The transmission and toxicity of polymer-bound doxorubicin-containing exosomes derived from human adenocarcinoma cells

Background: Exosomes are extracellular vesicles with the ability to encapsulate bioactive molecules, such as therapeutics. This study identified a new exosome mediated route of doxorubicin and poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA)-bound doxorubicin trafficking in the tumor mass. Materials & methods: Exosome loading was achieved via incubation of the therapeutics with an adherent human breast adenocarcinoma cell line and its derived spheroids. Exosomes were characterized using HPLC, nanoparticle tracking analysis (NTA) and western blotting. Results: The therapeutics were successfully loaded into exosomes. Spheroids secreted significantly more exosomes than adherent cells and showed decreased viability after treatment with therapeutic-loaded exosomes, which confirmed successful transmission. Conclusion: To the best of our knowledge, this study provides the first evidence of pHPMA-drug conjugate secretion by extracellular vesicles. Plain language summary Background: In cancer treatment, low-molecular-weight drugs (e.g., doxorubicin [DOX]) with a broad spectrum of side effects are commonly used. Through their conjugation with hydrophilic polymers - N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers - for example, most of the side effects can be reduced. These drug-polymer conjugates are delivered via bloodstream into the tumor. This study aimed to identify a new exosome-mediated route of DOX and polyHPMA(pHPMA)-DOX conjugates trafficking inside the tumor mass. Exosomes are small lipid membrane vesicles constitutively released from most of the cell types, including the tumor cells. Exosomes are able to encapsulate low-molecular-weight drugs. Methods: Exosomes were loaded with DOX and pHPMA-DOX in vitro via coincubation with cancer cells. Exosomes were isolated from the conditioned-cultivation medium after their release from cells and characterized (size, numbers, protein marker profiles). Results: The therapeutics were successfully loaded into exosomes and transmitted to the tumor cells. To the best of our knowledge, this is the first evidence of the pHPMA-drug conjugate secretion by exosomes.