33 targeting in PDX model. (F) Percentage of human CD45+CD33+ cells in the peripheral blood of PDX mice treated with miR‐125b ASO‐loaded RBCEVs with or without CD33 targeting. (G) White blood cells (WBC) count in mice peripheral blood (PB) on week 4. (H) Spleen size of PDX mice on week 4. (I) Weight of the spleens from PDX mice treated with miR‐125b ASO‐loaded RBCEVs with or without CD33 targeting. The graphs present the mean ± SEM. Student's one‐tailed t‐test: *p < .05, **p < .01, ***p < .001, ****p < .0001

Fig 4: Conjugation of RBCEVs with anti‐CD33 antibody enhances the delivery of FLT3‐ITD ASO and its effect in suppressing leukaemia progression. (A) SYBR Green qRT‐PCR analysis of FLT3‐ITD in MOLM13 cells treated with FLT3‐ITD ASO‐loaded CD33/non‐targeting RBCEVs, presented as fold change relative to the untreated group and normalized to GAPDH. (B) Apoptosis of MOLM13 cells following a 72 h treatment with FLT3‐ITD‐ASO‐loaded RBCEVs with or without CD33 targeting, determined by Annexin V and PI staining. (C) Percentage of Annexin−PI− viable cells and Annexin−PI+ necrotic cells among MOLM13 cells that were treated with FLT3‐ITD ASO‐loaded RBCEVs with or without CD33 targeting. (D) Outline of the treatment with FLT3‐ITD ASO‐loaded RBCEVs with or without CD33 targeting in leukaemia xenograft model. (E) Representative bioluminescent images of NSG mice implanted with MOLM13‐Luc‐GFP cells following RBCEV treatments. (F) Leukaemia progression in mice quantified using the average bioluminescent signals following treatment with FLT3‐ITD ASO‐loaded RBCEVs with or without CD33 targeting, relative to day 0. The graphs present the mean ± SEM (n = 4 mice). (G) Percentage of GFP‐positive leukaemia cells in the bone marrow, liver, and spleen. (H) Representative H&E staining images of the spleen sections from leukaemic mice treated with different conditions and non‐transplanted mouse. Scale bar, 75 μm. Student's one‐tailed t‐test and Two‐way ANOVA test (E): *p < .05, **p < .01, ***p < .001, ****p < .0001