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Co-delivery of docetaxel and disulphonate tetraphenyl chlorin in one nanoparticle produces strong synergism between chemo- and photodynamic therapy in drug-sensitive and -resistant cancer cells Elisa Gaio, Claudia Conte, Diletta Esposito, Giovanni Miotto, Fabiana Quaglia, Francesca Moret, and Elena Reddi Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00597 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018
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Molecular Pharmaceutics
Co-delivery of docetaxel and disulphonate tetraphenyl chlorin in one nanoparticle produces strong synergism between chemo- and photodynamic therapy in drug-sensitive and -resistant cancer cells
Elisa Gaio1, Claudia Conte2, Diletta Esposito2, Giovanni Miotto3, Fabiana Quaglia2, Francesca Moret1*, Elena Reddi1
1
Cell Biology Unit, Department of Biology, University of Padova, Padova, Italy
2
Drug Delivery Laboratory, Department of Pharmacy, University of Napoli Federico II, Napoli,
Italy 3
Department of Molecular Medicine, University of Padova, Padova, Italy
*Corresponding Author: e-mail:
[email protected]; Address: Department of Biology, Via Ugo Bassi 58/B, 35121 Padova, Italy. Telephone: +39 049 8276282.
Abstract Cancer therapies based on the combinations of different drugs and/or treatment modalities are emerging as important strategies for increasing efficacy and cure, decreasing unwanted toxicity and overcoming drug resistance, provided that optimized drug concentration ratios are delivered into the target tissue. To these purposes, delivery systems such as nanoparticles (NPs), offer the unique opportunity to finely tune the drug loading and the release rate of drug combinations in the target tissues. Here, we propose double-layered polymeric NPs for the delivery of the chemotherapeutic docetaxel (DTX) and the photosensitizer disulphonate tetraphenyl chlorin (TPCS2a) coated with hyaluronic acid (HA) which allows cell targeting via CD44 receptors. The simultaneous delivery of the two drugs aims at killing DTX-sensitive (HeLa-P, MDA-MB-231) and DTX–resistant (HeLa-R) cancer cells by combining chemotherapy and photodynamic therapy (PDT).
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Using the Chou and Talalay method that analyses drug interactions and calculates Combination Index (CI) using the median-effect principle, we compared the efficiency of DTX chemotherapy combined with TPCS2a-PDT for drugs delivered in the standard solvents, co-loaded in the same NP (DTX/TPCS2a-NP) or loaded in separate NPs (DTX-NPs + TPCS2a-NPs). Along with the drug interaction studies, we gained insight into cell death mechanisms after combo-therapy and into the extent of TPCS2a intracellular uptake and localization. In all cell lines considered, the analysis of the viability data revealed synergistic drug/treatment interaction especially when DTX and TPCS2a were delivered to cells co-loaded in the same NPs despite the reduced PS uptake measured in the presence of the delivery systems. In fact, while the combinations of the free drugs or drugs in separate NPs gave slight synergism (CI < 1) only at doses killing more than 50% of the cells, the combination of drugs in one NPs gave high synergism also at doses killing 10-20% of the cells. Furthermore, the DTX dose in the combination DTX/TPCS2a-NPs could be reduced by ~2.6 and 10.7 folds in HeLa-P and MDA-MB-231, respectively. Importantly, drug co-delivery in NPs was very efficient in inducing cell mortality also in DTX resistant HeLa-R cells over-expressing P-glycoprotein 1, in which the dose of the chemotherapeutic can be reduced by more than 100-times using DTX/TPCS2a-NPs. Overall, our data demonstrate that the protocol for the preparation of HA-targeted double layer polymeric NPs allows to control the concentration ratio of co-loaded drugs and the delivery of the transported drugs for obtaining a highly synergistic interaction combining DTX-chemotherapy and TPCS2aPDT.
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Molecular Pharmaceutics
Keywords: combination therapy, combination index, nanoparticles, docetaxel, disulphonate tetraphenyl chlorin, photodynamic therapy.
INTRODUCTION It is widely recognised that the treatments of aggressive tumours by one single modality, as for instance surgery, chemotherapy, radiotherapy or more recently photodynamic and photothermal therapy, are not sufficient to completely eradicate and cure the disease.1,2 Based on these observations, combinations of different chemotherapeutics and/or treatment modalities are increasingly considered as necessary strategies to improve the rate of tumour cure while decreasing systemic toxicity and circumventing drug-induced resistance.3-6 Recently, the combination of chemotherapy and photodynamic therapy (PDT) is being intensively investigated for the treatment of various types of solid tumours,7,8 being PDT minimally invasive and with reduced side effects compared with most of conventional therapies.9 PDT induces tumour ablation through the production of reactive oxygen species (ROS), after the activation of a light-activated photosensitizer (PS). This treatment is per se selective because only the PS previously taken up by the tumour is activated by the local irradiation with light of wavelength absorbed by the PS.10 PDT elicits a complex response in tumours, involving vascular damage, direct cancer cell killing, and the induction of innate and adaptive immune responses, whose relative contributions depend on the PDT treatment regimen.11 The combination with chemotherapy can maximise the effect of PDT and in turn PDT can potentiate the drug that can be administered at lower doses without losing treatment efficacy.12,13 The importance of delivering drugs at optimal concentration ratios for producing synergistic interactions has been repeatedly underlined for conventional chemotherapeutics but this appears important also for the combination of chemotherapy and PDT. In fact, chemotherapeutics and PSs, because of their different physico-chemical properties, exhibit different kinetics and efficiency of uptake in cancer cells both in vitro and in vivo. These differences highly complicate and make unlikely that the free drug combinations reach the target site at optimal ratios ACS Paragon Plus Environment
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guaranteeing synergistic effects. However, the use of multifunctional nanocarriers that accommodate in their structure more than one drug at a fixed ratio appears a unique opportunity to overcome this problem.14-18 In this perspective, here we decided to investigate on the possibility to obtain synergistic cancer cells killing through the combination of docetaxel (DTX)-based chemotherapy with a PDT treatment based on the disulfonated tetraphenyl chlorin (TPCS2a) and to assess whether co-delivery of the two molecules entrapped in a single nanoparticle (NP) could increase the synergism between the two treatment modalities. DTX is largely used for the treatments of several solid tumours including non-small cell lung, prostate, head and neck and breast cancers.19 However, its low water solubility
heavily
limits
bioavailability
that
together
with
interpatient
variability
of
pharmacokinetics and general toxicity suggests the need of DTX formulations providing more specific drug accumulation and cytotoxicity in cancer cells. Along this line, NP-based formulations of DTX are being investigated.20-22 TPCS2a (Fimaporfin) is an amphiphilic negatively-charged chlorin utilized in the photochemical internalisation (PCI) technology for the light-triggered cytosolic delivery of endocytosed drugs/toxins that are unable to penetrate the cell plasma membrane.23-25 Notably, the first phase 1 clinical trial in humans for the PCI of bleomycin showed that TPCS2a is safe and tolerable by the patients up to 1 mg/kg.26 Here we explore the possibility of using suboptimal doses of DTX together with low doses of TPCS2a-PDT for efficient killing of DTX-sensitive and -resistant cells in vitro. A comparative study is carried out co-delivering the two drugs in standard solvent formulations (free drugs) or loaded in double-layer nanoparticles (NPs) with a core-shell organization and ability to entrap and retain drugs of different solubility as already reported by us.27 The hydrophobic core made of poly(lactide-co-glycolide) (PLGA) efficiently accommodate DTX and is surrounded by a layer of the cationic polymer polyethyleneimine (PEI) that retains negatively-charged PSs through electrostatic interactions and an external layer of hyaluronan (HA). Furthermore, adsorbed HA acts as a targeting coating since it binds specific receptors including CD44, RHAMM (receptor for hyaluronan-mediated motility), HARE (HA ACS Paragon Plus Environment
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Molecular Pharmaceutics
receptor for endocytosis), and Toll-like receptors-2 and -4 that are overexpressed by various solid tumours such as breast, cervical, lungs, gastric, pancreatic and melanomas.28,29 Thus, HA receptorpositive cell lines MDA-MB-231 and HeLa (DTX -sensitive as well as resistant) were used to assess i) the occurrence of synergism when using DTX/TPCS2a-NPs vs. free drugs by applying the method of Chou and Talalay30 for the calculation of combination index (CI), ii) the mechanisms of cell death after single or combined therapy, iii) the extent of intracellular uptake and localization of the PS free or loaded in NPs.
MATERIALS AND METHODS
Materials. Meso-tetraphenyl chlorin disulphonate (TPCS2a) was provided by PCI Biotech AS (Oslo, Norway). DTX was purchased from LC Laboratories (USA). TPCS2a and DTX solutions were prepared by dissolving known amounts of powder in ethanol and DMSO, respectively. Poly (D,L-lactide-co-glycolide) (PLGA) (50:50 Resomer RG 502H inherent viscosity 0.16-0.24 dl/g) was purchased from Boehringer Ingelheim (Ingelheim, Germany). PEI (MW= 10-25 kDa branched) and Poloxamer 188 (Pluronic® F68) were purchased from Sigma-Aldrich. Acetonitrile and acetone were purchased from Carlo Erba Reagenti (Milan, Italy). HA (MW
