Modifying parent cells is a common method to obtain EVs with particular targeting properties and is superior to direct modification of exosomal surface in terms of the stability of the targeting moiety. In this method, a gene encoding the targeting proteins is inserted into donor cells, and the cells then release EVs carrying those proteins via the natural biogenesis pathways. If certain targeting moieties are not naturally found on EV membranes, cells can be made to express the desired moiety fused to an EV membrane component.

Lysosomal-associated membrane protein 2b (Lamp2b) is one of the most commonly used exosomal pedestals to attach guiding moieties [47,48,49,50]. For example, Tian et al. utilized Lamp2b fused to αv integrin-specific iRGD peptide to target αv integrin-positive breast cancer cells in vitro and in vivo [48]. A dramatic increase in cellular uptake was observed for iRGD-decorated exosomes compared to control exosomes (95.4% vs. 35.0%) [48]. Designer EVs can be used to overcome one of the main challenges faced by traditional therapeutics—crossing the BBB to deliver drugs to the brain. Again, Lamp2b acted as an exosomal pedestal to fuse brain targeting moiety in a study by Alvarez-Erviti et al. They accomplished the targeting of short interfering (si)RNA to the brain in mice by engineering dendritic cells to express Lamp2b conjugated to the neuron-specific rabies viral glycoprotein (RVG) peptide [47].

The designs of protein-targeting constructs that get incorporated into therapeutic EVs can become rather complicated. Ohno et al. used cloned tumour-targeting peptides (EGF and its less mitogenic alternative GE11) into pDisplay vector, which already contained hemagglutinin, myc-tag and platelet-derived growth factor receptor (PDGFR) [51]. Human embryonic kidney cell line 293 (HEK293) cells were then transfected with the pDisplay vector, thus allowing for the incorporation of targeting peptides into exosomes with the PDGFR acting as the carrying pedestal [51]. The targeting modifications with EGF and GE11 peptides increased the uptake of exosomes by breast cancer cells which typically overexpress EGF receptors [51]. At the same time, anti-hemagglutinin and anti-Myc-tag antibodies were used to confirm the expression of EGF and GE11 in exosomes [51]. Johnsen et al. have published a very comprehensive review of EVs as drug delivery vehicles, covering the use of targeting peptides to enhance the precision of EV-based therapeutics [44].

Besides targeting peptides, antibodies and nanobodies have been installed on EV surfaces to improve targeting. In one study, EV-producing cells were transfected with a vector encoding for anti-EGFR nanobodies fused to glycosylphosphatidylinositol (GPI) peptides to target cancer cells [52]. Lipid raft-associated lipids and proteins, including GPI, are naturally enriched on EV membranes, making them ideal for conjugating a targeting ligand [53]. Lactadherin was also used as an anchor for cancer-targeting moiety—single chain variable fragments (scFv) with an affinity to human epidermal growth factor receptor (EGFR) 2 overexpressed in breast cancer cells [54]. This approach was justified as lactadherin associates with the phosphatidylserine enriched in EV membranes.

While peptides and antibodies/nanobodies are the most popular choices for EV target guidance, other more imaginative approaches have also been employed. For example, pseudotyping—a method to change the tropism of viruses by packaging the genetic components of one virus into the envelope proteins of a different virus—has been applied to engineering targeted exosomes [55]. Meyer et al. expressed vesicular stomatitis virus glycoprotein (VSVG), which is frequently used for pseudotyping retroviruses and known for its broad tropism, in HEK293 cells [55]. The resulting VSVG-pseudotyped exosomes were incubated with multiple cell lines, and enhanced uptake by those cells was shown compared to controls [55].

An interesting direction in EV targeting is to use targeting moieties that also have a therapeutic effect. Jiang et al. induced overexpression of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in the membranes of donor cells which subsequently got incorporated into exosomes loaded with an anti-cancer agent triptolide [56]. In this study, TRAIL acted not only as a targeting ligand for death receptor 5, which is abundant in cancer cells, but also induced apoptosis in cancer cells, thus amplifying the therapeutic effect of triptolide loaded into exosomes [56]. In a different study, MHC-II, a major histocompatibility complex molecule normally only present on professional antigen-presenting cells, was overexpressed in murine melanoma cells [57]. The resulting MHC-II-enriched exosomes not only showed increased targeting towards T cells, but also enhanced the immunological response of the type 1 T helper cell (TH1) against cancer cells [57].

Supplying EVs with targeting properties via genetic modification of parent cells is an effective approach. However, it is not suitable for personalized medicine applications as it is difficult to apply this approach to patients’ own cells. It is also time-consuming, hard to scale for mass production and limiting as only genetically encodable targeting moieties may be used. Moreover, some targeting moieties tend to be expressed incorrectly and are quickly degraded in producer cells, affecting the resulting EVs’ targeting efficiency [58].

Direct chemical modification of EVs allows for a wider selection of targeting ligands and is more suited to personalized medicine applications, as EVs can be isolated from patient’s own body fluids and later decorated with chosen guiding moieties. Further, targeting ligands can be attached to isolated EVs in a very controlled manner which is not achievable with the parental cell modification approach.

Targeting peptides and antibodies/nanobodies are most commonly used to achieve efficient targeting of EVs post-isolation. An interesting approach was suggested by Ye et al., who used a multifunctional peptide to target EVs to glioblastoma cells [59]. The peptide contained a sequence which targeted it to the low-density lipoprotein receptor expressed on the glioblastoma cells, as well as an apoptosis-inducing sequence [59]. This therapeutic/targeting peptide amplified the effectiveness of the chemotherapy agent loaded into EVs and ensured successful delivery over the BBB in a mouse model [59]. Glioma cells were also targeted by Jia et al. via the conjugation of neuropilin-1-targeted peptide to exosomes via click chemistry [60]. The exosomes were loaded with superparamagnetic iron oxide nanoparticles and curcumin to enable both glioma imaging and treatment [60].

Anti-EGFR nanobodies fused to lactadherin, which bind to the phosphatidylserine in EVs post-isolation, have been used to target EGFR-positive tumour cells [61]. Similarly, anti-Her2 single-chain variable fragment fused to lactadherin was conjugated to phosphatidylserine-enriched EVs to target Her2-positive cancer cells [62]. A different approach to surface modification of EVs with nanobodies was adopted by Koojmans et al. who mixed EVs with micelles containing PEG and EGFR nanobodies [63]. This resulted in the emergence of PEGylated EVs targeted to EGFR-positive cancer cells [63].

More out-of-the-box approaches to direct modification of EVs for targeted drug delivery have also been tested. For instance, both DNA and RNA aptamers (single strands of nucleic acid that can bind to specific targets) have been used to direct EVs towards tumours. Cholesterol, naturally present in EV membranes, was used to conjugate AS1411, a DNA aptamer with a high affinity to nucleolin typically overexpressed in breast cancer cells [64]. AS1411-decorated EVs displayed better tumour-targeting abilities and had an added therapeutic effect as AS1411 is known to inhibit tumour activity [64]. An RNA aptamer targeted at prostate-specific membrane antigen (PSMA) has also been loaded into EV membranes to gain directional control [65]. Interestingly, the orientation of this arrow-shaped aptamer could be altered (with either the arrowhead or the arrow tail facing the membrane-anchoring cholesterol) to achieve either better targeting or better cargo loading into EVs [65].

Even actual magnets have been used to direct exosomes towards tumours. Qi at al. decorated transferrin receptors of blood-derived exosomes with magnetic nanoparticles and then guided them towards murine tumours using external magnets [66].

A niche and underexplored method for functionalizing EVs with targeting moieties is their fusion with liposomes decorated with targeting peptides or antibodies [67]. For example, Li et al. fused cancer exosomes with liposomes decorated with tumour-targeting peptide cRGD and loaded the resulting hybrid with a chemotherapy drug. The addition of the targeting peptide amplified the natural homing ability of cancer exosomes and resulted in the efficient delivery of the drug in vitro and in vivo [68]. A different study used an exosome-liposome hybrid to treat diabetic peripheral neuropathy. Singh et al. fused exosomes from bone marrow mesenchymal stromal cells with liposomes containing polypyrrole nanoparticles, thus combining stem cell therapy with electrical stimulation to achieve a therapeutic effect. Polypyrrole was selected because it is a conducting polymer and served to target the neurons’ electrical stimulation [69]. Despite some success, there are very few studies using fusion liposomes to achieve the targeting of exosomes.

Overall, the direct EV modification approach allows for a wider selection of targeting ligands and greater freedom in choosing chemical tools for their conjugation. These targeting moieties often have therapeutic and even diagnostic modalities that amplify the effect of loaded cargo drugs. Still, direct modification of EVs is challenging as the reaction conditions must be adapted to preserve EV membranes, prevent aggregation and ensure sufficient density of targeting ligands on EV surfaces.