Engineering strategies for apoptotic bodies

Abstract

Extracellular vesicles (EVs) are lipid bilayer vesicles containing proteins, lipids, nucleic acids, and metabolites secreted by cells under various physiological and pathological conditions that mediate intercellular communication. The main types of EVs include exosomes, microvesicles, and apoptotic bodies (ABs). ABs are vesicles released during the terminal stages of cellular apoptosis, enriched with diverse biological entities and characterized by distinct morphological features. As a result, ABs possess great potential in fields like disease diagnosis, immunotherapy, regenerative therapy, and drug delivery due to their specificity, targeting capacity, and biocompatibility. However, their therapeutic efficacy is notably heterogeneous, and an overdose can lead to side effects such as accumulation in the liver, spleen, lungs, and gastrointestinal system. Through bioengineering, the properties of ABs can be optimized to enhance drug-loading efficiency, targeting precision, and multifunctionality for clinical implementations. This review focuses on strategies such as transfection, sonication, electroporation, surface engineering, and integration with biomaterials to enable ABs to load cargoes and enhance targeting, providing insights into the engineering of ABs.

Keywords: apoptotic bodies; engineering; extracellular vesicles.

FIGURE 1

Schematic representation of the biogenesis and secretion of EVs and the “find‐me” and “eat‐me” signals. Exosomes originate from the inward budding of endosomal membranes and accumulate in MVBs. Some MVBs undergo direct lysosomal degradation, while others fuse with the plasma membrane to release exosomes. Microvesicles are formed directly by the budding of the cell membrane. ABs are produced when cells undergo a highly regulated series of processes such as plasma membrane blebbing, apoptotic membrane protrusion formation, and AB fragmentation during the final stages of apoptosis. “Find‐me” signals: Apoptotic cells release mediators such as ATP, UTP, and CX3CL1, establishing a chemotactic gradient to attract phagocytes. “Eat‐me” signals: PS on the apoptotic body surface binds to phagocytic cell receptors, inducing phagocytosis.

FIGURE 2

Current applications of ABs.

FIGURE 3

Common methods for separating and isolating ABs: (A) ultracentrifugation (represented by differential ultracentrifugation), (B) size‐based separation methods (represented by ultrafiltration), (C) affinity‐based separation methods (represented by ELISA), (D) precipitation, and (E) microfluidic‐based separation methods (represented by acoustic purification) (Reproduced with permission. Copyright 2015, American Chemical Society).

FIGURE 4

Schematic diagram of parent cell transfection. (A) The principle of parent cell transfection involves introducing the target nucleic acids into parent cells by various methods and then inducing apoptosis to generate ABs containing the target nucleic acids. (B) Cationic polymers can compress DNA structures and form stable complexes to protect DNA. (C) After cationic polymers encapsulate DNA, they enter the cell. Subsequently, the intracellular pH decreases, resulting in the dissociation of the polymer and consequent release of DNA inside the cell. (Reproduced with permission. Copyright 2015, The Royal Society of Chemistry).

FIGURE 5

(A) Schematic diagram of CPT and PR104 A enhancing drug penetration and whole tumor destruction through ApoBD‐mediated neighboring effect. (Reproduced with permission. Copyright 2021, The Authors, published by American Association for the Advancement of Science). (B) Schematics of the AuNR‐CpG/AB system for enhanced tumor photothermal immunotherapy. Donor cells were first fed with CpG‐modified AuNR and then irradiated with UV light to generate AB‐encapsulated AuNR‐CpG. After intravenous injection into tumor‐bearing C57BL/6 mice, AuNR‐CpG/AB could be rapidly phagocytized by circulating monocytes/macrophages (MAs). With the natural tumor‐homing tendency of MAs, AuNR‐CpG/AB could be efficiently delivered to the inner region of a tumor. Then, NIR laser irradiation was applied to ablate the tumor through the photothermal effect of AuNR. (C) Flow cytometric analysis of AuNR/ABs (red) and ABs (blue). AuNR was coated with RhB‐embedded silica. Both AuNR/ABs and ABs were stained with Annexin V‐FITC. Inset: corresponding CLSM image of AuNR/ABs. (D) Temperature–time curves of the medium with AuNR (red), AuNR/AB (blue), and DPBS (black) with NIR laser irradiation (808 nm, 2 W/cm2). The corresponding concentration of AuNR in each sample was fixed at 2.1 μg. (Reproduced with permission. Copyright 2020, American Chemical Society).

FIGURE 6

(A) In parent cells, target proteins are coupled to known exosomal conservative non‐specific membrane proteins, producing exosomes carrying target proteins. (B) Schematic representation of the modified Lamp2b protein. Targeting peptides are fused to the extra‐exosomal N terminus of murine Lamp2b. (SP, signal peptide; TP, targeting peptide; TM, transmembrane domain; CT, C terminus.) (C) Electron micrograph of phosphotungstic acid stained RVG exosomes. (Reproduced with permission. Copyright 2011, Springer Nature). (D) Directly modifying the surface of parent cells, followed by apoptosis induction, directly produces surface‐engineered ApoNVs. (E) Bright‐field images of NIH3T3 cells after the Ac4ManNAz treatment at various concentrations. Scale bars = 100 μm. (F) Confocal microscopic images showing the conjugation of NIH3T3 cells with dextran (green) and CHP (red). Blue indicates DAPI. Scale bars = 50 μm. (G) 3D fluorescent images and (H) the quantification data showing the attachment of NVs on the membrane of H9C2 cells 3 h after the NV treatment. Scale bars = 100 μm. White arrows indicate NVs (red) on the H9C2 cell membrane (green). (Reproduced with permission. Copyright 2023, The Authors, published by John Wiley and Sons).

FIGURE 7

Direct Manipulation of AB Content (co‐incubation, electroporation, sonication, saponin permeation, freeze‐thaw cycle, extrusion, construction of liposomes).

FIGURE 8

Schematic illustration of the ABs content emptying, membrane retrieval, and subsequent reloading. (A) Removal of ABs content through hypotonic treatment and ultrasonication, extraction of the membrane followed by fusion with silica nanoparticles carrying miR‐21 or curcumin, resulting in the formation of chimeric ABs. (B) Representative SEM images of ABs. Scale bars, 1 μm (a), 500 nm (b), and 100 nm (c, d). (C) Representative fluorescence images show the colocalization of MSNs and AB ghosts after manipulation of cABs. Scale bars, 20 (left) and 5 μm (right). (Reproduced with permission. Copyright 2020, The Authors, published by American Association for the Advancement of Science). (D) DFO‐nABs were obtained by mixing drugs along with sonication after hypotonic treatment. (Reproduced with permission. Copyright 2023, Elsevier).

FIGURE 9

Surface engineering using click chemistry techniques. (A) Copper‐catalyzed azide‐alkyne cycloaddition links the fluorescent molecule azide‐fluor 545 dye to the surface of exosomes. (Reproduced with permission. Copyright 2014, American Chemical Society). (B) Copper‐free click chemistry allows the coupling of injured vascular targeting peptide (DA7R) and the stem cell recruiting factor (SDF‐1) to the surface of EVs, recruiting NSCs and promoting differentiation at central nervous system injury sites. (Reproduced with permission. Copyright 2023, Elsevier).

FIGURE 10

(A) Hydrogels can enable the sustained release of ABs at the wound site. (B) Schematic illustration of the design and application of the AB‐laden HA hydrogel. The hydrogel consisted of ABs produced from apoptotic MSCs and was administered into the uterine cavity via in situ injection to promote endometrial regeneration and restore fertility. (C) Scanning electron microscopy (SEM) images of the AB‐laden HA hydrogel. (D) The cumulative protein‐releasing profile of ABs from the AB‐laden HA hydrogel in the presence of hyaluronidase. (E) Hematoxylin and eosin (H, E) staining and Masson's trichrome staining images of uteri under different treatments for 12 d. (F) Quantification of endometrial thickness and endometrial gland under different treatments for 12 d. (n = 11, p < 0.01). (Reproduced with permission. Copyright 2022, The Authors, published by Elsevier).