Polymeric nanocapsules as drug carriers for sustained anticancer activity of calcitriol in breast cancer cells
Graphical abstract
Introduction
Calcitriol (1,25-dihydroxyvitamin D3), the active form of vitamin D, has several biological activities involving its binding to the vitamin D receptor (VDR), a ligand-activated transcription factor of several genes (Deeb et al., 2007). Calcitriol is currently used in clinic for its activity on calcium and phosphorus metabolism. It is marketed for oral administration as capsules (0.25 or 0.50 µg) and solution (1 µg·mL−1). In addition, calcitriol is known for its anti-inflammatory properties, but this has not been translated into clinical practice (Di Rosa et al., 2011). Several studies concluded that low blood levels of calcitriol were associated with high cancer incidence and low survival, especially for breast cancer (Duffy et al., 2017, Feldman et al., 2014). The anticancer activity of calcitriol has been demonstrated in vitro on cancer-derived cell cultures, and in vivo upon either calcitriol administration or a vitamin D supplementation of the diet using various animal models (Feldman et al., 2014). Calcitriol exerts its anticancer effects through several mechanisms depending on experimental conditions and tumor types. They include growth inhibition, induction of apoptosis, and stimulation of differentiation (Deeb et al., 2007). The consistency of these findings across cell cultures and animal experiments suggests that calcitriol could be used for cancer treatment, notably in the case of hormone-dependent breast and prostate cancers (Ben-Eltriki et al., 2016, Krishnan et al., 2012).
The anticancer activity of calcitriol has been evaluated in clinical studies in combination with other anticancer agents, including docetaxel, paclitaxel and imatinib, showing additive to synergistic effects (Maj et al., 2015, Woloszynska-Read et al., 2011). However, efficient anticancer activity of calcitriol requires supraphysiological doses (1–1000 nM in vitro and >1 nM in vivo) that preclude clinical use because of the high risk of hypercalcemia-related side effects (Woloszynska-Read et al., 2011). Even with intermittent dose schedules proposed to limit hypercalcemia, the maximal tolerated dose of calcitriol was as low as 60 µg per os per week. In addition, no actual efficacy was observed in the last large clinical trial (ASCENT-2) conducted on 953 patients with metastatic castration-resistant prostate cancer receiving 45 µg of calcitriol per os per week in combination with docetaxel and dexamethasone (Scher et al., 2011). Calcitriol has a short half-life in bloodstream (10 h for the marketed oral dosage forms and around 5 h for intravenous forms) and a high protein binding ratio, which makes available dosage forms inconvenient for cancer treatment (Ben-Eltriki et al., 2016, Wagner et al., 2013). It is worth considering that actual efficiency relies on the concentration and lifetime of calcitriol inside cancer cells. Thus, the importance of intracellular calcitriol level was demonstrated in experiments on human prostate cancer cell line DU-145 that overexpresses 24-hydroxylase (Ly et al., 1999). Ly et al. (1999) showed that liarozole fumarate (CYP24A1 catabolism enzyme inhibitor) prolonged the intracellular half-life of calcitriol from 11 to 31 h and increased its antiproliferative activity.
For these reasons, it is considered that calcitriol encapsulation using NPs is expected suitable for cancer therapy regarding four physiological aspects: i) it may protect calcitriol against degradation or elimination in the bloodstream, ii) it may inhibit calcitriol systemic side effects, notably hypercalcemia, iii) NPs may increase tumor targeting, and iv) NPs may enhance calcitriol cellular delivery and prolong its intracellular residence time by limiting its catabolism and its exocytosis. Data supporting these starting hypotheses come from our previous work showing that encapsulated calcitriol into polymeric NCs had a rapid release profile and a similar antiproliferative activity over 10 days in vitro on MCF-7 cells as compared to free calcitriol (Almouazen et al., 2013). The efficacy of encapsulated calcitriol systems has also been reported in few studies and confirmed the interest of calcitriol nanoencapsulation regarding its anticancer activities (Ramalho et al., 2015, Sabzichi et al., 2017). Ramalho et al. demonstrated that encapsulation of calcitriol into PLGA-based nanospheres enhanced its antiproliferative activity on lung carcinoma cells as after 72 h of incubation with free and encapsulated calcitriol (3.2 µM), 20% and 45% of growth inhibition were observed, respectively (Ramalho et al., 2015).
Similarly, Sabzichi et al. found that calcitriol-loaded nanostructured lipid careers showed significant higher antiproliferative and cytotoxic activities on the human breast cancer cell line MCF-7 than free calcitriol after 24 h of exposure to the compounds (Sabzichi et al., 2017). However, sustained activity of encapsulated calcitriol could not be investigated in these early works, and remains an open question (Almouazen et al., 2013, Ramalho et al., 2015, Sabzichi et al., 2017). It is accepted that nanocapsules are a better choice than nanospheres for encapsulating hydrophobic drugs as reviewed in Mora-Huertas et al. (2010). However, the choice of the oil to form NCs core has not been extensively studied. It appears as a crucial parameter as encapsulation efficiency into NCs is correlated to drug solubility in the oily core. Indeed, slow release is correlated with a high affinity of the drug for the oily core (Larsen et al., 2002, Mora-Huertas et al., 2010). These issues are addressed in the present work.
We also evaluated the antiproliferative and cytotoxic activities of calcitriol-loaded NPs on the human breast adenocarcinoma cell line MCF-7. Breast cancers, in particular with positive estrogen receptors, have been well studied for calcitriol anticancer activity and were the subject of several clinical trials (Krishnan et al., 2012). MCF-7 is an estrogen-dependent well-differentiated cell line known to express VDR, which makes it interesting to evaluate nanodelivery systems (Welsh, 2011). Previous studies on MCF-7 highlighted the impact of experimental conditions on growth inhibitory effects of calcitriol in particular cell exposure time to the molecule (Chouvet et al., 1986, Simboli-Campbell et al., 1996). Chouvet et al. (1986) reported that a reduction of cell number was not detected before at least 3 days of exposure, and 7 days were needed to achieve statistical significance. They also found that cells grew rapidly after calcitriol removal from the culture medium. As long exposure times are required for a definite antiproliferative activity, long-lasting calcitriol delivery seems necessary for therapeutic uses. Sustained delivery can be achieved by encapsulation of calcitriol. Moreover, long exposure times in vitro do not allow an evaluation of the influence of sustained release from encapsulated dosage forms, and experimental protocols have to be adapted using shorter exposure times closer to in vivo conditions, where tumor cells receive punctual massive doses of drug at each administration.
In the present study, these two crucial points were investigated. The composition of the NP oily core and the polymer:oil ratio were modified in order to determine suitable NP formulation parameters allowing the development of a more efficient delivery system of calcitriol. The experimental protocol was optimized in terms of cell exposure time to allow the evaluation of a sustained antiproliferative activity of calcitriol-loaded NPs in vitro on MCF-7 cell line. Finally, the cytotoxic activity of the selected NP formulation was evaluated in vitro.
Section snippets
Chemicals
Calcitriol was purchased from Cayman Chemical Company (Ann Arbor, MI, USA) (purity ≥ 97%). Poly(d,l-lactic acid), a biodegradable polymer was used for NP formulations (PLA, Mw 20,000 g·mol−1, Evonik, Germany). Caprylic/capric triglycerides (Labrafac Lipophile® WL1349), caprylic/capric triglyceride PEG-4 esters (Labrafac Hydro® WL1219), apricot kernel oil PEG-6 esters (Labrafil® M1944CS) and PEG-8 caprylic/capric glycerides (Labrasol®) were kindly gifted by Gattefossé (Saint-Priest, France).
Impact of formulation parameters on calcitriol encapsulation
Five calcitriol-loaded NP formulations were prepared by changing the polymer:oil ratio: nanoemulsions (NE, polymer:oil ratio of 0:1), nanospheres (NS, polymer:oil ratio of 1:0), and nanocapsules (NC, polymer:oil ratios 1:8, 1:4 and 1:2) were obtained (Table 1). As nanoprecipitation is not a conventional method to obtain stable nanoemulsions, the size distribution and surface charge of NE were evaluated for the entire experimental period and confirmed their stability over 10 days (data not
Discussion
Calcitriol nanoencapsulation offers an interesting dosage form in cancer therapy to improve its efficacy while avoiding side effects, which compromises its current clinical use. NPs undergo endocytosis, leading to cellular internalization of the drug. NPs also protect the drug from premature inactivation in the bloodstream, which contributes to a higher bioavailability (Brigger et al., 2012). However, more investigations on formulation parameters are needed to improve the efficacy of such
Conclusion
The influence of formulation parameters such as the polymer:oil ratio and the polarity of the oil on the encapsulation of calcitriol and its release profile has been demonstrated. We demonstrated a relationship between the release profile of the encapsulated drug from NPs and its therapeutic activity. Limiting burst release from nanocapsules by optimizing polymer and oil contents improved drug activity. Such formulation-activity relationships allowed the development of a promising NP dosage
Acknowledgments
This work received financial support of the Ministère de l’Enseignement Supérieur et de la Recherche for the PhD scholarship (France).
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