Effective light-triggered contents release from helper lipid-incorporated liposomes co-encapsulating gemcitabine and a water-soluble photosensitizer

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Abstract

Triggered drug release is a promising strategy for delivering anticancer drugs to cancer cells and tissues. We found that liposomes co-encapsulating calcein (a water-soluble model drug and fluorescence marker) and talaporfin sodium (TPS, a water-soluble photosensitizer) released the drug upon irradiation with a near-infrared (NIR)-laser. The liposomes were composed of phospholipid (DSPC)/helper lipid (DOPE)/cholesterol/PEG-lipid (PEG2000-DSPE) at a molar ratio of 85/10/5/5 and released a large amount of drug (70%<, within 10 min) upon irradiation, but no drug in the absence of NIR-laser irradiation and/or TPS. NIR-laser-triggered drug release was facilitated by the incorporation of DOPE into the liposomes, and the amount of DOPE incorporated affected drug leakage in the absence of NIR-laser-irradiation at 37 °C (body temperature). Drug leakage was tuned by incorporating cholesterol into the liposomes. NIR-laser-triggered drug release from the liposomes was confirmed using the anticancer drug gemcitabine. NIR-laser treatment of liposomes co-encapsulating gemcitabine and TPS provided the maximum cytotoxic effect towards EMT6/P cells. These results suggest that these novel light sensitive liposomes may be useful for drug delivery to cancer cells.

Introduction

Liposomes are amongst the best-known drug carriers, exhibit many advantageous properties, have been widely investigated in clinical trials, and are currently marketed as drug carriers (Allen and Cullis, 2013, Tagami and Ozeki, 2017). Liposomes are primarily composed of phospholipids, which are also the main component of the cell membrane, and are biologically inert. Liposomal encapsulation masks the systemic side effects of drugs with a narrow therapeutic window. Liposomes whose residence time in blood is prolonged (e.g., through PEGylation) can passively accumulate in solid cancer tissue through the leaky cancer vasculature. This is termed the “enhanced permeability and retention (EPR) effect” (Maeda et al., 2013). Liposomal drugs have been used to treat many kinds of diseases, including cancer (e.g., Doxil/Caelyx as liposomal doxorubicin) (Barenholz, 2012, Dawidczyk et al., 2014, Samad et al., 2007). However, the therapeutic effect of liposomal anticancer drugs acting via the EPR effect remains limited. Incomplete drug release from stabilized liposomes results both in low bioavailability of the drug in cancer tissue and adverse effects derived from liposomal drugs, such as hand-foot syndrome. These negative characteristics limit the maximum tolerated dose of liposomal drugs.

Triggered release is a promising strategy to improve the bioavailability and therapeutic effect of current liposomal and nanoparticulate drugs in solid cancer tissue (Mura et al., 2013, Zhou et al., 2017). The physical and chemical triggers are various kinds of stimuli such as heat (Kneidl et al., 2014), ultrasound (Sirsi and Borden, 2014), light (Miranda and Lovell, 2016, Shum et al., 2001), pH (Kanamala et al., 2016), and enzymes (Andresen et al., 2010). All these stimuli have been used for efficient drug release and drug delivery to the target site, such as cancer tissue. ‘Smart carriers’ have the following attributes: they release the drug at higher concentration into cancer tissue, respond quickly to the trigger, and retain the drug in the liposome in normal tissue. For example, thermosensitive liposomes and heating the solid cancer tissue by radiofrequency thermal ablation, microwave ablation, or high-intensity focused ultrasound is used as a multimodal therapy. A thermosensitive liposome formulation (ThermoDOX) is currently in clinical trial (Phase 3, ClinicalTrials.gov Identifier: NCT02112656). The delivery of liposomal drugs via triggered drug release (e.g., thermosensitive liposomes) supports basic cancer therapy (e.g. radiofrequency thermal ablation).

Porphyrin-based compounds such as hemoglobin in the human body and chlorophyll in plants have long been studied as photosensitizers. Photosensitizers are used for photodynamic therapy against cancers because they can generate reactive oxygen species (ROS) to kill cancer cells via a photochemical reaction triggered by light at a specific wavelength range (600 nm - 1000 nm), including near-infrared (NIR) (Debele et al., 2015). The first generation of photosensitizer (i.e., porfimer sodium) was slowly cleared from the skin and the patients had to avoid light for a long period of time to avoid phototoxicity after treatment. In contrast, the next generation of photosensitizer, talaporfin sodium (TPS), addressed this problem. TPS is excreted from kidney faster and has a higher absorption peak at a given wavelength than porfimer sodium. The combination of TPS, a diode laser, and NIR wavelengths has been applied clinically for the photodynamic treatment of lung cancer (Usuda et al., 2006), brain cancer (Akimoto, 2016), and persistent/recurrent esophageal cancer.

Here we show a model of NIR-laser-triggered drug release from a novel liposome. Several groups have reported that the incorporation of a functional lipid into liposomes containing a porphyrin-based compound triggers drug release upon irradiation by a NIR-laser, resulting in an anticancer effect in vivo (Luo et al., 2016a, Sine et al., 2015). However, there is little information regarding the mechanism of drug release, and what information there is does not seem to be systemically integrated. We anticipated that liposomes containing a drug and TPS would have an unstable liposomal membrane and would thus release the drug efficiently. In this study, we identified a novel liposome formulation that quickly releases the encapsulated drug upon NIR-laser-irradiation. To our knowledge, this is the first report of the simultaneous encapsulation of a drug and TPS into a liposome that subsequently releases the drug upon irradiation with a NIR-laser; furthermore, the incorporation of a helper lipid into the liposome could facilitate drug release. The combination of anticancer chemotherapy and photodynamic therapy, called ‘chemophototherapy’, have been expecting for the treatment option to improve the therapeutics effect of monotherapy (Luo et al., 2016b). For example, Miranda et al., reported the porphyrin-lipid incorporated liposome containing oxaliplatin to control the intratumor drug delivery (Miranda et al., 2017). In this study, novel liposomes co-encapsulating TPS and anticancer drug, gemcitabine (GEM) were fabricated. We also investigated the cytotoxic effect of the liposome formulation and NIR-laser-irradiation on a cancer cell line in vitro.

Section snippets

Materials

1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from NOF Corporation (Tokyo, Japan). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (PEG2000-DSPE) was generously donated by NOF Corporation. Cholesterol (CHOL) and chloroform were purchased from Wako Pure Chemical (Osaka, Japan). TPS was obtained from Meiji Seika Pharma (Tokyo, Japan). Calcein was purchased from Dojindo (Kumamoto, Japan). 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC)

Drug release by NIR-laser-irradiation of liposomes containing a water-soluble sensitizer and the effect of DOPE-incorporation into the liposomes

We first demonstrated that our novel liposome formulation can release a model drug (calcein, a fluorescence marker) with high sensitivity upon NIR-laser-irradiation (Fig. 1). The liposomes were composed of DSPC/DOPE/CHOL/PEG2000-DSPE (=85/10/5/5, molar ratio). These components were included as main component of liposome (DSPC), to assist drug release (DOPE), to stabilize liposome (CHOL) and to prolong blood circulation time in future in vivo studies (PEG2000-DSPE), respectively. The

Discussion

Drug delivery systems responsive to physical and chemical stimulations, including light, have been studied intensely to improve the therapeutic effects of the encapsulated drug and decrease its side effects. In the present study, a novel liposome formulation co-encapsulating a drug and TPS showed remarkable drug release upon stimulation with a NIR-laser, although the mechanism of drug release from our liposomes remains unclear.

Typical light sensitive liposomes and nanocarriers often utilize a

Conclusion

A novel NIR-laser-sensitive liposome formulation containing TPS and an anticancer drug was developed in the current study. TPS functioned not only as the trigger to release the anticancer drug but also as the agent for photodynamic therapy. The incorporation of a helper lipid enhanced the sensitivity of the liposomes to NIR-laser-irradiation. Although further in vivo study is necessary, the strategy of combining photodynamic therapy and efficient delivery of an anticancer drug is useful for

Acknowledgements

This research was partly supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (16K18952). We thank Dr. Hiromitsu Yamamoto and Dr. Noriko Ogawa (Aichi gakuin Univ) for the assistance of measurement of zeta potentials.

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