*Review* **Strategies for Delivery of siRNAs to Ovarian Cancer Cells**

**Rossella Farra 1,**†**, Matea Maruna 1,**†**, Francesca Perrone 1, Mario Grassi 2, Fabio Benedetti 3, Marianna Maddaloni 1, Maguie El Boustani 4,5, Salvo Parisi 4,5, Flavio Rizzolio 4,6, Giancarlo Forte 7, Fabrizio Zanconati 8, Maja Cemazar 9,10, Urska Kamensek 10, Barbara Dapas 1,\* and Gabriele Grassi <sup>1</sup>**


Received: 20 September 2019; Accepted: 18 October 2019; Published: 22 October 2019

**Abstract:** The unmet need for novel therapeutic options for ovarian cancer (OC) deserves further investigation. Among the different novel drugs, small interfering RNAs (siRNAs) are particularly attractive because of their specificity of action and efficacy, as documented in many experimental setups. However, the fragility of these molecules in the biological environment necessitates the use of delivery materials able to protect them and possibly target them to the cancer cells. Among the different delivery materials, those based on polymers and lipids are considered very interesting because of their biocompatibility and ability to carry/deliver siRNAs. Despite these features, polymers and lipids need to be engineered to optimize their delivery properties for OC. In this review, we concentrated on the description of the therapeutic potential of siRNAs and polymer-/lipid-based delivery systems for OC. After a brief description of OC and siRNA features, we summarized the strategies employed to minimize siRNA delivery problems, the targeting strategies to OC, and the preclinical models available. Finally, we discussed the most interesting works published in the last three years about polymer-/lipid-based materials for siRNA delivery.

**Keywords:** ovarian cancer; siRNA; polymer; lipid; delivery

#### **1. Introduction**

Effective therapeutic approaches are lacking for ovarian carcinoma (OC), the most lethal gynecological neoplasm. Thus, the development of novel strategies is of the utmost urgency. In this review, we focused our attention on the use of small interfering RNAs (siRNAs), short double stranded RNA molecules with the ability to downregulate the expression of virtually any gene causing disease in humans [1,2]. However, due to their fragile nature in the biological environment, siRNAs, as many other nucleic acid based molecules [3–5], need to be complexed with adequate delivery materials. Among these, we focused herein on polymers and lipids, which show high biocompatibility and the ability to deliver siRNAs. Other reviews have covered additional delivery systems [6–9].

For the development of effective delivery strategies and novel therapeutic drugs for OC, different aspects should be considered. The first is the biological/anatomical features of OC. For this reason, we have dedicated specific sections to this topic (Sections 1.1–1.3). The second aspect is related to the specific nature of siRNAs, as well as the problems of their delivery in general and in particular to OC cells (Sections 2.1 and 2.2). The third element involves the features of polymer- and lipid-based delivery materials, the general characteristics of which have been described (Sections 3.1 and 3.2). A fourth aspect relates to the possibility of generating siRNA delivery complexes able to target OC cells (Section 4.1) and/or OC-specific gene products (Section 4.2) to minimize side effects and improve effectiveness. The fifth aspect relates to the employment of models adequate to produce data with potential value in humans (Sections 5.1–5.3). Finally, we have presented the most recent works (from the last three years, Section 6) related to the delivery of siRNAs, also making methodological considerations (Section 7).

#### *1.1. Ovarian Carcinoma*

Ovarian carcinoma (OC), the seventh most common cause of death among women in industrialized countries, is the most lethal gynecological neoplasm [10]. In women under 40 years of age, OC is rare; however, the risk increases above age 40, peaking in the late 70s [10]. Annually, 240,000 new diagnoses are registered worldwide, with a higher incidence in the Caucasian population in the countries of northwestern Europe and in the USA compared to Asian, African, and South American countries [11]. As OC does not have a specific symptomatology in the initial phases, about 70% of OCs are diagnosed in advanced stages when there are already abdominal metastases [12]. For the advanced forms, the combined association of surgical removal of cancer and chemotherapy has a five-year survival rate of 25% [13]. Moreover, disease recurrence within two years of diagnosis is frequent [14], and when this occurs, OC very frequently displays a high resistance to chemotherapy. Together, these factors explain why the five-year survival in OC has remained almost stable (38% 1990–1994; 41% 2000–2004) in recent years.

#### *1.2. Classification and Biology of OCs*

From a histological point of view, OCs can be divided into three subtypes based on the cells of origin. Epithelial tumors, representing more than 90% of OCs, originate from the epithelial cells that line the surface of the ovaries or the fimbriae. Germ cell tumors, accounting for just 5% of OCs, originate from germ cells and are almost exclusively found in juvenile populations. Finally, stromal tumors, constituting about 4% of OCs, originate from the gonadal stroma, which supports ovary tissue. OCs of epithelial origin have been further subdivided into (Figure 1): type I tumors, which contain well-differentiated cells (such as low grade serous carcinoma and endometrioid carcinoma) and are characterized by low malignity; type II tumors, which are rather aggressive and arise directly from the epithelial tissue of the organ without going through a precancerous phase (typically represented by the high-grade serous ovarian carcinomas—HGSOCs) [15]. Gene expression profiling has revealed the existence of five molecular subtypes of HGSOCs: mesenchymal; proliferative, with the worst overall

survival; differentiated, with an intermediate prognosis; anti-mesenchymal; and immunoreactive, with a better prognosis [16–18].

**Figure 1.** Histological classification of epithelial ovarian carcinoma.

HGSOC is the most common (70%) and most aggressive OC histotype [19]. The survival for early-stage and advanced-stage patients 10 years from diagnosis is 55%, and 15%, respectively [20]. In contrast to many other tumors, HGSOC does not require the blood or lymph to metastasize, as it can spread by direct contact to the neighboring organs within the peritoneal cavity or through cell exfoliation from the primary tumor [19]. Although, in principle, every organ within the peritoneal cavity may be involved in tumor dissemination, the involvement of a part of the neighboring peritoneum named omentum is the most frequent. The peritoneum is a large serous membrane extending from the stomach and covering the intestines (Figure 2), which contains energy-dense adipocytes. It has been proposed that HGSOC predilection for the omentum is due to cancer cell metabolism, which requires fatty-acid β-oxidation [21].

#### *1.3. Current Therapies*

The primary intervention for HGSOC is based on the use of surgery with the aim to remove the macroscopic lesions including metastasis [22], which are, as mentioned above, predominantly localized in the omentum. Notably, because of the specific biology of HGSOC, metastasis removal can improve overall survival. After surgery, the vast majority of patients undergo chemotherapy, which is the same for any type of OC [23]. Current chemotherapy is based on the use of the combined systemic administration of cisplatin–paclitaxel (PTX) [24]. More recently, intra-peritoneal (IP) administration of drugs has been proposed. As HGSOC is mostly confined to the peritoneal cavity, the IP route enables a higher local concentration [24]. Notably, a recent meta-analysis showed that IP administration significantly improved progression-free survival in advanced HGSOC patients compared to systemic administration [25]. However, significant toxicity is still an issue to be solved.

About 70% of patients treated for HGSOC respond to the first-line, PTX-based chemotherapy [26]. However, about 80% of these relapse soon or later [22]. Within relapsing patients, about 50% are

still responsive to PTX, even though persistent side effects from previous treatment may become problematic. For PTX-resistant patients, alternative options are limited and poorly effective [22]. Novel therapeutic approaches include the use of poly (ADP-Ribose) polymerase (PARP) inhibitors. PARP mediates base excision repair of single-strand breaks to resolve spontaneous DNA damage [27]. Thus, PARP inhibition can expose the cancer cell to increased DNA damage, eventually leading to cell death.

**Figure 2.** (**A**) Anatomical distribution of the peritoneum; the blue dotted line indicates the peritoneum. (**B**) Cellular structure of the peritoneum.
