Surface-engineered extracellular vesicles for targeted delivery of therapeutic RNAs and peptides for cancer therapy

Abstract

The advent of novel therapeutics in recent years has urged the need for a safe, non-immunogenic drug delivery vector capable of delivering therapeutic payloads specifically to diseased cells, thereby increasing therapeutic efficacy and reducing side effects. Extracellular vesicles (EVs) have garnered attention in recent years as a potentially ideal vector for drug delivery, taking into account their intrinsic ability to transfer bioactive cargo to recipient cells and their biocompatible nature. However, natural EVs are limited in their therapeutic potential and many challenges need to be overcome before engineered EVs satisfy the levels of efficiency, stability, safety and biocompatibility required for therapeutic use. Here, we demonstrate that an enzyme-mediated surface functionalization method in combination with streptavidin-mediated conjugation results in efficient surface functionalization of EVs. Surface functionalization using the above methods permits the stable and biocompatible conjugation of peptides, single domain antibodies and monoclonal antibodies at high copy number on the EV surface. Functionalized EVs demonstrated increased accumulation in target cells expressing common cancer associated markers such as CXCR4, EGFR and EpCAM both in vitro and in vivo . The functionality of this approach was further highlighted by the ability of targeting EVs to specifically deliver therapeutic antisense oligonucleotides to a metastatic breast tumor model, resulting in increased knockdown of a targeted oncogenic microRNA and improved metastasis suppression. The method was also used to equip EVs with a bifunctional peptide that targets EVs to leukemia cells and induces apoptosis, leading to leukemia suppression. Moreover, we conducted extensive testing to verify the biocompatibility, and safety of engineered EVs for therapeutic use, suggesting that surface modified EVs can be used for repeated dose treatment with no detectable adverse effects. This modular, biocompatible method of EV engineering offers a promising avenue for the targeted delivery of a range of therapeutics while addressing some of the safety concerns associated with EV-based drug delivery.

Small Extracellular Vesicles and Their Involvement in Cancer Resistance: An Up-to-Date Review

“…Based on the analyzed research and our observations [ 14 , 30 , 166 , 167 ], it must be noted that both small and large EVs are the future of personalized medicine, including not only in the diagnosis and prognosis of cancer patients but also as a treatment strategy. Such an application of our knowledge of exosomes can rely on two potential activities: (1) inhibiting the release of exosomes [ 168 ] and (2) using exosomes as a platform for chemotherapeutic agent transport [ 169 , 170 ] and as a ‘freighter’ for therapeutic RNAs and peptides [ 171 ]. Here, advanced techniques of biomedical engineering come to our aid [ 172 , 173 , 174 , 175 ].…”

Advances in Drug Delivery Systems Based on Red Blood Cells and Their Membrane-Derived Nanoparticles

“…Possessing a nanosized dimension and a favorable safety profile, RBCEVs represent a promising therapeutic delivery platform (Table ). With a continued growing interest in generating an efficient and versatile DDS, our group has developed a robust delivery platform using RBCEVs for chemotherapy drugs and RNA drugs in various cancer models. − ,, We have demonstrated that treatment with RBCEVs loaded with antisense oligonucleotides (ASOs) against the oncogenic miR-125b leads to inhibition of tumor growth with minimal toxicity in leukemic and breast cancer models . Taking advantage of the natural accumulation of RBCEVs in the liver, Zhang et al loaded RBCEVs with ASOs of microRNA-155 to decrease inflammation and ameliorate acute liver failure in a mouse model .…”

“…This engineering approach is biocompatible and site-specific while maintaining a high copy number of engineered molecules per EV. Since then, many studies have expanded on this approach via the use of cyclic bifunctional peptides with higher copy number and the use of avidin–biotin interactions to conjugate larger proteins on the EV surface efficiently. ,, These studies have demonstrated that enzymatic ligation mediated by OaAEP1 C247A ligase is stable, irreversible, and does not alter the biocompatibility or physicochemical characteristics of the EVs.…”

Extracellular Vesicles as New Players in Drug Delivery: A Focus on Red Blood Cells-Derived EVs

“…In 2022, Jayasinghe and co-workers conjugated RBCEVs with several peptides and/or antibodies for targeted delivery of cargoes to cancer cells [ 200 ]. They conjugated RBCEVs with a cyclic peptide to specifically target CXCR4 or with a monoclonal antibody anti-CD33 to promote the specific binding and uptake of the conjugated EVs by leukemia cells expressing the corresponding receptors.…”

“…CXCR4-conjugated RBCEVs were loaded with the pro-apoptotic peptide KLA, demonstrating that these were able to significantly suppress leukemia burden and increase survival in a leukemia xenografted mouse model. Antibody-conjugated RBCEVs were also used to deliver RNA antisense oligonucleotides to knock down FLT3 and miR-125b in cell lines and in patient-derived xenograft models of leukemia [ 200 ]. This study demonstrated for the first time that peptide/antibody-conjugated RBCEVs are biocompatible and non-immunogenic and can be used for targeted delivery of therapeutic peptides and RNAs for potential clinical applications.…”