Cellular uptake and in vivo distribution of mesenchymal-stem-cell-derived extracellular vesicles are protein corona dependent.

Extracellular vesicles (EVs) derived from mesenchymal stem cells are promising nanotherapeutics in liver diseases due to their regenerative and immunomodulatory properties. Nevertheless, a concern has been raised regarding the rapid clearance of exogenous EVs by phagocytic cells. Here we explore the impact of protein corona on EVs derived from two culturing conditions in which specific proteins acquired from media were simultaneously adsorbed on the EV surface. Additionally, by incubating EVs with serum, simulating protein corona formation upon systemic delivery, further resolved protein corona-EV complex patterns were investigated. Our findings reveal the potential influences of corona composition on EVs under in vitro conditions and their in vivo kinetics. Our data suggest that bound albumin creates an EV signature that can retarget EVs from hepatic macrophages. This results in markedly improved cellular uptake by hepatocytes, liver sinusoidal endothelial cells and hepatic stellate cells. This phenomenon can be applied as a camouflage strategy by precoating EVs with albumin to fabricate the albumin-enriched protein corona-EV complex, enhancing non-phagocytic uptake in the liver. This work addresses a critical challenge facing intravenously administered EVs for liver therapy by tailoring the protein corona-EV complex for liver cell targeting and immune evasion.

Serum-derived extracellular vesicles from breast cancer patients contribute to differential regulation of T-cell-mediated immune-escape mechanisms in breast cancer subtypes.

Background: Intracellular communication within the tumour is complex and extracellular vesicles (EVs) have been identified as major contributing factors for the cell-to-cell communication in the local and distant tumour environments. Here, we examine the differential effects of breast cancer (BC) subtype-specific patient serum and cell-line derived EVs in the regulation of T cell mediated immune responses. Methods: Ultracentrifugation was used to isolate EVs from sera of 63 BC patients, 15 healthy volunteers and 4 human breast cancer cell lines. Longitudinal blood draws for EV isolation for patients on neoadjuvant chemotherapy was also performed. Characterization of EVs was performed by Nanoparticle Tracking Analysis (NTA), transmission electron microscopy (TEM) and immunoblotting. CD63 staining was performed on a tissue microarray of 218 BC patients. In-house bioinformatics algorithms were utilized for the computation of EV associated expression scores within The Cancer Genome Atlas (TCGA) and correlated with tumour infiltrating lymphocyte (TIL) scores. In vitro stimulation of PBMCs with EVs from serum and cell-line derived EVs was performed and changes in the immune phenotypes characterized by flow cytometry. Cytokine profiles were assessed using a 105-plex immunoassay or IL10 ELISA. Results: Patients with triple negative breast cancers (TNBCs) exhibited the lowest number of EVs in the sera; whilst the highest was detected in ER+HER2+ cancers; reflected also in the higher level of CD63+ vesicles found within the ER+HER2+ local tumour microenvironment. Transcriptomic analysis of the TCGA data identified that samples assigned with lower EV scores had significantly higher abundance of CD4+ memory activated T cells, T follicular cells and CD8 T cells, plasma, and memory B cells; whilst samples with high EV scores were more enriched for anti-inflammatory M2 macrophages and mast cells. A negative correlation between EV expression scores and stromal TIL counts was also observed. In vitro experiments confirmed that circulating EVs within breast cancer subtypes have functionally differing immunomodulatory capabilities, with EVs from patients with the most aggressive breast cancer subtype (TNBCs) demonstrating the most immune-suppressive phenotype (decreased CD3+HLA-DR+ but increased CD3+PD-L1 T cells, increased CD4+CD127-CD25hi T regulatory cells with associated increase in IL10 cytokine production). In depth assessment of the cytokine modulation triggered by the serum/cell line derived exosomes confirmed differential inflammatory cytokine profiles across differing breast cancer subtypes. Studies using the MDA-231 TNBC breast cancer cell-line derived EVs provided further support that TNBC EVs induced the most immunosuppressive response within PBMCs. Discussion: Our study supports further investigations into how tumour derived EVs are a mechanism that cancers can exploit to promote immune suppression; and breast cancer subtypes produce EVs with differing immunomodulatory capabilities. Understanding the intracellular/extracellular pathways implicated in alteration from active to suppressed immune state may provide a promising way forward for restoring immune competence in specific breast cancer patient populations.

Spatiotemporal tracking of gold nanorods after intranasal administration for brain targeting.

Intranasal administration is becoming increasingly more attractive as a fast delivery route to the brain for therapeutics circumventing the blood-brain barrier (BBB). Gold nanorods (AuNRs) demonstrate unique optical and biological properties compared to other gold nanostructures due to their high aspect ratio. In this study, we investigated for the first time the brain region-specific distribution of AuNRs and their potential as a drug delivery platform for central nervous system (CNS) therapy following intranasal administration to mice using a battery of analytical and imaging techniques. AuNRs were functionalized with a fluorescent dye (Cyanine5, Cy5) or a metal chelator (diethylenetriaminepentaacetic dianhydride, DTPA anhydride) to complex with Indium-111 via a PEG spacer for optical and nuclear imaging, respectively. Direct quantification of gold was achieved by inductively coupled plasma mass spectrometry. Rapid AuNRs uptake in mice brains was observed within 10 min following intranasal administration which gradually reduced over time. This was confirmed by the 3 imaging/analytical techniques. Autoradiography of sagittal brain sections suggested entry to the brain via the olfactory bulb followed by diffusion to other brain regions within 1 h of administration. The presence of AuNR in glioblastoma (GBM) tumors following intranasal administration was also proven which opens doors for AuNRs applications, as nose-to-brain drug delivery carriers, for treatment of a range of CNS diseases.

PET Imaging of Small Extracellular Vesicles via [89Zr]Zr(oxinate)4 Direct Radiolabeling.

Exosomes or small extracellular vesicles (sEVs) are increasingly gaining attention for their potential as drug delivery systems and biomarkers of disease. Therefore, it is important to understand their in vivo biodistribution using imaging techniques that allow tracking over time and at the whole-body level. Positron emission tomography (PET) allows short- and long-term whole-body tracking of radiolabeled compounds in both animals and humans and with excellent quantification properties compared to other nuclear imaging techniques. In this report, we explored the use of [89Zr]Zr(oxinate)4 (a cell and liposome radiotracer) for direct and intraluminal radiolabeling of several types of sEVs, achieving high radiolabeling yields. The radiosynthesis and radiolabeling protocols were optimized for sEV labeling, avoiding sEV damage, as demonstrated using several characterizations (cryoEM, nanoparticle tracking analysis, dot blot, and flow cytometry) and in vitro techniques. Using pancreatic cancer sEVs (PANC1) in a healthy mouse model, we showed that it is possible to track 89Zr-labeled sEVs in vivo using PET imaging for at least up to 24 h. We also report differential biodistribution of intact sEVs compared to intentionally heat-damaged sEVs, with significantly reduced spleen uptake for the latter. Therefore, we conclude that 89Zr-labeled sEVs using this method can reliably be used for in vivo PET tracking and thus allow efficient exploration of their potential as drug delivery systems.

Exosome-mediated RNAi of PAK4 prolongs survival of pancreatic cancer mouse model after loco-regional treatment.

With a dismal survival rate, pancreatic cancer (PC) remains one of the most aggressive and devastating malignancies, predominantly due to the absence of a valid biomarker for diagnosis and limited therapeutic options for advanced diseases. Exosomes (Exo) as cell-derived vesicles, are widely used as natural nanocarriers for drug delivery. P21-activated kinase 4 (PAK4) is oncogenic when overexpressed, promoting cell survival, migration and anchorage-independent growth. Herein we validated PAK4 as a therapeutic target in an in vivo PC tumour mouse model using Exo-mediated RNAi following intra-tumoural administration. PC derived Exo were firstly isolated by ultracentrifugation on sucrose cushion and characterised for their surface marker expression, size, number, purity and morphology. SiRNA was encapsulated into Exo via electroporation and dual uptake of Exo and siRNA was investigated by flow cytometry and confocal microscopy. In vitro siPAK4 silencing in PC cells following uptake was assessed by flow cytometry, western blotting, and in vitro scratch assay. In vivo efficacy (tumour growth delay and mouse survival) of siPAK4 was evaluated in PC bearing NSG mouse model. Ex vivo tumours were examined using Haematoxylin and eosin (H&E) staining and immunohistochemistry. Results showed high quality PC-derived PANC-1 Exo were obtained. SiRNA was incorporated in Exo with 16.5% encapsulation efficiency. In vitro imaging confirmed Exo and siRNA co-localisation in cells. PAK4 knockdown was successful with 30 nM Exo-siPAK4 at 24 h post incubation in vitro. Intra-tumoural administration of Exo-siPAK4 (0.03 mg/kg siPAK4 and 6.1 × 1011 Exo, each dose, two doses) reduced PC tumour growth in vivo and enhanced mice survival (p < 0.001), with minimal toxicity observed compared to polyethylenimine (PEI) used as a commercial transfection reagent. H&E staining of tumours showed significant tissue apoptosis in siPAK4 treated groups. PAK4 knockdown prolongs survival of PC-bearing mice suggesting its potential as a new therapeutic target for PC. PANC-1 Exo demonstrated comparable efficacy but safer profile than PEI as in vivo RNAi transfection reagent.

Defined serum-free three-dimensional culture of umbilical cord-derived mesenchymal stem cells yields exosomes that promote fibroblast proliferation and migration in vitro.

Stem cell-derived exosomes are emerging as novel and clinically relevant cell-free therapeutics for regenerative therapy. This work focused on investigating the stimulation of fibroblasts by exosomes derived from umbilical cord-derived mesenchymal stem cells (ucMSC) in a defined serum-free three-dimensional (3D) culture. 3D culture of ucMSC was carried out in medium supplemented with KnockOut serum replacement (KO-medium) using the Aggrewell system. ucMSC in KO-medium formed spheroids with maintained size and integrity throughout culture. This enabled the isolation of vesicles from ucMSC spheroids in KO-medium with sizes that fall within the exosomal size range and were positive for the expression of canonical exosomal markers CD63, CD9, CD81, Alix, and TSG101. The ucMSC-derived exosomes (ExoucMSC ) were shown to significantly increase the migration and proliferation of murine fibroblasts in vitro. To conclude, 3D culture of ucMSC in defined serum-free KO-medium formed viable spheroids which enabled the isolation of ExoucMSC with the potential of accelerating wound healing.

Design of experiment (DoE)-driven in vitro and in vivo uptake studies of exosomes for pancreatic cancer delivery enabled by copper-free click chemistry-based labelling.

Exosomes (Exo)-based therapy holds promise for treatment of lethal pancreatic cancer (PC). Limited understanding of key factors affecting Exo uptake in PC cells restricts better design of Exo-based therapy. This work aims to study the uptake properties of different Exo by PC cells. Exo from pancreatic carcinoma, melanoma and non-cancer cell lines were isolated and characterised for yield, size, morphology and exosomal marker expression. Isolated Exo were fluorescently labelled using a novel in-house developed method based on copper-free click chemistry to enable intracellular tracking and uptake quantification in cells. Important factors influencing Exo uptake were initially predicted by Design of Experiments (DoE) approach to facilitate subsequent actual experimental investigations. Uptake of all Exo types by PC cells (PANC-1) showed time- and dose-dependence as predicted by the DoE model. PANC-1 cell-derived exosomes (PANC-1 Exo) showed significantly higher uptake in PANC-1 cells than that of other Exo types at the longest incubation time and highest Exo dose. In vivo biodistribution studies in subcutaneous tumour-bearing mice similarly showed favoured accumulation of PANC-1 Exo in self-tissue (i.e. PANC-1 tumour mass) over the more vascularised melanoma (B16-F10) tumours, suggesting intrinsic tropism of PC-derived Exo for their parent cells. This study provides a simple, universal and reliable surface modification approach via click chemistry for in vitro and in vivo exosome uptake studies and can serve as a basis for a rationalised design approach for pre-clinical Exo cancer therapies.