**1. Introduction**

Cancer is still the second leading cause of death in the world after cardiovascular diseases and, despite the continued efforts of scientists, its incidence and mortality rates have not yet been stopped [1]. Therefore, pursuing novel strategies and less toxic cancer treatments still remains a major challenge for the scientific community.

Researchers, to improve the selectivity and bioavailability of chemotherapeutic agents, have developed several drug delivery systems (DDSs) designed to allow secure transport to the selected target, thus providing molecules protection against elimination while minimizing drug toxicity to non-target cells [2–6]. During their administration, anticancer agents are generally distributed non-specifically throughout the body, affecting both tumor and healthy cells, resulting in inefficient treatment due to excessive side effects and low internalization of anticancer agents into tumor tissues [7]. On the contrary, among several advantages over traditional chemotherapeutic administration, DDSs can deliver a drug

**Citation:** Massironi, A.; Marzorati, S.; Marinelli, A.; Toccaceli, M.; Gazzotti, S.; Ortenzi, M.A.; Maggioni, D.; Petroni, K.; Verotta, L. Synthesis and Characterization of Curcumin-Loaded Nanoparticles of Poly(Glycerol Sebacate): A Novel Highly Stable Anticancer System. *Molecules* **2022**, *27*, 6997. https:// doi.org/10.3390/molecules27206997

Academic Editor: Nour Eddine Es-Safi

Received: 12 September 2022 Accepted: 12 October 2022 Published: 18 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

more selectively to a specific target site with less frequent and less concentrated dosing [5]. Moreover, in cancer cells, the passive diffusion is maximized due to the presence of abnormal vascular architecture necessary to serve fast-growing cancer cells. Such phenomenon is named enhanced permeation and retention effect (EPR effect) [6,8]. Accordingly, DDSs may exploit the EPR effect thanks to the nanometer size of the carrier which enhances their penetration and thus incorporation into the tumor target thanks to leaky vasculature [8].

Thanks to their unique physicochemical versatility, biodegradability and biocompatibility, biodegradable polymers are widely investigated for the preparation of micro- and nanoparticles as drug carriers for anticancer agents [3,9]. Among the polymers that can be used for this purpose, poly(glycerol sebacate) (PGS) has received notable attention due to its unique physical features [10]. Both monomers used for PGS synthesis via polycondensation, i.e., glycerol and sebacic acid, are bio-based, biocompatible and approved by the Food and Drug Administration. PGS synthesis is inexpensive, and the obtained product is generally soft and has flexible mechanical properties that make it suitable for working with soft tissue and organs in a mechanically dynamic environment [11]. Originally designed as a biodegradable polymer with improved elastic mechanical properties and biocompatibility, research on PGS-based medical applications has uncovered several unique properties that have bolstered its use as a biomaterial [12]. Nowadays, PGS is commonly exploited to develop 3D structures such as scaffolds for tissue engineering [13] but its possible application as a nanodelivery system has been explored less [14,15]. Moreover, several studies demonstrate that the hydrophobicity degree of a carrier is one of the major determinants to achieve correct and complete drug delivery in specific environments (e.g., lymphatic system) [4,14–16]. In this context, the aim of the present study was the development of highly stable PGS nanostructures designed to ensure secure loading of curcumin extract (standardized as 95% in curcuminoids composed by: curcumin 85%, demethoxycurcumin 14% and bis-demethoxycurcumin 1%) (Figure 1) as a novel anticancer system. Curcumin, [1,7-bis(4 hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], a naturally occurring polyphenolic phytochemical isolated from the powdered rhizome of *Curcuma longa*, was selected as an anticancer agent in virtue of its strong chemotherapeutic activity against different cancers types combined with good cytocompatibility against healthy cells [17–19].

**Figure 1.** Curcuminoids structures (**a**) curcumin, (**b**) demethoxycurcumin and (**c**) bis-demethoxycurcumin.

Several studies indicate that curcumin can modulate all kinds of cancer hallmarks, including uncontrolled cell proliferation, cancer-associated inflammation, cancer cell death, signaling pathways, cancer angiogenesis, and metastasis [20]. However, its possible exploitation as a novel chemotherapeutic is hampered by its low water solubility [21–23], which results in poor absorption and low bioavailability upon oral administration; its possible encapsulation within PGS nanoparticles represents a novel and promising administration alternative combining its strong anticancer activity with the biocompatibility and high hydrophobicity of the polymer. Nanoprecipitation was selected as the formulation technique since it offers several advantages for producing smaller nanoparticles with narrow unimodal size distribution [24]. Furthermore, it is scalable and rapid to perform [25]. The obtained NPs were characterized through dynamic light scattering and transmission electron microscopy analysis to evaluate nanoparticle size and morphology and monitoring their stability over time.

Cervical cancer is the leading cause of death for cancer in women resulting in over 340,000 deaths worldwide [26]. Most cases of cervical cancer can be attributed to persistent human papilloma virus (HPV) infection, which is preventable thanks to safe and effective anti-HPV vaccination. Screening programs allow the identification of cervical pre-cancer lesions, thus allowing prompt treatment and cure of early stage cervical cancer with surgical interventions, chemotherapy and/or radiotherapy [27]. However, recurrent cervical cancer due to resistance to chemotherapy still represents a major challenge. In this context, polyphenols, such as nanoencapsulated curcumin, are currently viewed as a possible adjuvant therapy for overcoming chemoresistance in cancer cells, since they affect multiple targets, including cell death [28]. Therefore, the biological activity of the PGS nanosystem has been investigated in vitro against human cervical cancer HeLa cells to evaluate the pro-apoptotic anticancer activities of curcumin-loaded PGS-NPs compared to free curcumin. Our results show that curcumin-loaded PGS-NPs display higher cytotoxicity, anti-HPV activity and can activate apoptosis in HeLa cervical cancer cells.
