Usion, and pinocytosis [137]. The endocytic pathway could be the principal route by which exosomes enter the cell, release the contents, and exert their biological effects. Even so, direct delivery of exosomes, such as IA or subcutaneous injections, is associated with swift clearance in vivo and restricted effective period [138]. Chondrocyte-targeted drug delivery is much more challenging because of the biological barrier formed by a dense matrix of proteoglycans, collagen, and highly negatively charged glycosaminoglycans within the cartilage [66], which calls for a lot more exosomes inside a greater concentration. To enhance yield, elongate retention time, and optimize therapy effects, quite a few approaches have already been proposed and studied, which include the improvement of exosome-mimetic nanovesicles (EMNVs), alteration of the culture condition, membrane surface modification, and controlled release with TrkC Proteins web biomaterial platforms [103,139]. As pointed out above, acceptable cell culture situations promote the production of exosomes. As an example, UC-MSCs grown in 3D microcarrier-based scaffolds yielded 20-fold a lot more exosomes than 2D cultures. If combined with tangential flow filtration (TFF) for exosome extraction, the production of exosomes could be further enhanced 7-fold greater than 3D cultures [140]. A rotary cell culture system (RCCS) simultaneously offers shear stress, hydrostatic stress, and buoyancy force, Cystatin F Proteins Accession generating an atmosphere of microgravity that positive aspects cell adhesion, proliferation, and aggregation; exosome secretion by UC-MSCs was substantially promoted at 36 rpm/min within 196 h [42]. EMNVs are one more system to achieve a large-scale production of exosomes. The generation of EMNVs through serially extruding cells by means of micro-sized filters boosted the yield of exosomes by more than one hundred folds and kept the biological functions equivalent to na e exosomes [141,142]. When applying EMNVs, focus should be paid towards the changed lipid species as well as altered membrane compositions compared with na e exosomes, as such changes may well have an effect on the PK/PD behavior of EMNVs in vivo [143]. Quite a few approaches modifying exosomal surface structures happen to be place forward to improve the entry of exosomes to cells that could be applied in OA research. As an example, chondrocyte-targeting exosomes had been ready by fusing the lysosome-associated membrane glycoprotein 2b (Lamp2b) protein present on the exosome surface using the chondrocyte-affinity peptide (CAP). These exosomes correctly encapsulated miR-140 and specifically entered chondrocytes to provide the cargoes in vitro [47]. Equipping exosomes with cell-penetrating peptides (CPPs), including arginine-rich CPPs (e.g., octa-arginine peptides, oligoarginine peptides, and human immunodeficiency virus variety 1 Tat (480) peptide), facilitated exosome entry in to the cell by stimulating cell micropinocytosis [144]. Coating exosomes with all the amphiphilic cationic CHP (cCHP) nanogel particles is often a polymerbased surface engineering strategy to facilitate exosome content material delivery and raise the encapsulation of large-size nucleic acids (e.g., plasmid) [145]. 1 challenge concerning hybrid exosomes is their comparable cytotoxicity as liposomes (Lipofectamine). Consequently, further investigation is essential to create liposomes with less toxicity [146]. Increasing the efficiency of fusion among exosomes plus the targeted cells is one more approach. Studies have shown that an increased fusion efficiency between recipient cells and exosomes was achieved by enhancing membrane r.