Extracellular Vesicle (EV) biohybrid systems for cancer therapy: Recent advances and future perspectives.

Extracellular vesicles (EVs) are a class of cell-derived lipid-bilayer membrane vesicles secreted by almost all mammalian cells and involved in intercellular communication by shuttling various biological cargoes. Over the last decade, EVs - namely exosomes and microvesicles - have been extensively explored as next-generation nanoscale drug delivery systems (DDSs). This is in large due to their endogenous origin, which enables EVs to circumvent some of the limitations associated with existing cancer therapy approaches (i.e. by preventing recognition by the immune system and improving selectivity towards tumor tissue). However, successful translation of these cell-derived vesicles into clinical applications has been hindered by several factors, among which the loading of exogenous therapeutic molecules still represents a great challenge. In order to address this issue and to further advance these biologically-derived systems as drug carriers, EV-biohybrid nano-DDSs, obtained through the fusion of EVs with conventional synthetic nano-DDSs, have recently been proposed as a valuable alternative as DDSs. Building on the idea of "combining the best of both worlds", a combination of these two unique entities aims to harness the beneficial properties associated with both EVs and conventional nano-DDSs, while overcoming the flaws of the individual components. These biohybrid systems also provide a unique opportunity for exploitation of new synergisms, often leading to improved therapeutic outcomes, thus paving the way for advancements in cancer therapy. This review aims to describe the recent developments of EV-biohybrid nano-DDSs in cancer therapy, to highlight the most promising results and breakthroughs, as well as to provide a glimpse on the possible intrinsic targeting mechanisms of EVs that can be bequeathed to their hybrid systems. Finally, we also provide some insights in the future perspectives of EV-hybrid DDSs.

Dual-Targeting Dual-Action Platinum(IV) Platform for Enhanced Anticancer Activity and Reduced Nephrotoxicity.

A novel and highly efficient dual-targeting platform was designed to ensure targeted in vivo delivery of dual-action PtIV prodrugs. The dual targeting was established by liposomal encapsulation of PtIV complexes, thereby utilizing the enhanced permeability and retention (EPR) effect as the first stage of targeting to attain a high accumulation of the drug-loaded liposomes in the tumor. After the release of the PtIV prodrug inside cancer cells, a second stage of targeting directed a portion of the PtIV prodrugs to the mitochondria. Upon intracellular reduction, these PtIV prodrugs released two bioactive molecules, acting both on the mitochondrial and on the nuclear DNA. Our PtIV system showed excellent activity in vitro and in vivo, characterized by a cytotoxicity in a low micromolar range and complete tumor remission, respectively. Notably, marked in vivo activity was accompanied by reduced kidney toxicity, highlighting the unique therapeutic potential of our novel dual-targeting dual-action platform.

EXOPLEXs: Chimeric Drug Delivery Platform from the Fusion of Cell-Derived Nanovesicles and Liposomes.

Cell-derived nanovesicles (CDNs) have been recently investigated as novel drug delivery systems (DDSs), due to the preservation of key features from the cell membrane of their precursor cells, which are responsible for an efficient cellular uptake by target cells. However, CDNs suffer from low drug loading efficiencies as well as challenges in functionalization compared to conventional DDS like liposomes. Here, we describe the first study proposing the fusion of CDNs with liposomes to form EXOPLEXs. We report the preservation of cell membranes from precursor cells similarly to CDNs, as well as high loading efficiencies of more than 65% with doxorubicin hydrochloride, a model chemotherapeutic drug. The doxorubicin-loaded EXOPLEXs (DOX-EXO) also demonstrated a higher in vitro cell killing effect than liposomes, while EXOPLEXs alone did not show any remarkable cytotoxicity. Taken together, these results illustrate the potential of EXOPLEXs as a novel DDS for targeted delivery of chemotherapeutics.

Synthesis and biodistribution studies of (3)H- and (64)Cu-labeled dendritic polyglycerol and dendritic polyglycerol sulfate.

Dendritic polyglycerol sulfate (dPGS) is a biocompatible, bioactive polymer which exhibits anti-inflammatory activity in vivo and thus represents a promising candidate for therapeutic and diagnostic applications. To investigate the in vivo pharmacokinetics in detail, dPGS with a molecular weight of approx. 10 kDa was radiolabeled with (3)H and (64)Cu, and evaluated by performing biodistribution studies and small animal positron emission tomography (PET). (3)H-labeling was accomplished by an oxidation-reduction process with sodium periodate and [(3)H]-borohydride. (64)Cu-labeling was achieved by conjugation of isothiocyanate- or maleimide-functionalized copper(II)-chelating ligands based on 1,4-bis(2-pyridinylmethyl)-1,4,7-triazacyclononane (DMPTACN) to an amino functionalized dPGS scaffold, followed by reaction with an aqueous solution containing (64)CuCl2. Independent biodistribution by radioimaging and PET imaging studies with healthy mice and rats showed that the neutral dPG was quantitatively renally eliminated, whereas the polysulfated analogues accumulated mainly in the liver and spleen. Small amounts of the dPGS derivatives were slowly excreted via the kidneys. The degree of uptake by the reticuloendothelial system (RES) was similar for dPGS with 40% or 85% sulfation, and surface modification of the scaffold with the DMPTACN chelator did not appear to significantly affect the biodistribution profile. On the basis of our data, the applicability of bioactive dPGS as a therapeutic agent might be limited due to organ accumulation even after 3 weeks. The inert characteristics and clearance of the neutral polymer, however, emphasizes the potential of dPG as a multifunctional scaffold for various nanomedical applications.

Detection of endogenous matrix metalloprotease-12 active form with a novel broad spectrum activity-based probe.

Matrix metalloproteases (MMPs) have attracted considerable attention as critical mediators of pathological tissue remodeling processes. However it remains an unresolved challenge to detect their active forms in biological samples. To prove the efficacy of a recently developed MMP activity-based probe, we examined the content in MMP active forms of bronchoalveolar lavage fluids (BALf) from male C57BL/6 mice exposed to ultrafine carbon black nanoparticles, a model of chronic obstructive pulmonary disease. This probe was shown to label proteins, mostly expressed in BALf of mice exposed to nanoparticles. Using competition assays with a selective MMP-12 inhibitor as well as MMP-12 knock-out mice, one of these proteins was identified as the active form of the catalytic domain of MMP-12. This new probe can detect the active form of MMP-12 down to a threshold of 1 fmol. Radioactive counting showed the concentration of the active form of MMP-12 to be around 1 fmol/µl in BALf from nanoparticle-treated mice. A less sensitive probe would therefore not have detected MMP-12. As the probe can detect other MMPs in the femtomolar range, it is a potentially powerful tool for monitoring the levels of MMP active forms in various diseases.

Effects of selective MMP-13 inhibition in squamous cell carcinoma depend on estrogen.

Matrix metalloproteinases like MMP-13 cleave and remodel the extracellular matrix and thereby play a crucial role in tumor progression in vivo. Using a highly selective inhibitor to block MMP-13 protein activity, we demonstrate a striking inhibitory effect on invasive tumor growth and vascularization in murine skin squamous cell carcinoma (SCC). Therapy outcome critically depends on animal age in C57Bl/6 mice and was successful in old female but not in young female mice. Treatment success was recovered by ovariectomy in young and abolished by 17ß-estradiol supplementation in old mice, suggesting a hormone dependent inhibitor effect. Responsiveness of the tumorigenic keratinocytes BDVII and fibroblasts to 17ß-estradiol was confirmed in vitro, where MMP-13 inhibitor treatment led to a reduction of cell invasion and vascular endothelial growth factor (VEGF) release. This correlated well with a less invasive and vascularized tumor in treated mice in vivo. 17ß-estradiol supplementation also reduced invasion and VEGF release in vitro with no additional reduction on MMP-13 inhibitor treatment. This suggests that low 17ß-estradiol levels in old mice in vivo lead to enhanced MMP-13 levels and VEGF release, allowing a more effective inhibitor treatment compared to young mice. In our study, we present a strong link between lower estrogen levels in old female mice, an elevated MMP-13 level, which results in a more effective MMP-13 inhibitor treatment in fibroblasts and SCC cells in vitro and in vivo.

Crystallization of bi-functional ligand protein complexes.

Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects.

Simple pseudo-dipeptides with a P2' glutamate: a novel inhibitor family of matrix metalloproteases and other metzincins.

A series of pseudo-peptides with general formula X-l-Glu-NH(2) (with X corresponding to an acyl moiety with a long aryl-alkyl side chain) have been synthesized, evaluated as inhibitors of matrix metalloproteases (MMPs), and found to display remarkable nanomolar affinity. The loss in potency associated with a substitution of the P(2)' l-glutamate by a l-glutamine corroborates the importance of a carboxylate at this position. The binding mode of some of these inhibitors was characterized in solution and by x-ray crystallography in complex with various MMPs. The x-ray crystal structures reveal an unusual binding mode with the glutamate side chain chelating the active site zinc ion. Competition experiments between these inhibitors and acetohydroxamic acid, a small zinc-binding molecule, are in accord with the crystallographic results. One of these pseudo-dipeptides displays potency and selectivity toward MMP-12 similar to the best MMP-12 inhibitors reported to date. This novel family of pseudo peptides opens new opportunities to develop potent and selective inhibitors for several metzincins.

A pan photoaffinity probe for detecting active forms of matrix metalloproteinases.

A photoaffinity probe based on the scaffold of a potent broad-spectrum phosphinic peptide inhibitor of matrix metalloproteinases (MMPs) has been developed. A photolabile diazirine group for covalent modification of MMP active forms was incorporated at the P(1) ' position, and a tritium radioactive label for the sensitive detection of MMP covalent adducts by radioimaging was attached. The probe was characterized on seven catalytic domains of human MMPs (MMP-2, -3, -8, -9, -12, -13 and -14) and was found to display nanomolar affinities towards this set of MMPs, covalently modifying them with crosslinking yields varying from 12 to 58 %, thus leading to highly sensitive detection of these MMPs. In a complex proteome complemented with four recombinant MMPs (MMP-2, -9, -12 and -13), this probe enabled their simultaneous detection with a threshold of few femtomoles and low background labelling. Those properties should make this new pan-activity-based MMP probe a valuable tool for the detection of MMP active forms from biological fluids or tissue extracts.

Cell-mimicking polyethylene glycol-diacrylate based nanolipogel for encapsulation and delivery of hydrophilic biomolecule.

Lipid based nanoparticulate formulations have been widely used for the encapsulation and sustain release of hydrophilic drugs, but they still face challenges such as high initial burst release. Nanolipogel (NLG) emerges as a potential system to encapsulate and deliver hydrophilic drug while suppressing its initial burst release. However, there is a lack of characterization of the drug release mechanism from NLGs. In this work, we present a study on the release mechanism of hydrophilic Dextran-Fluorescein Isothiocyanate (DFITC) from Poly (ethylene glycol) Diacrylate (PEGDA) NLGs by using different molecular weights of PEGDA to vary the mesh size of the nanogel core, drawing inspiration from the macromolecular crowding effect in cells, which can be viewed as a mesh network of undefined sizes. The effect is then further characterized and validated by studying the diffusion of DFITC within the nanogel core using Fluorescence Recovery after Photobleaching (FRAP), on our newly developed cell derived microlipogels (MLG). This is in contrast to conventional FRAP works on cells or bulk hydrogels, which is limited in our application. Our work showed that the mesh size of the NLGs can be controlled by using different Mw of PEGDA, such as using a smaller MW to achieve higher crosslinking density, which will lead to having smaller mesh size for the crosslinked nanogel, and the release of hydrophilic DFITC can be sustained while suppressing the initial burst release, up to 10-fold more for crosslinked PEGDA 575 NLGs. This is further validated by FRAP which showed that the diffusion of DFITC is hindered by the decreasing mesh sizes in the NLGs, as a result of lower mobile fractions. These findings will be useful for guiding the design of PEGDA NLGs to have different degree of suppression of the initial burst release as well as the cumulative release, for a wide array of applications. This can also be extended to other different types of nanogel cores and other nanogel core-based nanoparticles for encapsulation and release of hydrophilic biomolecules.

Electron beam-irradiated donor cornea for on-demand lenticule implantation to treat corneal diseases and refractive error.

The cornea is the major contributor to the refractive power of the eye, and corneal diseases are a leading cause of reversible blindness. The main treatment for advanced corneal disease is keratoplasty: allograft transplantation of the cornea. Examples include lenticule implantation to treat corneal disorders (e.g. keratoconus) or correct refractive errors. These procedures are limited by the shelf-life of the corneal tissue, which must be discarded within 2-4 weeks. Electron-beam irradiation is an emerging sterilisation technique, which extends this shelf life to 2 years. Here, we produced lenticules from fresh and electron-beam (E-beam) irradiated corneas to establish a new source of tissue for lenticule implantation. In vitro, in vivo, and ex vivo experiments were conducted to compare fresh and E-beam-irradiated lenticules. Results were similar in terms of cutting accuracy, ultrastructure, optical transparency, ease of extraction and transplantation, resilience to mechanical handling, biocompatibility, and post-transplant wound healing process. Two main differences were noted. First, ∼59% reduction of glycosaminoglycans resulted in greater compression of E-beam-irradiated lenticules post-transplant, likely due to reduced corneal hydration-this appeared to affect keratometry after implantation. Cutting a thicker lenticule would be required to ameliorate the difference in refraction. Second, E-beam-sterilised lenticules exhibited lower Young's modulus which may indicate greater care with handling, although no damage or perforation was caused in our procedures. In summary, E-beam-irradiated corneas are a viable source of tissue for stromal lenticules, and may facilitate on-demand lenticule implantation to treat a wide range of corneal diseases. Our study suggested that its applications in human patients are warranted. STATEMENT OF SIGNIFICANCE: Corneal blindness affects over six million patients worldwide. For patients requiring corneal transplantation, current cadaver-based procedures are limited by the short shelf-life of donor tissue. Electron-beam (E-beam) sterilisation extends this shelf-life from weeks to years but there are few published studies of its use. We demonstrated that E-beam-irradiated corneas are a viable source of lenticules for implantation. We conducted in vitro, in vivo, and ex vivo comparisons of E-beam and fresh corneal lenticules. The only differences exhibited by E-beam-treated lenticules were reduced expression of glycosaminoglycans, resulting in greater tissue compression and lower refraction suggesting that a thicker cut is required to achieve the same optical and refractive outcome; and lower Young's modulus indicating extra care with handling.

Cell-derived nanovesicles from mesenchymal stem cells as extracellular vesicle-mimetics in wound healing.

Wound healing is a dynamic process that involves a series of molecular and cellular events aimed at replacing devitalized and missing cellular components and/or tissue layers. Recently, extracellular vesicles (EVs), naturally cell-secreted lipid membrane-bound vesicles laden with biological cargos including proteins, lipids, and nucleic acids, have drawn wide attention due to their ability to promote wound healing and tissue regeneration. However, current exploitation of EVs as therapeutic agents is limited by their low isolation yields and tedious isolation processes. To circumvent these challenges, bioinspired cell-derived nanovesicles (CDNs) that mimic EVs were obtained by shearing mesenchymal stem cells (MSCs) through membranes with different pore sizes. Physical characterisations and high-throughput proteomics confirmed that MSC-CDNs mimicked MSC-EVs. Moreover, these MSC-CDNs were efficiently uptaken by human dermal fibroblasts and demonstrated a dose-dependent activation of MAPK signalling pathway, resulting in enhancement of cell proliferation, cell migration, secretion of growth factors and extracellular matrix proteins, which all promoted tissue regeneration. Of note, MSC-CDNs enhanced angiogenesis in human dermal microvascular endothelial cells in a 3D PEG-fibrin scaffold and animal model, accelerating wound healing in vitro and in vivo. These findings suggest that MSC-CDNs could replace both whole cells and EVs in promoting wound healing and tissue regeneration.

A selective matrix metalloproteinase-12 inhibitor retards atherosclerotic plaque development in apolipoprotein E-knockout mice.

OBJECTIVE: Matrix metalloproteinase (MMP)-12 has been implicated in plaque progression and instability and is also amenable to selective inhibition. In this study, we investigated the influence of a greater than 10-fold selective synthetic MMP-12 inhibitor on plaque progression in the apolipoprotein E knockout mouse model of atherosclerosis. METHODS AND RESULTS: A phosphinic peptide (RXP470.1) that is a potent, selective murine MMP-12 inhibitor significantly reduced atherosclerotic plaque cross-sectional area by approximately 50% at 4 different vascular sites in male and female apolipoprotein E knockout mice fed a Western diet. Furthermore, RXP470.1 treatment resulted in less complex plaques with increased smooth muscle cell:macrophage ratio, less macrophage apoptosis, increased cap thickness, smaller necrotic cores, and decreased incidence of calcification. Additional in vitro and in vivo findings indicate that attenuated monocyte/macrophage invasion and reduced macrophage apoptosis probably underlie the beneficial effects observed on atherosclerotic plaque progression with MMP-12 inhibitor treatment. CONCLUSIONS: Our data demonstrate that a selective MMP-12 inhibitor retards atherosclerosis development and results in a more fibrous plaque phenotype in mice. Our study provides proof of principle to motivate translational work on MMP-12 inhibitor therapy in humans.

Synthesis and biological evaluation of a new triazole-oxotechnetium complex.

A new triazole oxotechnetium chelating agent was synthesized via a 'Click-to-Chelate' strategy. In vivo evaluation of the corresponding (99m)Tc complex shows that the tracer exhibits very interesting properties for molecular imaging.

Novel mechanism of inhibition of human angiotensin-I-converting enzyme (ACE) by a highly specific phosphinic tripeptide.

Human ACE (angiotensin-I-converting enzyme) has long been regarded as an excellent target for the treatment of hypertension and related cardiovascular diseases. Highly potent inhibitors have been developed and are extensively used in the clinic. To develop inhibitors with higher therapeutic efficacy and reduced side effects, recent efforts have been directed towards the discovery of compounds able to simultaneously block more than one zinc metallopeptidase (apart from ACE) involved in blood pressure regulation in humans, such as neprilysin and ECE-1 (endothelin-converting enzyme-1). In the present paper, we show the first structures of testis ACE [C-ACE, which is identical with the C-domain of somatic ACE and the dominant domain responsible for blood pressure regulation, at 1.97Å (1 Å=0.1 nm)] and the N-domain of somatic ACE (N-ACE, at 2.15Å) in complex with a highly potent and selective dual ACE/ECE-1 inhibitor. The structural determinants revealed unique features of the binding of two molecules of the dual inhibitor in the active site of C-ACE. In both structures, the first molecule is positioned in the obligatory binding site and has a bulky bicyclic P(1)' residue with the unusual R configuration which, surprisingly, is accommodated by the large S(2)' pocket. In the C-ACE complex, the isoxazole phenyl group of the second molecule makes strong pi-pi stacking interactions with the amino benzoyl group of the first molecule locking them in a 'hand-shake' conformation. These features, for the first time, highlight the unusual architecture and flexibility of the active site of C-ACE, which could be further utilized for structure-based design of new C-ACE or vasopeptidase inhibitors.

A Highly Conducting Polymer for Self-Healable, Printable, and Stretchable Organic Electrochemical Transistor Arrays and Near Hysteresis-Free Soft Tactile Sensors.

A stretchable and self-healable conductive material with high conductivity is critical to high-performance wearable electronics and integrated devices for applications where large mechanical deformation is involved. While there has been great progress in developing stretchable and self-healable conducting materials, it remains challenging to concurrently maintain and recover such functionalities before and after healing. Here, a highly stretchable and autonomic self-healable conducting film consisting of a conducting polymer (poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate), PEDOT:PSS) and a soft-polymer (poly(2-acrylamido-2-methyl-1-propanesulfonic acid), PAAMPSA) is reported. The optimal film exhibits outstanding stretchability as high as 630% and high electrical conductivity of 320 S cm-1 , while possessing the ability to repair both mechanical and electrical breakdowns when undergoing severe damage at ambient conditions. This polymer composite film is further utilized in a tactile sensor, which exhibits good pressure sensitivity of 164.5 kPa-1 , near hysteresis-free, an ultrafast response time of 19 ms, and excellent endurance over 1500 consecutive presses. Additionally, an integrated 5 × 4 stretchable and self-healable organic electrochemical transistor (OECT) array with great device performance is successfully demonstrated. The developed stretchable and autonomic self-healable conducting film significantly increases the practicality and shelf life of wearable electronics, which in turn, reduces maintenance costs and build-up of electronic waste.

Plant-derived extracellular vesicles: Recent advancements and current challenges on their use for biomedical applications.

Extracellular vesicles (EVs) represent a diverse class of lipid bilayer membrane vesicles released by both animal and plant cells. These ubiquitous vesicles are involved in intercellular communication and transport of various biological cargos, including proteins, lipids, and nucleic acids. In recent years, interest in plant-derived EVs has increased tremendously, as they serve as a scalable and sustainable alternative to EVs derived from mammalian sources. In vitro and in vivo findings have demonstrated that these plant-derived vesicles (PDVs) possess intrinsic therapeutic activities that can potentially treat diseases and improve human health. In addition, PDVs can also act as efficient and biocompatible drug carriers. While preclinical studies have shown promising results, there are still several challenges and knowledge gaps that have to be addressed for the successful translation of PDVs into clinical applications, especially in view of the lack of standardised protocols for material handling and PDV isolation from various plant sources. This review provides the readers with a quick overview of the current understanding and research on PDVs, critically analysing the current challenges and highlighting the immense potential of PDVs as a novel class of therapeutics to treat human diseases. It is expected that this work will guide scientists to address the knowledge gaps currently associated with PDVs and promote new advances in plant-based therapeutic solutions.

Insights from selective non-phosphinic inhibitors of MMP-12 tailored to fit with an S1' loop canonical conformation.

After the disappointment of clinical trials with early broad spectrum synthetic inhibitors of matrix metalloproteinases (MMPs), the field is now resurging with a new focus on the development of selective inhibitors that fully discriminate between different members of the MMP family with several therapeutic applications in perspective. Here, we report a novel class of highly selective MMP-12 inhibitors, without a phosphinic zinc-binding group, designed to plunge deeper into the S(1)' cavity of the enzyme. The best inhibitor from this series, identified through a systematic chemical exploration, displays nanomolar potency toward MMP-12 and selectivity factors that range between 2 and 4 orders of magnitude toward a large set of MMPs. Comparison of the high resolution x-ray structures of MMP-12 in free state or bound to this new MMP-12 selective inhibitor reveals that this compound fits deeply within the S(1)' specificity cavity, maximizing surface/volume ratios, without perturbing the S(1)' loop conformation. This is in contrast with highly selective MMP-13 inhibitors that were shown to select a particular S(1)' loop conformation. The search for such compounds that fit precisely to preponderant S(1)' loop conformation of a particular MMP may prove to be an alternative effective strategy for developing selective inhibitors of MMPs.

Oxorhenium-mediated assembly of noncyclic selective integrin antagonists: a combinatorial approach.

The parallel oxorhenium-mediated assembly of 288 noncyclic RGD analogues is reported. All complexes contain a NS(2) +S chelating motif that enables the unambiguous coordination of the oxorhenium and oxotechnetium cores. In this study, "modules S" contain a variety of pending guanidinium groups whereas the "NS(2) modules" are made of a series of N-acylated amino acids. Combination of sets of "NS(2) " and "S modules" together with tetrabutylammonium tetrachlorooxorhenate gave the corresponding oxorhenium complexes in good yields and satisfactory purities. Evaluation of these metalloconstructs towards integrins α(V) ß(3) , α(IIb) ß(3) , and α(V) ß(5) led to the identification of micromolar and submicromolar antagonists of theses integrins. These compounds exhibit interesting selectivities and promise attractive applications for the molecular imaging of integrin-dependent pathologies.

Cell-Derived Nanovesicles as Exosome-Mimetics for Drug Delivery Purposes: Uses and Recommendations.

Cell-derived Drug Delivery Systems (DDSs), particularly exosomes, have grown in popularity and have been increasingly explored as novel DDSs, due to their intrinsic targeting capabilities. However, clinical translation of exosomes is impeded by the tedious isolation procedures and poor yield. Cell-derived nanovesicles (CDNs) have recently been produced and proposed as exosome-mimetics. Various methods for producing exosome-mimetics have been developed. In this chapter, we present a simple, efficient, and cost-effective CDNs production method that uses common laboratory equipment (microcentrifuge) and spin cups. Through a series of extrusion and size exclusion steps, CDNs are produced from in vitro cell culture and are found to highly resemble the endogenous exosomes. Thus, we envision that this strategy holds great potential as a viable alternative to exosomes in the development of ideal DDS.