In the absence of an effective nanocarrier vehicle, traditional cancer treatment, tumor imaging, and immunotherapy remain ineffective. Artificial liposomes have been used to encapsulate bioactive compounds and release the payload continuously and stimuli-responsively, but they suffer from multiple drawbacks. Extracellular vesicles (EVs) are natural endogenous macromolecule transporters used for drug delivery, but adequate and repeatable loading of the EVs with therapeutic or imaging agents still limits their application as drug delivery systems. EV-liposome hybrids are a novel nanocarrier platform that can solve the challenges existing with individual nanocarrier systems and provide precise cancer detection and tailored treatment
Effective cell-to-cell communication is essential for normal physiological homeostasis in multicellular organisms like humans. All bodily functions involve the coordinated interaction of cells. Extracellular vesicles (EVs) are one of the several processes through which cells interact with one another. EVs are extracellular, membrane-limited, mobile, cell-derived vesicles discharged in the extracellular space and, based on their biosynthesis, size, and membrane makeup, they can be categorized into three broad categories: micro-vesicles microvesicles MVs), apoptotic bodies (ABs), and exosomes. EVs harbor the respective parent cell's physiological state-specific proteins (such as transcription factors, surface receptors, heat-shock proteins), lipids, and nucleic acids (DNA, messenger RNA or mRNA, microRNA or miRNA, and long non-coding RNA or lncRNA), circulate in the blood and other bodily fluids. Once EVs attach to or are absorbed by other cells, the EV payload is released, facilitating information transfer-mediated pleiotropic physiological effect and establishing cell-cell communication. EVs are released by most cells, including the tumor cells. Nonetheless, little is understood about the specific mechanisms and intricate signaling pathways underlying EV-mediated cell-cell communication, especially in disease conditions. Hence, current research focuses on gaining a greater understanding of the biogenesis and function of EVs, highlighting the difference of the EV cargo in normal vs. disease conditions, and engineering EVs to exploit their potential therapeutic applicability in diagnostics, therapeutic delivery, and theranostics. EVs also play a critical role in cancer diagnosis and therapy.
Over the last two decades, scientists and researchers devoted to revolutionizing cancer therapy have acquired a vast amount of information about cancer and have realized that early diagnosis has the potential to revolutionize cancer management. Tissue biopsies depend on invasively resected representative tumor tissue sections and are fundamental for diagnosis, pathological interpretation, treatment planning, and response prediction to targeted therapy. Though tissue biopsies remain the gold standard for confirming a cancer diagnosis, they suffer from multiple challenges and remain unrealistic for early cancer detection. Liquid biopsy or fluid phase biopsy is a simple and non-invasive alternative to tissue biopsy and identifies and evaluates liquid-state biological matter (often blood, urine, saliva, or other fluids) for diagnosis, monitoring, and treatment surveillance of cancers. The foundation of liquid biopsy is the discovery and characterization of tumor-derived exosomes (TEX)/ extracellular vesicles (EVs), circulating tumor cells (CTCs), and circulating tumor DNA (ctDNA). Among the three types of liquid biopsy targets, the wild-type target is in far higher quantities than the tumor-derived components, making it challenging to use [4]. The advent of advanced state-of-the-art technologies such as single molecular array, Surface-enhanced Raman spectroscopy, Luminex, AlphaLISA, droplet-sequencing, and Electroluminescence ELISA has sparked great enthusiasm and optimism regarding the faster and less intrusive identification of liquid biopsy-based disease biomarkers at an early stage. The discovery of cancer-associated mutational changes and tumor antigens in EVs has offered the potential to use EV-based liquid biopsy as a cancer diagnostic and therapeutic-targeting platform and can complement the tissue biopsy approach. Several publications have established the role of EVs in cancer immunology, which can also be used for immunomodulation and suppression of tumor growth. EVs are key players not only in cancer diagnostics but also in therapeutic delivery.
Traditional chemotherapy has shown some efficacy, but its primary drawbacks include limited absorption, large dose requirements, and development of multiple drug resistance, unexpected side effects, low therapeutic indices, and non-specific targeting. In order to overcome such hurdles, scientists are in pursuit of an effective drug delivery vehicle with efficient delivery and targeting potential. Several artificial nanotechnology (NT)-based formulations (e.g., liposomes, polymeric nanoparticles (NP), albumin NP, and inorganic NP) have exhibited promise, and few of them have reached clinical application. Various research teams have also examined the therapeutic potential of EV-mediated drug delivery due to their endogenous cellular origin, minimal immunogenicity, and intrinsic capacity to penetrate the blood - brain barrier, enhanced target selectivity, and significant biocompatibility. EV surface proteins influence their absorption/uptake by tumor cells and can modulate therapeutic delivery and dispersion. EV-based delivery of small molecule drugs, pathway inhibitors, plasmids, proteins, siRNA, and miRNA have successfully attempted to ameliorate tumor growth and promote antitumor immunity. Many EV-based formulations are under clinical investigation in light of their vast range of cancer-specific applications and are summarized in other reviews. Although significant progress has been achieved with respect to EVs, other NT-based products, especially liposomes, are already in clinical use.
Among all the nanotechnology-based delivery vehicles, liposomes are the most well-characterized and successful, and several clinical trials are undergoing. Liposomes are artificially synthesized lipid nano-vesicles delivery vehicles, with a lipid bilayer (can enclose hydrophobic therapeutics) enclosing a hydrophilic aqueous core (for hydrophilic small molecules/ biotherapeutics). In addition, liposomes protect the cargo, are nano-sized, exhibit colloidal stability, are biocompatible, have enhanced bioavailability, permeate cell membrane, exhibit minimal toxicity, and can be surfacefunctionalized to be cell-type specific. A wide range of bioactive chemicals can be transported via liposomes, including anti-cancer small molecules, genetic materials, peptides, hormones, enzymes, proteins, CRISPR/Cas9, and vaccines. Currently, over seventeen separate liposomal formulations are available for treating a wide range of pathological causes, and of these, many are currently used for targeting various cancers. The FDA-approved liposomal trade names are Marqibo, Doxil, LipoDox, Onco TCS, Onivyde Caelyx, Myocet, DepoCyt, Mepact, Daunoxome, Vyxeos, and Lipusu. In recent years, cancer immunotherapy has made considerable advances, and liposomal formulations for the delivery of immunotherapeutics, cancer vaccines, and immunomodulators are being tested in preclinical and clinical trials. Hence, it is eminent that nanocarriers such as EVs and liposomes have the potential to improve conventional cancer therapy, immunotherapy, and combination therapy significantly. Both EV and liposome platforms have benefits and limitations, and hybrids may assist conventional and cancer immunotherapies in overcoming major obstacles.
Due to the short retention in blood, poor specificity of tumor cells, and fast clearance by the mononuclear phagocyte system, it is tough to deliver cancer therapeutics to the tumor. EV-liposome hybrids created by membrane fusion, sonication, and freeze-thaw cycles are an attractive next-generation platform that combines the strengths of both the EVs and the liposomes. Figure 1 presents the outlying concept of the EV-liposome hybrid. A distinct advantage of using the hybrid platform is the modulation of the membrane lipid composition of the EV-liposome hybrid for better celltype- specific uptake, enhanced stability, biocamouflage, long-term systemic circulation, and reduced immunogenicity. Using the knowledge of membrane fusion, a fundamental biological process, we can generate merged membrane architecture by fusing two distinct lipid bilayers. This opens up an exciting domain of membrane engineering that may be a potential method for designing rationally tailored hybrid nanocarriers for improved drug delivery and theranostics. A few examples are discussed in the following section.
A significant paper in this direction was published by Sato et al. in 2016, where they made exosome-liposome hybrids and evaluated cellular uptake. Recently macrophage-derived immunoexosome were hybridized with synthetic liposomes to administer doxorubicin to target and treat breast cancer. Cheng et al. created hybrid nanovesicles with gene-engineered exosomes and drug-loaded thermosensitive liposomes, which blocked the CD47 "don't eat me" signal limiting the tumor cell's ability of immunological escape using combined photothermal therapy and immunotherapy. Li et al. have used similar hybrids to co-deliver triptolide (a natural product with an anti-cancer effect) and miR497 to attack chemoresistance in ovarian cancer. Another study used engineered, exosomes-thermosensitive liposomes hybrid NPs to target metastatic peritoneal cancer. Belhadj et al. used hybrid c(RGDm7)-LS vesicles to studynanomedicine tumor accumulation and uptake both in vitro and in vivo. A recent preprint paper by Liu et al. used paclitaxel-loaded hybrid exosome to target triple-negative breast cancer and showed that the liposome-EV hybrids infiltrated the tumor and attenuated tumor proliferation with no signs of systemic toxicity. New approaches are also being tested to generate intelligent EV-liposome hybrids using polyethylene glycol-mediated fusion. Studies with EV-liposome hybrids showed that engineered EVs hold great promise in establishing an approach to better diagnostics, dramatically increasing anti-cancer treatment effects, and helping tackle the existing challenges of conventional delivery systems.
It is anticipated that several avenues for hybrid-engineered EV-liposome nanovesicle will become available shortly and include clinical research translation. An important aspect is the source of EVs, and the hybrid production method also needs considerable attention. Novel surface bioengineering design strategies of EV-liposome hybrids may include stealth methodology for avoiding phagocytosis and long-term circulation of therapeutic hybrids. In addition to the surface alterations, the physical features of the hybrids should be carefully considered, including size, shape, and total charge of NPs, and can critically impact systemic clearance, cellular internalization, and immune activation. Research should also be focused on hybrids that can aid and monitor the effects of immunotherapy in cancer patients. Further investigation is required to study the EV-liposome hybrids in creating theranostic applications and personalized tailored medicine. Moreover, other artificial platforms can also be hybridized with the EVs to generate novel systems and need further investigation. NP-based cancer diagnosis, treatment, and theranostics have snowballed, emphasizing the need for absorption, distribution, metabolism, and excretion (ADME) studies for FDA clearance.
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Manash K. Paul works as a Scientist, Principal Investigator at UCLA, USA. He has contributed significantly to stem cell biology, extracellular vesicles, early diagnostics, and lung cancer. He has published in over 65 peer-reviewed journals and is the recipient of many awards, including the UCLA Vice-Chancellor’s award, AAISCR-R Vijayalaxmi Award, and a senior member of the IEEE.
