Aspirin-loaded nanoexosomes as cancer therapeutics

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Abstract

The long history of discovery and recently encouraging studies of the anti-cancer effect of aspirin promise a closer step to widely used aspirin-based medication in cancer therapy. To resolve the poor water-solubility of aspirin and low encapsulation efficiency of exosomes for further developing a new delivery of aspirin as anti-cancer treatment, our nanoamorphous exosomal delivery platform was established. In this study, the anti-tumour effects of nanoamorphous aspirin-loaded exosomes with exosomes derived from breast and colorectal cancer cells, were comprehensively studied using both in vitro and in vivo models. These exosomes displayed enhanced cellular uptake via both clathrin-dependent and -independent endocytosis pathways, and significantly improved cytotoxicity of aspirin to breast and colorectal cancer cells, accompanied by the enhanced apoptosis and autophagy. Remarkably, this nanoamorphous exosomal platform endowed aspirin with the unprecedented cancer stem cell eradication capacity. Further animal study demonstrated that this developed exosomal system was able to efficiently deliver aspirin to in vivo tumours. The active targeting of these exosomes to tumour was further improved by conjugating an aptamer specifically targeting EpCAM protein. Hence, this nanoamorphous structured exosome system effectively transformed aspirin into a potential cancer stem cell killer with distinguished properties for clinical translation.

Introduction

Aspirin is one of the mostly used nonsteroidal anti-inflammatory drugs originally developed in 1899 for the symptomatic treatment of fever, headaches and muscle pain. In 1974, with the discovery of the role of aspirin in the secondary prevention of death from cardiovascular diseases such as heart attack and stroke (Elwood et al., 1974), the clinical application of aspirin was greatly extended. Later in 1991, one hundred years after its invention, the anti-cancer benefits of aspirin were firstly revealed (Thun et al., 1991). Recent clinical results suggested that aspirin could increase the survival period of cancer patients by 20% and greatly improve the treatment outcomes of patients with bowel, breast or prostate cancer (Elwood et al., 2016). Latest studies suggested that the anti-cancer mechanism of aspirin is mediated by a COX-1-dependent manner (Dovizio et al., 2012, Lichtenberger et al., 2017), or a rapid perturbation of the epidermal growth factor (EGF) and EGF receptor (EGFR) internalization (Bashir et al., 2019). Further investigations of the anti-cancer mechanism of aspirin would be performed to conclude the most accurate of the mechanism or conclude the combined mechanisms actually drive this effect of aspirin. Even so, aspirin does open a new avenue for the development of low-cost and low-toxicity anti-cancer therapeutics. To secure further clinical translation, the first barrier, the poor water-solubility of aspirin (pKa of 3.5 (Voelker and Hammer, 2012)), needs to be solved. Otherwise, insoluble crystalline grains of aspirin are likely to occur in the liquid dosage forms (Bakar and Niazi, 1983), resulting in reduced concentration of aspirin in the systemic circulation and at the diseased site. However, for effective anti-cancer treatment, it is necessary for therapeutic drugs to maintain a prolonged circulation time with high-concentration at specific tumour sites (Wang et al., 2017c). Thus, the poor water-solubility, together with the short plasma half-life (20 min) of aspirin (Altman et al., 2004), detrimentally affect the clinical translation of aspirin as a potent anti-cancer reagent.

The recently developed exosome-based drug delivery systems provide opportunities to develop nano-sized aspirin preparations with superior pharmaceutical properties for anti-cancer treatment. Exosomes are extracellular vesicles with diameter between 50 and 150 nm. In both physical and pathological conditions, exosomes can be released from virtually all types of cells upon fusion of the endosome-originated multivesicular bodies with plasma membrane (Meldolesi, 2018). Widely distributed in body fluids and cell culture with the content of crucial proteins and genetic materials of their parental cells, exosomes have been intensively exploited in non-invasive diagnosis of various types of diseases including cancer (Whiteside, 2018). Furthermore, the great potential of exosomes in targeted anti-cancer drug delivery has been drawing increasing attention (Gomari et al., 2018, Wang et al., 2017b). Indeed, the unique physical and biological properties of exosomes make them ideal carriers for cancer therapeutics. Exosomes can be easily isolated from various body fluids or cell cultures and the structure of exosomes is characterized by aqueous core (hydrophilic) and the lipid bilayer membrane (hydrophobic), which provides great opportunities for efficient encapsulation of both hydrophilic and hydrophobic cargoes. Of note, apart from being granted the enhanced permeability and retention (EPR) effect for passive tumour targeting due to the nanosize range, exosome-based delivery systems also display a natural targeting property to tumour. In line with various investigations, exosomes can specifically bind to their parental tumour cells following a mechanism named homing effect (Jiang and Gao, 2017). In addition, exosomes are endogenously produced by cells, which endorses them with enhanced stability (Luan et al., 2017), minimized immunogenicity (Edgar, 2016), and prolonged circulation time (Ha et al., 2016, Luan et al., 2017). As a result, exosomes are increasingly becoming one of the most studied drug delivery systems in current oncological field.

However, the encapsulation efficiency (EE) of exosomes is still far from being satisfactory. According to our preliminary study, the EE of exosomes to aspirin reached merely 18% with the conventional incubation method (Tran et al., 2019), in accordance with the 20% of EE observed by other independent studies (Banizs et al., 2014, Gilligan and Dwyer, 2017, Hood, 2016). The reason lies in two factors: (1) the reservoir of exosomes is occupied by various endogenous protein and genetic contents, which limits the accumulation of another loading cargo, especially with the commonly used incubation method (whereby the cargo is simply mixed with the exosomes) (Batrakova and Kim, 2015, Zhuang et al., 2011); (2) the poor water-solubility of aspirin strongly impedes its complete loading into both the aqueous core and membrane of exosomes, resulting in the low amount of the drug at the membrane only and the quick release of drug from this site, which is not suitable for the course of a long-term treatment. To overcome these limitations, recently we have introduced a new strategy using two FDA approved materials, amphiphilic poloxamer 407 (POX 407) and D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS), for developing a new platform to transform poorly water-soluble drugs into a hydrophilic-hydrophobic nanoamorphous structure (Tran et al., 2019). According to our assessment, this nanoamorphous form can be embedded in both hydrophilic and hydrophobic sections of the exosomes for enhanced EE. Consequently, the resulted aspirin loaded-nanoamorphous preparation demonstrated not only unprecedented 80% EE, but also significantly increased drug release rate (Tran et al., 2019). In the current study, the nanoamorphous aspirin-loaded exosomes using exosomes derived from both MDA-MB-231 breast cancer and HT29 colorectal cancer cells, were used to harness the homing effect-mediated native tumour targeting effect of exosomes for breast and colorectal cancer treatment. Via both in vitro and in vivo studies, the biological activities in terms of the potential anti-cancer effect of the resulted ESD3b (breast cancer exosome) and ESD3c (colorectal cancer exosome) were systematically investigated. In addition, the active tumour targeting effect of these reagents was further exploited by fabricating the existing ESD3b and ESD3c with aptamer (serving as a chemical antibody), the rising star of targeting technology in the past decade.

Section snippets

Materials

Aspirin (Cat. No. A2093), poloxamer 407 (POX 407) (Cat. No.16758), and D-α-Tocopheryl polyethylene glycol succinate (TPGS) (Cat. No. 57668) were from Sigma Aldrich (USA). All solvents used were HPLC grade. All other analytical grade chemicals were used as received without further purification.

Cell culture

The human colorectal adenocarcinoma cell line HT29 and human metastatic breast cancer cell line MDA-MB-231 were from the American Type Culture Collection (ATCC, Manassas, USA). The cell lines were

Nanoamorphous aspirin-loaded exosomes promote enhanced cellular uptake

To provide the most effective cancer treatment, following cell surface attachment, the drug delivery system has to be efficiently internalized inside of cancer cells. As a result, the cellular uptake of the nanoamorphous aspirin-loaded exosomes were investigated. To this end, breast cancer (MDA-MB-231) and colorectal cancer (HT29) cells were incubated with PKH26 (a fluorescent marker)-labelled ESD3b and ESD3c, followed by cell surface fluorescence quenching (to specifically investigate

Conclusions

In this study, the potent anti-cancer effect of the novel exosome-based nanoamorphous platform was comprehensively studied. Apart from the previously reported enhanced dissolution and encapsulation efficiency capability, this nanoamorphous-loaded aspirin exosomal delivery system can facilitate the penetration of the exosomes to subcellular compartments resulting in increased cellular uptake. As a consequence, the exosomes drastically reduced the proliferation of breast and colorectal cancer

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

Dr. Phuong Ha Lien Tran is the recipient of Australian Research Council's Discovery Early Career Researcher Award (project number DE160100900).

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