Exosomes are naturally occurring nanosized vesicles that have attracted considerable attention as drug delivery vehicles in the past few years. Exosomes are comprised of natural lipid bilayers with the abundance of adhesive proteins that readily interact with cellular membranes. We posit that exosomes secreted by monocytes and macrophages can provide an unprecedented opportunity to avoid entrapment in mononuclear phagocytes (as a part of the host immune system), and at the same time enhance delivery of incorporated drugs to target cells ultimately increasing drug therapeutic efficacy. In light of this, we developed a new exosomal-based delivery system for a potent antioxidant, catalase, to treat Parkinson’s disease (PD). Catalase was loaded into exosomes ex vivo using different methods: the incubation at room temperature, permeabilization with saponin, freeze-thaw cycles, sonication, or extrusion. The size of the obtained catalase-loaded exosomes (exoCAT) was in the range of 100 - 200 nm. A reformation of exosomes upon sonication and extrusion, or permeabilization with saponin resulted in high loading efficiency, sustained release, and catalase preservation against proteases degradation. Exosomes were readily taken up by neuronal cells in vitro. A considerable amount of exosomes was detected in PD mouse brain following intranasal administration. ExoCAT provided significant neuroprotective effects in in vitro and in vivo models of PD. Overall, exosome-based catalase formulations have a potential to be a versatile strategy to treat inflammatory and neurodegenerative disorders.

Exosomes have recently come into focus as “natural nanoparticles” for use as drug delivery vehicles. Our objective was to assess the feasibility of an exosome-based drug delivery platform for a potent chemotherapeutic agent, paclitaxel (PTX), to treat MDR cancer. Herein, we developed and compared different methods of loading exosomes released by macrophages with PTX (exoPTX), and characterized their size, stability, drug release, and in vitro antitumor efficacy. Reformation of the exosomal membrane upon sonication resulted in high loading efficiency and sustained drug release. Importantly, incorporation of PTX into exosomes increased cytotoxicity more than 50 times in drug resistant MDCKMDR1 (Pgp+) cells. Next, our studies demonstrated a nearly complete co-localization of airway-delivered exosomes with cancer cells in a model of murine Lewis Lung Carcinoma pulmonary metastases, and a potent anticancer effect in this mouse model. We conclude that exoPTX holds significant potential for the delivery of various chemotherapeutics to treat drug resistant cancers.

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Lysosomes are a promising therapeutic target for induction apoptosis in cancer cells due to lysosomal membrane permeabilization (LMP) leading to leakage of hydrolytic enzymes, especially the cathepsins, into the cytoplasm. We hypothesized that with the modification of the ceramide-loaded liposomes with transferrin (Tf), we would achieve both tumor targeting and increased delivery of lysosome-destabilizing agents, such as ceramides to lysosomes, to initiate LMP-induced apoptosis. We prepared Tf-modified (TL) and plain (PL) liposomes and loaded with short (C6)- or long (C16) N-acyl chain ceramides. Uptake, intracellular localization of liposomes, stability of the lysosomal membrane and release of cathepsin D were investigated on Hela cells by fluorescence microscopy and flow cytometry. Apoptosis was evaluated by binding of fluorescently-labeled Annexin V. Antitumor and pro-apoptotic effects of C6Cer-loaded Tf-liposomes were demonstrated in vivo in an A2780-ovarian carcinoma xenograft mouse model. TL were internalized specifically via the TfR-dependent endocytic pathway and localized within the endosome-lysosomal compartment. Ceramide-loaded Tf-liposomes significantly increased apoptosis compared with ceramide-free and ceramide-loaded non-modified liposomes. The treatment of cancer cells with TL led to increased LMP and cytoplasmic relocation of the intralysosomal cathepsin D. A strong antitumor and pro-apoptotic effect of C6Cer-loaded TL was also demonstrated in vivo in an A2780-ovarian carcinoma xenograft mouse model. The lysosomal accumulation of ceramides delivered by Tf-liposomes initiates the permeabilization of the lysosomal membranes required for the release of lysosomal cathepsins into the cytoplasm and initiation of the cancer cell apoptosis both in vitro and in vivo.

Purpose Platinum-based therapies are the first line treatments for most types of cancer including ovarian cancer. However, their use is associated with dose-limiting toxicities and resistance. We report initial translational studies of a theranostic nanoemulsion loaded with a cisplatin derivative, myrisplatin and pro-apoptotic agent, C6-ceramide. Methods The surface of the nanoemulsion is annotated with an endothelial growth factor receptor (EGFR) binding peptide to improve targeting ability and gadolinium to provide diagnostic capability for image-guided therapy of EGFR overexpressing ovarian cancers. A high shear microfludization process was employed to produce the formulation with particle size below 150 nm. Results Pharmacokinetic study showed a prolonged blood platinum and gadolinium levels with nanoemulsions in nu/nu mice. The theranostic nanoemulsions also exhibited less toxicity and enhanced the survival time of mice as compared to an equivalent cisplatin treatment. Conclusions Magnetic resonance imaging (MRI) studies indicate the theranostic nanoemulsions were effective contrast agents and could be used to track accumulation in a tumor. The MRI study additionally indicate that significantly more EGFR-targeted theranostic nanoemulsion accumulated in a tumor than non-targeted nanoemulsuion providing the feasibility of using a targeted theranostic agent in conjunction with MRI to image disease loci and quantify the disease progression.