Extracellular vesicles as natural therapeutic agents and innate drug delivery systems for cancer treatment: Recent advances, current obstacles, and challenges for clinical translation.

As cancer poses a significant threat to the well-being of humans on a global scale, many researchers have embarked on the search for effective anticancer therapeutic agents. In recent years, many drugs have been shown to have extraordinary anticancer effects. However, in a lot of cases the treatment is accompanied by undesirable side effects due to some intrinsic properties linked to the therapeutic agents, such as poor targeting selectivity and short half-life in the circulation. In this regard, extracellular vesicles (EVs), a diverse family of natural cell-derived vesicles, steal the show as potential anticancer immunotherapy or delivery vectors of anticancer agents since they are an innate mechanism of intercellular communication. Here, we describe some of the most hotly-debated issues regarding the use of EVs as anticancer therapeutics. First, we review the biology of EVs providing the most up-to-date definition of EVs as well as highlighting their circulation kinetics and homing properties. Next, we share our views on popular methods reported for EV isolation, characterization, and functional analysis. Pioneering and innovative reports along with emerging challenges in the field of EV imaging and EV drug loading strategies are then discussed. Finally, we examine in detail the therapeutic application of EVs in cancer treatment, including their role in cancer immunotherapy and as natural delivery systems for anticancer agents including natural compounds such as paclitaxel and doxorubicin. We consider standardised protocols and proper analytical approaches to be crucial in improving the reproducibility and rigor in EV research and ensuring the successful translation of EVs as anticancer therapeutics.

The Role of Platelets in the Tumor-Microenvironment and the Drug Resistance of Cancer Cells.

Besides the critical functions in hemostasis, thrombosis and the wounding process, platelets have been increasingly identified as active players in various processes in tumorigenesis, including angiogenesis and metastasis. Once activated, platelets can release bioactive contents such as lipids, microRNAs, and growth factors into the bloodstream, subsequently enhancing the platelet⁻cancer interaction and stimulating cancer metastasis and angiogenesis. The mechanisms of treatment failure of chemotherapeutic drugs have been investigated to be associated with platelets. Therefore, understanding how platelets contribute to the tumor microenvironment may potentially identify strategies to suppress cancer angiogenesis, metastasis, and drug resistance. Herein, we present a review of recent investigations on the role of platelets in the tumor-microenvironment including angiogenesis, and metastasis, as well as targeting platelets for cancer treatment, especially in drug resistance.

Complex formation with plasmid DNA increases the cytotoxicity of cationic liposomes.

Cationic liposomes (CL) are one of the most widely studied non-viral vectors for gene delivery. It is well-known that CL induces cytotoxicity following lipofection. However, little is known regarding the mechanism involved in the cytotoxicity. In this study, the in vitro cytotoxicity of CL and its complex with pDNA (lipoplex) was investigated, and a part of the mechanism of induction as well. While free pDNA did not show any cytotoxicity, pDNA increased the cytotoxicity of CL via the formation of lipoplex. In addition, the lipoplex-induced cytotoxicity increased in a lipoplex dose-dependent manner, irrespective of the type of pDNA, cell line and the absence or presence of serum. An assay showed that apoptosis was largely induced by treatment with the lipoplex (lipofection), but not with CL alone, in the tested range of concentration of CL and pDNA. Furthermore, following treatment with lipoplexes, the cells exhibited the morphological features of apoptosis and DNA fragmentation. A cDNA microarray study showed that the lipofection up-regulated 45 genes related to apoptosis, transcription regulation and immune response. These results clearly indicate that pDNA in the lipoplex increases the cytotoxicity of CL as a result of inducing apoptosis. The fundamental principle for gene therapy is to deliver gene-based therapeutics to target cells for specific gene targeting with minimal cytotoxicity. Our results suggest the possibility that cytotoxicity induced by lipofection, accompanied by gene changes, could intrinsically exacerbate, attenuate or even mask the desired effects of gene-based therapy.

Gene expression in primary cultured mouse hepatocytes with a cationic liposomal vector, TFL-3: comparison with rat hepatocytes.

We recently reported that a cationic liposomal vector, TFL-3, could be used to achieve significant gene expression in primary cultured rat hepatocytes (Nguyen et al., Biol. Pharm. Bull., 26, 880-885 (2003)). A combination of hepatocyte transplantation and hepatocyte-targeted gene transfer represents a potentially important strategy for expanding treatment options for liver disease. A widely applied approach to support cross-species is necessary before human applications can be realized. Therefore, in this study, we examined the utility of TFL-3 in another species of rodent hepatocytes, namely mouse hepatocytes. Gene expression in mouse hepatocytes by TFL-3 was successful and the level was higher than those in rat hepatocytes that we recently reported on. Interestingly, it appears that both the degree and rate of gene expression were dependent on the incubation time prior to lipofection as well as on the density of cells per dish, but these parameters were independent of the amount of pDNA associated with the cells. These significantly suggest that the culture time prior to and following lipofection, which are related to the biological condition of the cells, may be one of major factors that affect gene expression in hepatocytes and non- or less dividing cells.

Culture time-dependent gene expression in isolated primary cultured rat hepatocytes by transfection with the cationic liposomal vector TFL-3.

The development of a carrier system that enables the transfer of a functional exogenous gene to non- or less frequently dividing mammalian cells is essential for increasing the available options for the treatment of various diseases. The issue of whether TFL-3, a recently developed cationic liposome, can be successfully used to achieve gene expression in primary cultured rat hepatocytes was examined. The hepatocytes were transfected for 4 h with plasmid DNA (pDNA) in TFL-3 at various time points after 4-h preculture. The transfection efficiency was determined at various times posttransfection with pDNA coding for chloramphenicol acetyltransferase (CAT), luciferase, or beta-galactosidase. The amount of intranuclear pDNA present, as a consequence of the lipofection, was also quantitatively determined. Successful lipofections were observed for all pDNA tested, and the efficiencies were superior to that of commercially available LIPOFECTAMINE under our experimental conditions. The degree and rate of gene expression were dependent on incubation time prior to lipofection as well as on the density of the cells per dish, but this relationship did not hold for the amount of gene delivered to the nuclei. These results indicate that TFL-3 could be a useful vector for achieving sufficient gene expression in rat hepatocytes and suggest that the culture time prior to and following lipofection, which is related to the biological condition of the cells, may be one major factor affecting efficient gene expression in nondividing cells.