Sclareol-loaded hyaluronan-coated PLGA nanoparticles: Physico-chemical properties and in vitro anticancer features
Graphical abstract
Introduction
The concept of nanoencapsulation has often been used in recent decades in order to modulate the biopharmaceutical properties of various active compounds [[1], [2], [3]]. Polymeric nanosystems, for example, were found to favor the administration of lipophilic and water-soluble compounds in physiological environments, acting as suitable pharmacokinetic biomodulators [4,5]. The encapsulation of lipophilic drugs within biocompatible materials, in fact, achieves greater therapeutic efficacy besides bringing about a decrease in the most common side effects that were due to the use of organic solvents for their dissolution; this was true, for instance, in the case of taxanes before the development of nab-technology [6,7]. Sclareol (labd‑14‑ene‑8,13‑diol), another natural lipophilic compound belonging to the labdane-type diterpenes, has exhibited significant cytostatic and cytotoxic effects on various human cancer cell lines [8]. Its anticancer features are based on the induction of cell apoptosis promoted by p53-independent mechanisms; in particular, cell death occurs upon the activation of caspases-8 and -9, followed by the activation of caspase-3, and the subsequent degradation of PARP [8,9]. Due to its physico-chemical properties and structural analogy to cholesterol, the drug was encapsulated within liposomes in order to favor its administration in aqueous media. The vesicular formulation evidenced a variable cytotoxicity as a function of its cell line characteristics, besides assuming a different localization in the subcellular compartments with respect to the free form [10,11]. In particular, SCL was totally localized in the cytosol while the liposomal formulation was distributed in the cell membranes, influencing its pharmacological activity as a consequence of its slow release profile from the various bilayers [10]. The aim of this experimental work was to investigate the influence of SCL on the physico-chemical properties of biocompatible polymeric nanoparticles. The polymer used to develop the system able to retain the drug was polyester poly‑lactic‑co‑glycolic acid (PLGA), due to its biodegradability and because it has the approval of the FDA and EMA for pharmaceutical application and human administration [12]. Moreover, the possibility of increasing the cell targeting of the nanosystems by using suitable agents may represent a useful strategy able to maximize the anticancer efficacy of SCL [13,14]; this is why the PLGA nanoparticles were coated with hyaluronan (HA) in order to obtain specific targeting against the cells expressing HA-receptors [15,16]. HA is a glycosaminoglycan made up of repeated units of d‑glucuronic acid and N‑acetyl‑d‑glucosamine linked by alternating β-1,3 and β-1,4 glycosidic bonds; it can be used as a targeting agent against various receptors such as the cluster determinant 44 (CD44), the receptor for hyaluronate-mediated motility (RHAMM), the HA receptor for endocytosis (HARE) and the lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) [[17], [18], [19]]. It has been shown that high-molecular-weight HA is involved in maintaining cell integrity and water content in the extracellular matrix, while low-molecular-weight HA modulates receptor-mediated intracellular signaling besides being responsible for angiogenesis and inflammatory phenomena [20,21]. The PLGA nanosystems were coated with high-molecular-weight HA (1500 kDa) in order to increase the delivery of the SCL against the HA+ cells as well as prolong the circulation of the particles after systemic administration, avoiding the use of PEG-derivatives [19,20]; the amount of HA adsorbed on the particle surface was investigated by analytical testing and the biological features of the resulting nanocarrier were evaluated.
Section snippets
Materials
Poly(d,l‑lactide‑co‑glycolide) (PLGA) 75:25 (molecular weight 66.000–107.000 Da), cholesterol (chol), sclareol (SCL), 3‑[4,5‑dimethyl thiazol‑2‑yl]‑3,5‑diphenyltetrazolium bromide salt (used for MTT-tests), dimethyl sulfoxide (DMSO), amphotericin B solution (250 μg/ml) and phosphate saline tablets (for the preparation of phosphate buffer solution pH ~7.4) were purchased from Sigma Chemicals Co. (Milan, Italy). High-molecular-weight hyaluronic acid (HA, sodium salt, 1500 kDa, purity of 95%) was
Physico-chemical characterization of PLGA nanoparticles
The first step of the characterization was focused on the investigation of the physico-chemical properties of the nanosystems as a function of the amount of SCL used; namely, empty nanoparticles had a diameter of ~140 nm, while the addition of the drug during the preparation procedure favored a decrease in the colloidal size (up to 100 nm when 3 mg of SCL were used, Table 1) probably as a consequence of the condensing properties exerted by the active compound. Moreover, the polydispersity index
Conclusions
Many plant-derived chemicals (phytochemicals) are useful compounds able to prevent and treat cancer-related diseases [39]. Diterpene sclareol exhibited significant anticancer activity against a wide range of tumors, and due to its lipophilic characteristics it was entrapped within vesicular and lipid-based carriers in order to make its administration in biological environments possible [[40], [41], [42], [43]]. The integration of the drug within a polymeric matrix made up of PLGA was performed
Author contributions
D.C. and M.F. designed the experiments, analyzed data and prepared the manuscript. D.C. and R.M. performed the experiments of cell viability and contributed to the analysis of the results. R.M. and D.P. prepared the nanosystems and evaluated their physico-chemical characteristics. C.S. performed the TEM analysis. F.C. performed the HPLC experiments. D.C. performed the CLSM experiments. D.P. and R.M. acquired data, provided a critical revision. All authors discussed the results and approved the
Acknowledgments
This paper was financially supported by funds from the Department of Health Sciences.
The authors are grateful to Lynn Whitted for her language revision of this article.
Conflicts of interest
The authors declare no conflicts of interest in this work.
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