MicroRNA are non-coding RNA species that act as negative regulators of gene expression. We have demonstrated that hsa-miR-101-3p is upregulated in serum exosomes of Alzheimer’s disease (AD) patients and in exosomes isolated from post-mortem brain tissue from Alzheimer’s brains compared to controls. Using TargetScan, hsa-miR-101 is predicted to target the amyloid precursor protein (APP) and is therefore potentially involved in the pathology of AD. Exosomes are now being used for personalized nanomedicine therapy to deliver siRNA based therapies, however, cell culture exosomes produce low yield, poor purity and tedious purification methods. To address these issues, this study aims to investigate the use of exosome mimetic nanovesicles to deliver miRNA silencers/mimics to recipient cells in order to restore imbalances of mRNA transcription and protein synthesis. Exosome-mimetic nanovesicles were generated using an Avanti mini-extruder by performing serial extrusion of cells over-expressing miRNA through polycarbonate filters of gradual decreasing pore size. Here, we characterise these exosome-mimetic nanovesicles loaded with hsa-miR-101-3p and compare vesicle yields with exosomes secreted from Human neuroblastoma SH-SY5Y cell lines over-expressing hsa-miR-101-3p. These exosome-mimetic nanovesicles will be a useful tool to study the functionality and transfer of exosomal microRNA cargo in recipient cells in order to observe whether they can be used as a therapeutic tool to restore deregulation of genes associated with AD in cell culture models.