Nanoparticle based siRNA therapeutics for ovarian cancer overcoming drug resistance and future directions

Abstract

Recently, nanomedicine has made significant advancements, opening exciting new possibilities for treating a wide range of diseases. In this field, novel drug delivery systems (DDSs) are among the most noteworthy developments. The primary objective of DDSs is to ensure that treatments reach the intended targets while minimising adverse effects. In this context, nanoparticle (NP)-based DDSs have shown remarkable potential in oncology, particularly for ovarian cancer (OC), the deadliest type of gynecological cancer due to its mortality rate and the occurrence of treatment resistance. In this review, we provide a comprehensive description of the different types of NPs being explored for OC treatment, with a special emphasis on their involvement in delivering small interfering RNA (siRNA) treatments. We review various NP platforms shedding light on how they enhance drug stability, enable controlled release, and reduce toxicity. We also explore the techniques used to synthesise these NPs, emphasizing how modifying their physical and chemical properties can improve their ability to target cancer cells effectively. We also discuss the importance of 3D-tumor models, which more accurately replicate the complexity of real tumors. This enables us to examine the ability of NPs to penetrate tumors and consequently therapies are delivered in a setting that really resembles real-life situations. Recent advances in RNA-based therapeutics through DDS offer a highly targeted approach to shutting down oncogenes and drug resistance mechanisms, making them a powerful strategy to complement conventional treatments. The analysis of clinical trials results indicate a requirement for further studies in order to refine the clinical applications of drugs based on siRNAs. Despite the ongoing challenges, NP-based DDSs are paving the way for more precise and personalized OC treatments.

Graphical abstract

Introduction

Ovarian carcinoma (OC), is globally recognized as the most lethal gynaecological cancer, often called the “silent killer” due to its symptomless progression until reaching advanced stages [1]. Despite advancements in disease management, particularly in surgical techniques, neoadjuvant chemotherapy (aiming at reducing tumor bulk and allowing optimal cytoreduction surgery), adjuvant chemotherapy (aiming at reducing the recurrence risk after surgery) and palliative systemic therapy, in the majority of nations OC patients continue to exhibit a five-year survival rate that remains below 50% [2]. This is largely due to late-stage diagnosis, driven by non-specific symptoms and the absence of early detection methods, along with the frequent development of drug resistance, which leads to disease recurrence [3, 4]. The pathogenesis of such cancer involves a complex interplay of genetic and environmental factors, including mutations in BRCA1/2 genes, which significantly increase the risk [5].

OC exhibits considerable heterogeneity, with multiple histological subtypes impacting on treatment response and clinical outcomes, the most prevalent subtype being high-grade serous over endometrioid, clear cell and mucinous carcinomas [1].

The therapeutic management of OC primarily relies on the disease stage, with tumor histology, molecular profile, and the patient’s medical information represent a pivotal element in the decision-making phase [2]. Cytoreductive surgery is typically used as the initial line of treatment, and intravenous chemotherapy is thereafter administered. This includes a platinum agent (cisplatin or carboplatin) combined with a taxane (paclitaxel or docetaxel) [6, 7]. Radiotherapy is typically reserved for palliative care [8]. Furthermore, in advanced or recurrent cases, additional therapies such as the angiogenesis inhibitor bevacizumab, small molecule PARP inhibitors, antibody-drug conjugates that act on the alpha folate receptor, such as mirvetuximab soravtansine, and hormonotherapy agents are employed (applied). However, the effectiveness of these treatments is often compromised by toxicity, poor drug delivery efficiency and the emergence of resistance mechanisms, especially during disease recurrence, suggesting the urgent need for more targeted and personalized therapeutic strategies [9]. In resistant ovarian cancer, traditional drug efficacy is often compromised due to shared underlying mechanisms of resistance, such as altered drug transport, impaired DNA repair, disrupted signaling pathways, and epigenetic changes [10].

In the preceding decade, molecularly targeted therapy has emerged as a particularly salient strategy for addressing the challenges posed by cancers and, among them, by OC [11]. This is evidenced by the advent of small interfering RNA and microRNA-based approaches. The development of small interfering RNA (siRNA) as a potential therapeutic agent can be attributed to its capacity to target and silence specific genes that are associated with diverse pathologies, included neoplasm. Over the past several decades, scientists have focused on developing siRNA-based treatments to target cancer-related genes. These include proto-oncogenes and antiapoptotic genes, which have been found to have crucial roles in the pathophysiology of tumors. miRNAs have been investigated in various pathologies, including OC, to gain insights into disease mechanisms and improve patient diagnosis and treatment. Regarding OC, these small RNA molecules have been demonstrated to exert a regulatory influence on gene expression with the capacity to act as either oncogenes or tumor suppressors [12].

In this context, nanotechnology offers a potential solution by enabling the targeted delivery of therapeutic agents, thereby enhancing their efficacy while minimizing side effects. The use of nanoparticles (NPs) as delivery system, which possesses a variety of advantages including small size and the potential to adjust surface properties through the attachment of targeting ligands such as antibodies or small molecules, is a feasible strategy for the targeted delivery of therapeutic drugs to cancer cells. This method can lead to an increased local concentration of drug, with concomitant reduction in systemic toxicity.

In this review, we conducted an extensive analysis of the current literature on NPs-DDSs specifically developed for the transport of siRNA in OC. Our aim is to provide an overview of cutting-edge strategies addressing the limitations of conventional therapies, particularly drug resistance, a major barrier to effective treatment. We examined a broad spectrum of nanocarriers, including lipid-based, polymeric, inorganic, and hybrid systems, focusing on their structural characteristics, mechanisms of cellular uptake and intracellular trafficking in the context of OC. For each type of nanoparticle, we discuss their advantages and limitations to provide readers with a clear understanding of their current applications and potential new opportunities. A key focus of this review is on siRNA-loaded nanoparticles targeting genes also involved in chemoresistance. Additionally, we reviewed various production techniques, highlighting microfluidics as a particularly advantageous approach due to its precise control over particle size, exceptional reproducibility, and scalability, all key factors for the successful translation of these technologies from bench to clinic.

Overall, this review not only summarizes existing knowledge but also identifies current challenges and emerging opportunities in siRNA nanodelivery for OC.

Nanotechnology-based approaches in ovarian cancer treatment

In the treatment of OC, nanotechnology has emerged as a promising frontier by offering novel strategies to overcome the limitations of traditional therapies, such as drug resistance and nonspecific toxicity. Various NP platforms have been developed for OC therapy, including lipid-based NPs, polymeric NPs and inorganic NPs, such as gold and silica. The encapsulation of hydrophilic and hydrophobic drugs within lipid-based NPs, such as liposomes and solid lipid NPs (SLNs), represents a particularly promising area of research due to potential for protecting the drugs from degradation, and enhancing their accumulation in tumor tissues by exploiting the EPR effect [13].

One of the most innovative applications of nanotechnology in OC treatment is the development of multifunctional nanosystems capable of simultaneously delivering multiple therapeutic agents, imaging tumor lesions and monitoring the treatment response. The integration of diagnostic and therapeutic functions into a single platform, enabled by these “theranostic” NPs facilitates real-time tracking of drug delivery and treatment efficacy [14, 15].

Preclinical research on innovative NP-based DDSs has highlighted their potential to improve treatment outcomes. For instance, studies examining the use of multifunctional NPs in combination therapy, which combines chemotherapeutic agents with targeted therapies, have shown increased effectiveness in preclinical OC models [16].

Diverse studies have shown that miRNA deregulation is a key process in the pathogenesis of multiple OC subtypes. Additionally, the dysregulation of specific miRNAs has been linked to OC development, progression, and resistance to chemotherapy [12].

In fact, preclinical investigations have shown the potential of miRNA mimics and inhibitors in the treatment of OC. This is evidenced by the restoration of normal gene expression patterns, inhibition of tumour cell’s proliferation, and increased sensitivity of cancer cells to alkylating drugs, such as cisplatin [17].

Instead, siRNA-based therapeutic approach targets genes that play pivotal roles in tumor progression, helping to reduce cancer growth and spread [18]. Due to their high specificity and their ability to target genes previously deemed “undruggable”, siRNA therapies hold promise in overcoming many limitations of conventional treatments, particularly chemotherapy and radiotherapy. In OC, siRNAs have been designed to silence oncogenes and drug resistance-associated genes, such as Bcl-2, MDR1 and KRAS, which are frequently overexpressed in OC cells. It has been established that these genes are responsible for the malignant phenotype and for the resistance exhibited by the subjects to conventional therapies [19]. Importantly, siRNA-based therapies have progressed to clinical trials in OC patients. Two prominent siRNA agents, TKM-080301 and CALAA-01, have been investigated. Both utilize nanotechnology-based delivery systems and target key mediators of tumor progression, i.e., PLK1 (polo-like kinase 1) and the M2 subunit of ribonucleotide reductase (RRM2) [ClinicalTrials.gov].

The first agent, TKM-080301, is a PLK1-targeting siRNA formulated in lipid NPs (LNPs). PLK1 plays a critical role in cell cycle regulation and cytokinesis, making it an attractive therapeutic target. Preclinical studies demonstrated potent anti-proliferative activity, gene-specific silencing, and significant anti-tumor effects in xenograft models. Due to the study’s encouraging results, a Phase I clinical trial [ClinicalTrials.gov ID: NCT01437007] has been carried out for patients with colorectal, pancreatic, gastric, breast, and ovarian cancers that are unresectable, and oesophageal sites that had metastasised to the liver. TKM-080301 had a favourable toxicity profile at the tested doses, showed signs of efficacy (23% of patients achieved a partial response of disease) and insufficient data to justify further exploration [20].

Similarly, CALAA-01, a siRNA designed to reduce RRM2 expression, was tested in a Phase I clinical trial for solid tumors [ClinicalTrials.gov, ID: NCT00689065]. By inhibiting RRM2, a key regulator of DNA synthesis, CALAA-01 disrupts tumor growth through RNA interference. This siRNA was encapsulated in a stabilized NP delivery system, protecting it from nuclease degradation and ensuring targeted delivery to tumor cells. The study illustrated that targeted nanoparticles carrying siRNA were successfully delivered to tumors in all 24 patients [21] and in three patients, with effective RRM2 knockdown confirmed in biopsies, demonstrating the feasibility of systemic siRNA therapy for cancer [22]. However, the study with CALAA-01 was terminated, primarily due to disease progression in 29% of patients and two patients experienced dose-limiting toxicities [21]. The lack of subsequent follow-up studies suggests that the scientific community has shifted its focus toward alternative therapeutic strategies.

Nanoparticle-based drug delivery systems for targeted cancer therapy

A long-standing challenge in the management of cancer treatment has been the precise administration of drugs to the desired site of action; indeed, many of the conventional medication available in therapy suffered from short half-life, poor biodistribution, and a series of adverse side effects which hinder their use [23]. Furthermore, the key to successful fight tumor is to create novel drug delivery systems (DDSs) that selectively address tumor cells, increasing the therapeutic efficacy and shielding normal cells from damage [24]. According to the extant studies drug-loaded NPs, to be effective, they should exhibit properties that facilitate stability in the bloodstream until the desired site is reached, thereby avoiding clearance by the reticuloendothelial system (RES) and mononuclear phagocyte system (MPS). Selective accumulation within the tumor microenvironment (TME), minimal interaction with adjacent healthy tissues, is therefore essential [25]. More specifically, NPs could use passive and/or active targeting techniques to reach their target sites [26]. One of the most important players in passive targeting is the TME; in fact, its metabolic changes could have a significant impact on targeting mechanisms because the variations in environmental pH facilitates the accumulation and release of drugs from pH-sensitive NPs [27]. Besides, the tumor-related phenomena of hypoxia and inflammation contribute to turning the endothelium more permeable [24, 28]. In addition, the neoangiogenesis associated with solid tumors leads to the formation of new blood vessels, which have been observed to be characterized by large fenestrations (200–2000 nm), in contrast with healthy conditions [28]. The extravasation of NPs from the bloodstream to the tissues affected by pathology is facilitated by the aforementioned alterations. This facilitates the NPs’ selective accumulation via a mechanism known as the “enhanced permeability and retention” (EPR) effect [29]. On the other hand, directing molecules labeled ligands are used to decorate the surfaces of NPs in order to accomplish active targeting. In this scenario, the targeted NPs reach the target tissue, whereupon the specific ligand-receptor interaction subsequently triggers the internalisation process of the nanocarrier via endocytosis [30]. For instance, transferrin acts as one of the most popular ligands suitable for stimulating active targeting in solid tumors where it is estimated that there is an expression of the transferrin receptor (TfR1) up to 100 times higher than in healthy tissues [31]. The next sections summarize different classes of nanocarriers, with a particular focus on their structure and their cellular uptake and trafficking mechanisms in OC.

Liposomes

Definition and properties

Liposomes are described as nanoscale carriers consisting of lipid bilayers enclosing an aqueous center. Chemically, they are typically characterized by the presence of phospholipids, which, as amphiphilic molecules, self-assemble in an aqueous solution to form closed vesicles containing cholesterol [32]. This component is particularly important since it stabilizes the phospholipid bilayer, impacting both chemical-physical properties and morphological traits. Indeed, due to the amphiphilic nature of the molecule, cholesterol has a tendency to arrange itself in space such that the hydroxyl group is oriented toward the polar heads of the phospholipids and the aromatic chains located at the alkyl tails [33]. By creating a degree of controlled disorder with enhanced fluidity at the core and greater packing at the polar heads, this intrusion into the phospholipid bilayer causes liposomes’ structure to be sufficiently stable in an aqueous phase and prevent water penetration. Nonetheless, since alkyl chains are more fluid, drugs can be integrated into them, making liposomes an effective drug delivery tool.

Moreover, the classification of liposomes is dependent on the lamellarity of their lipid structure, whereby liposomes can be categorised into three distinct classes: small unilamellar vesicles (SUVs, diameter < 100 nm), large unilamellar vesicles (LUVs, diameter 100–1000 nm), and giant unilamellar vesicles (GUVs, diameter > 1 μm). Generally, classical production leads to the formation of multilamellar vesicles (MLVs), with a complex multilayer structure [32]. Moreover, liposomes colloidal stability in solution is dependent on the specific surface charge (ζ-potential) that they exhibit. Indeed, the presence of lipids with polar heads that have a negative surface charge enhances dispersive interactions and prevents lipid self-aggregation [34].

Relevance in ovarian cancer

Liposomal gene therapy has shown promise in overcoming drug resistance in OC models. Liposomes delivering siRNA or antisense nucleotides targeting MDR1 (and BCL2 in some cases), in combination with chemotherapeutic agents like paclitaxel or doxorubicin, significantly reduced tumor growth, enhanced apoptosis-related gene expression, and drastically lowered the effective drug dose, as reducing doxorubicin IC₅₀ by up to 15-fold [35].

These results provided conclusive evidence of the ability of liposome­mediated targeting to enhance drug potency and reduce systemic toxicity and other adverse effects caused by antitumoral drugs.

Specific siRNA delivery studies

Recent years have seen significant advancements in the development of novel lipid-based delivery mechanisms for siRNAs [36]. It is evident that unmodified siRNAs are intrinsically unstable in biological fluids and are incapable of traversing cell membranes independently; therefore, the incorporation of lipid components is imperative to facilitate cellular uptake and ensure efficient transportation to the target cells [37], thereby safeguarding them from nuclease deterioration mechanism and kidney clearance [38].

It is notable that neutral nanoliposomes, such as 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), have been observed to demonstrate a high degree of loading efficacy, thereby affording control over the administration of chemotherapy. Guillermo et al., demonstrated the suppression of neoplastic cell’s proliferation and the reduction of macrophages’ quantity expressing specific surface markers. This result was achieved through the administration of hMCP1 siRNA-DOPC NPs to ovarian cancer samples, obtained from murine that have been subjected to chronic stress [39].

Given the elevated anionic charge exhibited by small interfering ribonucleic acids, the efficacy of cationic lipid-based carriers is enhanced [40]. In line with this, Zhao et al. revealed that inhibition of Notch homolog 1 translocation-associated gene in the SKOV-3 cell line through the use of siRNAs, in combination with a cationic cholesterol molecule, has the faculty to impede mechanisms of replication and cell death [41].

The development of a procedure for extremely efficient in vivo gene silencing was achieved by William M. Merritt and his team. siRNAs of proinflammatory chemokines, involved in tumor progression processes such as IL-8, were incorporated into neutral liposomes composed of DOPC. The experiments conducted on murine models of ovarian cancer showed that IL-8 siRNA-DOPC reduced the mean tumor weight by 32% and 52% in the HeyA8 and SKOV-3ip1 mouse models, respectively [42].

Advantages and limitations

Due to their dual nature, liposomes are capable of incorporating both hydrophilic and hydrophobic compounds, positioned within the aqueous core and lipid bilayer, correspondingly [32, 43].

The size of these NPs is a critical factor in determining their purpose; in fact, those in the 100–150 nm dimensional range experience cellular uptake and the EPR effect, whilst those in the 50–100 nm, are able to avoid the MPS clearance [44].

Moreover, coating the nanosystems’ surface with polyethylene glycol (PEG) is one of the most popular methods for extending the stability and half-life of liposomes in the bloodstream. The hydration layer, that is formed around the NPs due the PEGylation, enables them to be identified as stealth, thus avoiding fast clearance mechanisms [32, 45].

Solid lipid nanoparticles (SLNs) and nanostructured lipid nanoparticles (NLCs)

Definition and properties

The SLNs are NPs characterized by a solid structure at room temperature consisting of glycerides, fatty acids, or waxes. The addition of a surfactant guarantees the stability of these formulations in the aqueous environment [46]. Following the first description, these NPs structures evolved into NLCs, which included a combination of solid lipids and oils in the organic phase to improve the system fluidity while making it less challenging to incorporate drugs within [47].

Relevance in ovarian cancer

In a recent report, the production and optimisation of gemcitabine (GEM) and oxaliplatin (OXA)-loaded SLNs (GEM: OXA-SLN) was described as potential treatment for OC [48]. In accordance with the findings of in vitro research, this study demonstrated that GEM: OXA-SLN prevented the proliferation of OC cells by triggering autophagy, reducing the synthesis of Hsp90, and blocking the MDR efflux pump [48].

Specific siRNA delivery studies

To the best of our knowledge, there are currently no established delivery systems for siRNAs utilizing SLNs or NLCs.

Advantages and limitations

Due to their simplicity of production and the non-toxic and more biocompatible nature of the lipids utilized, these nanosystems show several benefits, including sustained drug release over days to weeks without the problems associated with fluctuation in plasma levels [49]. Moreover, due to the solid nature of the lipidic core, SLNs could be the optimal delivery system for compounds prone to degradation as well as peptides and enzymes. In fact, the great protection provided by the solid core from biological pH and plasmatic degradative enzymes may be the functional key to proper deliver biotechnological drugs, alongside the possibility to enhance the surface’s capacity for active targeting through the strategic incorporation of ligands [50]. Furthermore, SLNs measuring approximately 120–200 nm with a hydrophilic coating exhibit enhanced physiological competence in prolonging their presence within the bloodstream, thereby circumventing hepatic and splenic filtration processes while avoiding RES recognition [49].

Lipid-nanoparticles (LNPs)

Definition and properties

LNPs are a subclass of lipid-based NPs that have recently undergone development in response to the COVID-19 pandemic and have been promptly authorized for clinical use by the FDA due to their promising application for nucleic acid encapsulation [51]. LNPs are characterised by a specific chemical composition, which includes the presence of an ionizable lipid, a helper lipid, cholesterol and PEG-lipid conjugates arranged in a defined ratio [52].

Relevance in ovarian cancer and specific siRNA delivery studies

In a recent study, Singh et al. exploited the use of hyaluronic acid (HA)-LNPs to target various cellular pathways crucial to the development of OC by encapsulating a combination of siRNAs, PLK1 and eukaryotic translation-initiation factor 3c (eIF3c). They demonstrated that the combination of siRNA-LNPs achieved robust gene silencing in the target tumor of xenografted mice with an orthotopic ovarian metastasis model, as well as in adriamycin-resistant ovarian cancer cell lines, resulting in significantly improved survival compared to both untreated mice and those treated with single siRNA-loaded LNPs [53].

Michael S. Goldberg and colleagues, reported a protocol for the administration of siRNAs, by intraperitoneal injection, which were targeted against the DNA repair gene Parp1. The objective of their in vivo study was to demonstrate the efficacy of the lipidoid-formulated nanoparticle, containing the antisense oligonucleotides, in prolonging murine survival [54].

Advantages and limitations

The ionizable lipid interacts electrostatically with the nucleic acid due to the difference in charge at low pH ensuring the complete complexation of genetic material. The helper lipid contributes to the stability and the release of the payload within cells while cholesterol stabilizes the structure of the nanosystem. Finally, the presence of PEGylated lipids makes the LNPs stealthy, preventing their uptake by the RES and allowing them to remain in the bloodstream for a longer time [52, 55].

Polymeric NPs

Definition and properties

Polymeric NPs are nanostructured carriers produced starting from monomers or pre-formed polymers, in which the active cargo could be embedded in the polymer matrix or could be chemically linked to the polymer or NPs surface [56]. The two most prevalent categories of polymeric NPs are solid matrix systems, otherwise referred to as nanospheres and nanocapsules. The presence of a hollow region which is surrounded by a polymeric shell is a hallmark of these. Within these two broad groups, NPs are further classified by morphologic features into polymersomes, dendrimers, and micelles [26].

Synthetic vesicles known as polymersomes are constructed from amphiphilic unit copolymer membranes; these structures appear similar to liposomes, with an improved stability and drug encapsulation efficacy [57]. Dendrimers are a class of polymer that possess a complex three-dimensional structure. The drug has the capacity to be incorporated into the polymer, or alternatively, to undergo a chemical linkage with the active functional groups on the nanostructure surface. Thus, dendrimers can transport a variety of cargoes, although they are most commonly considered for the delivery of small molecules and nucleic acids [58]. Finally, polymeric micelles are nanospheres with an hydrophilic core and hydrophobic outer structure, enabling the protection and retention of the encapsulated cargo [59].

Relevance in ovarian cancer and Specific siRNA delivery studies

Drug resistance has been overcome by using polymeric nanoparticles. In fact, to provide an explanatory example, Risnayanti and colleagues have developed poly(lactic-co-glycolic) acid (PLGA) NPs involving both Bcl-2 and MDR1 siRNAs with the aim to simultaneously inhibit cell death defence pathways and drug efflux mechanisms, responsible for drug resistance in OC. As a result, pretreatment of siRNA@PLGA NPs enhanced the therapeutic effects of cisplatin and paclitaxel on MDR OC cells. The IC₅₀ values of the SKOV-3-TR and A2780-CP20 cells were found to decrease significantly by 4.46 and 7.65 fold, respectively [60].

Steg and colleagues demonstrated that targeting of Jagged1, with Chitosan(CS)/siRNA NP led to sensitization to docetaxel and decreased tumor burden in orthotopic OC mouse models [61]. In their work Kim et al. employed siRNA to target Src, a transmembrane receptor tyrosine kinase that is integrated into CS-NP. This approach led to a demonstrable a reduction in cell proliferation, angiogenesis and an enhancement of apoptosis in taxane-resistant OC cell lines. The same results were observed in vivo [62].

In their study Ma and colleagues [63] explored the potential of synthetic polymers, including dendrimers for the delivery of siRNAs. The employment of siRNAs directed against P70s6k, a kinase protein that plays a pivotal role in the process of metastasis and tumour progression in OC, was a key component of the experimental design. The experimental findings have demonstrated a reduction in proliferation and expansion of tumor cells in vitro. Moreover, emphasis has been placed on the decline in migration and invasion of cancer stem cell in vivo [63]. PI3K/Akt represents a further significant signalling pathway in the context of pathophysiology of OC, with the potential to serve as a target for therapeutic intervention. In a recent study Shashwati Kala and colleagues utilised a triethanolamine-core poly(amidoamine) dendrimer to facilitate the transportation of siRNA directed against the Akt gene in conjunction with paclitaxel. This type of dendrimer has been found to exhibit optimal compatibility due to its inherent characteristics of openness and flexibility.

Consequently, this combination has demonstrated a heightened response in comparison to monotherapy, as evidenced by both in vivo experiments and in vitro drug-resistant models such as SKOV-3 cell line [64].

Advantages and limitations

These nanosystems have been widely explored as drug delivery systems for cancer treatment due to their high storage stability, water solubility, biodegradability, and potential to overcome drug resistance. Nevertheless, in comparison with lipid-based nanocarriers, polymeric NPs may be associated with an increased risk of particle aggregation and toxicity [26].

Inorganic NPs

Definition and properties

The employment of inorganic materials in generation of inorganic NPs has been extensively documented, with such applications including theranostics and imaging. Elements as gold, iron, and silica, have been studied for the production of NPs in the technological form of nanospheres, nanoshell and nanocages [65].

Relevance in ovarian cancer and specific siRNA delivery studies

Among inorganic NPs, gold NPs (AuNPs) have shown promise in gene delivery for drug-resistant cancers such as OC, due to their exceptional surface plasmon resonance and easily controllable size. In this context, Arvizo and colleagues, developed positively charged AuNPs that were loaded with specific siRNA molecule designed to target the MICU1 gene. MIUC1 is a key regulator of the mitochondrial calcium uniporter, an important component of the process that enables cells to resist the effects of cytotoxic agents. The team then proceeded to assess the efficacy of these AuNPs in a series of in vitro tests utilising human OC cell lines including OVCAR5, OV167 and OV202. By silencing MICU1, these NPs have been demonstrated to reduce the expression of the Bcl-2 gene and simultaneously increase the level of free calcium ions within the cell. Silencing MICU1, these NPs were able to reduce the expression of Bcl-2 and simultaneously increase the level of cytosolic calcium. These alterations have been demonstrated to trigger the mitochondrial pathway of programmed cell death [66] .

In a recent study, delivery of siRNAs in OC by using selenium-based NPs (SeNPs) was reported [67]. Natural selenium is skilled to stimulate immune system activity, whilst SeNPs have been shown to be low in toxicity, easily adaptable, and highly biocompatible. In this specific study, Se-NPs were functionalized with the cationic peptide RGDfC to electrostatically complex the MEF2D-siRNA. Thus, it has been established that the addition of the cationic peptide boosted R-Se@MEF2D-siRNA uptake by SKOV-3 cells primarily via clathrin-associated endocytosis. This peptide is also responsible for the enhanced release of MEF2D siRNA in the acidic lysosomal microenvironment (pH 4.5) compared to physiological condition (pH 7.4). Furthermore, the efficiency of silencing MEF2D mRNA in SKOV-3 cell line was established, demonstrating a relevant apoptotic effect. Lastly, in vivo investigation showed that R-Se@MEF2D-siRNA enhanced anticancer activity while controlling adverse effects, developing a promising treatment strategy to face OC [67].

Advantages and limitations

The versatility of inorganics materials allows to design NPs with a wide range of dimensions, geometries, and shapes. Moreover the starting materials, due to their intrinsic properties, enable the production of NPs with unique magnetic and electrical features [26].

Biofunctionalized targeted NPs

Definition and properties

Biofunctionalized targeted nanoparticles are engineered nanosystems designed to improve the specificity of drug delivery through surface modification with biological or synthetic targeting moieties. These nanoparticles are typically composed of organic, inorganic, or hybrid materials and are functionalized with ligands such as antibodies, peptides, or small molecules. Surface biofunctionalization allows controlled interaction with cellular receptors and biological barriers. Their physicochemical properties, including size, surface charge, and composition, can be finely tuned during formulation. As a result, these NPs exhibit well-defined structural and biological characteristics suitable for biomedical applications [68].

Relevance in ovarian cancer

In the field of oncology a study demonstrated that transferrin-decorated polymeric micelles loaded with paclitaxel and tariquidar were able to penetrate deeper into 3D OC models, reverting the MDR phenotype in SKOV-3-TR cells and increasing cytotoxicity in both single and double drug-loaded micelles as respect to plain polymeric micelles, demonstrating the effectiveness of such active targeting [69]. Additionally, the functionalization with transferrin was also found to be useful in reducing cytotoxic effects. Indeed, a study conducted by Yuan and Zhu demonstrated that transferrin­modified docetaxel-loaded liposomes greatly decreased the systemic toxicity of docetaxel and efficiently reduced tumor growth in A2780 and SKOV-3 °C cells compared to docetaxel-loaded liposomes [70].

It has been demonstrated that the majority of primary human ovarian tumours in addition to the vascular endothelial cells of neoplastic origin, exhibit an expression of CD44 proteins. Since HA is these proteoglycans’ natural ligand, Wang and Jia in their work developed a HA-based paclitaxel (PTX)-loaded lipid NPs demonstrating that such functionalized nanosystems had increased therapeutic efficacy for OC chemotherapy in both in vitro and in vivo tests when compared to PTX-loaded cationic nanostructured lipid NPs (PTX-NLCs) [71].

Specific siRNA delivery studies

Given that chemoresistance is a major contributor to the poor prognosis of ovarian cancer, adoptive cell therapy using CAR T cells has emerged as a potential alternative treatment for OC. In the present context, exploration has been undertaken of strategies that are based on siRNAs. To illustrate this point, the work of Pei Yun Teo and colleagues may be cited: this study highlighted that delivering PD-L1 siRNAs to luciferase-expressing SKOV-3 ovarian cancer cells improved the sensitization to T cell-mediated death by disrupting PD-1/PD-L1 interactions. In order to enhance the uptake of siRNAs by such cells, which have high level of folate receptors, polyethyleneimine (PEI) on the surface was substituted with FA or PEG-FA. The study demonstrate that FA-conjugated polymers enhanced the uptake of siRNAs into SKOV-3-luc cells and diminished the uptake of siRNA into monocytes. This resulted in a 40–50% decrease in PD-L1 protein expression. Additionally, it was noted that SKOV-3-Luc cells treated with PEI-FA or PEI- polyethylene glycol (PEG)-FA/PD-L1 siRNA complexes showed increased susceptibility to T cell-mediated, reaching up to twofold more susceptible compared to the control group treated with scrambled siRNAs [72].

Moreover, Jingguo Li and his group synthesized a ternary copolymer linkages for siRNA delivery conjugating a single chain monoclonal antibody (Herceptin) to the carrier as a targeting ligand for the Her2/neu receptor. This resulted in a significant increase in the transfection efficiency of the nanocomplex in the SKOV-3 cell line. The present study was conducted with the objective of ascertaining the effectiveness of Her2-targeted delivery systems in downregulating the X-linked inhibitor of apoptosis protein gene (XIAP). This was accomplished by increasing cancer cell apoptosis and improving therapeutic interventions both in vitro and in vivo [73].

Advantages and limitations

Biofunctionalised targeted nanoparticles allow selective interactions with specific biological targets through surface-bound ligands, contributing to controlled engagement with defined cell populations and improved biodistribution leading to better therapeutic outcomes. Their surface chemistry can be adjusted to modulate interactions with biological environments. However, the introduction of targeting moieties may increase formulation complexity and require additional optimization steps. Variability in functionalization parameters can also influence reproducibility and in vivo performance, highlighting the need for careful design and characterization [74].

Biomimetic NPs

Definition and properties

Pharmaceutical research about novel DDSs has recently focused on the production of biomimetic NPs. These innovative biosystems, which are composed of elements including lipids, polymers, or inorganic metallic compounds coupled with biological components, represent a great novelty as they have experienced tremendous growth in the fields of nucleic acid delivery and cancer therapy due to the need to address selectivity issues that classical nanosystems may face [75]. Thus, one of the most recent advances in pharmaceutical technology is the development of cell membrane camouflaged NPs by using biological components from tumors, leukocytes, platelets and erythrocytes [76]. The hybridized nature of biomimetic NPs retains the proteins and antigens from the original cancer cells on the NPs surface, effectively mimicking the tumor’s biological identity [77, 78]. From a technological standpoint, as illustrated in Fig. 1, biomimetic NPs fall into two types based on their structure: biomimetic hybrid NPs and fused biomimetic hybrid NPs [76]. In the first case, cell membrane is applied to preformed NPs or more basic materials as polymers and lipids, resulting in a coating that increases biocompatibility and targeting while masking non-biological components [79]. In the case of hybrid fused biomimetic NPs, proteins and/or cell membrane fragments, as well as other biological components, are integrated into the developing NPs via fusion, resulting in the formation of a new biohybrid entity [80].

Fig. 1.

Explanatory diagram of the different structure and production phases in the preparation of biomimetic NPs. Reproduced with permission from [76]

In recent research, a range of cell membranes, encompassing those derived from cancer cells, stem cells and blood cells, have been utilised to encapsulate drug through a process of biomimetic nanodrug delivery. This method involves the use of an extrusion technique to formulate the encapsulation [81].

Relevance in ovarian cancer

Ye and colleagues developed an arsenic trioxide (ATO)-based nano-delivery system to combine starvation therapy, chemodynamic therapy, and chemotherapy for improving treatment of OC. In particular, they wrapped NPs with the cell membrane of SKOV-3 °C cells (AMGNPs@SKOV-3) with the aim of increasing the uptake by tumor cells. These study demonstrated that the homologous targeting of the coated NPs resulted in increased uptake by tumor cells, achieving the strongest anti-tumor effect with good biosafety and few side effects [82].

Tianbao Chen and colleagues prepared chemodrug-gene NPs (Mito-Her2 NPs) by electrostatic interaction coself-assembly of mitoxantrone and Her2 antisense oligonucletide (Her2 ASO). Then, these NPs are coated by a hybrid membrane consisting of the red blood cell membrane (RBC-M) and the SKOV-3 °C cell membrane to produce biomimetic chemodrug-gene NPs for combination therapy of OC. The results showed that these NPs effectively degraded to release Her2 ASO and mitoxantrone simultaneously, producing a high apoptosis rate of 75.7% in vitro, and a high tumor suppression rate in vivo, without significant damage of normal tissues [83].

Jianqiang Xiong and co-workers in their studies fabricated a hybrid consisting of murine-derived ID8 OC cell membrane (ID8-M) with RBC-M and coated them onto indocyanine green-loaded magnetic NPs (Fe3O4-ICG) for synergistic photothermal-immunotherapy of OC. These elaborately synthesized NPs (Fe3O4-ICG@IRM) have shown highly specific self-recognition of ID8 cells in vitro and in vivo, prolonged circulation time in blood, and they have determined the release of whole-cell tumor antigens by photothermal-induced tumor necrosis. This last event enhanced the antitumor immunotherapy for primary and metastatic tumor by activating CD8⁺ cytotoxic T cells. In summary, the biomimetic Fe3O4-ICG@IRM NPs showed synergistic photothermal-immunotherapy for OC [78].

As a further example, Lei Zhang and co-workers developed a fusion cell membrane (FCM) nano-vaccine FCM-NPs, which was prepared by fusing dendritic cells (DCs) with OC cells ID8 and coating the FCM on the PLGA NPs loaded with the immune adjuvant CpG-oligodeoxynucleotide (CpG-ODN). FCM-NPs exhibited a strong immuno-activating effect both in vitro and in vivo, and it was capable of delay the growth and inhibit the metastasis of OC [84].

Specific siRNA delivery studies

In the context of OC, these biomimetic NPs could be engineered to carry gene-silencing agents, such as siRNAs or miRNAs, or a combination of these molecules with chemotherapeutic drugs. Integrating both therapeutic strategies within a single NP would enable a synergistic effect, enhancing anticancer activity while ensuring highly selective delivery to the relevant target. An example outside the OC and in the condition known as Diminished Ovarian Reserve (DOR), characterized by a reduced number and/or quality of eggs, impacting female fertility, is the study by Guo in which the authors demonstrated that biomimetic PLGA-based nanoparticles loaded with siPAI-1 effectively prevent the pathology by enhancing siRNA delivery, silencing PAI-1 expression, reducing ovarian fibrosis, and promoting follicle preservation [85].

Advantages and limitations

Due to their increased stability, resistance to immune system activation, ability to prevent nonspecific absorption, and selective targeting properties, studies in the literature demonstrate the benefits that biomimetic NPs can offer [86]. This “biomimicry” not only improves the targeting of the NPs, but also reduces their recognition and clearance by the immune system. This approach consequently produces longer circulation time and greater accumulation in the TME [77].

Nanoparticle manufacturing: traditional methods and microfluidic approach

The challenge of synthetic feasibility remains one of the main obstacles to the widespread use of novel formulations in healthcare. In practice, the large-scale production of NPs with unique chemical-physical properties remains a major challenge. Previously, formulations have been developed in academia using traditional synthetic techniques, which are now inadequate to ensure consistent, safe, and reproducible production. As a result, the in-flow production technique has gained increasing attention in recent years, paving the way for industrial scale-up and broader applications.

Conventional strategies of NPs production

The conventional methodologies employed for synthesizing NPs can be categorised into two primary classifications: the top-down method, which uses a series of degradative processes to form NPs from starting materials, and the bottom-up method, which involves atoms coalescing and/or aggregating to form nanostructured entities [87]. Another approach to classification based on the type of synthetic process involved. These processes can be categorised as follows: physical synthesis, chemical synthesis, and bio-assisted synthesis [24, 87]. Identifying the appropriate approach is essential taking into account both the type of NPs to be synthesized as well as the starting material. More in detail, liposomes are usually produced by chemical processes. The thin film hydration method, the depletion method, the solvent injection method and the reverse phase method are the four principal techniques under consideration [32]. The produced nanosystems, in the form of multi lamellar vesicles, need to be downsized via the use of physical processes as extrusion, sonication, and high-pressure homogenization [32]. By applying these post-productional methods, nanosized monodisperse liposomes could be achieved; however, the quality of the formulation relies on the type of lipid, the extruding pores and filter [88]. If considering lipid-based NPs, the formation is usually based on nanoprecipitation process occurring when an ethanolic lipid solution encounter an aqueous phase while mixing [89]. Even in this case, due to the impossibility of controlling all the forces that drive the bulk method, there is a lack of reproducibility and dimensional range over 100 nm [89, 90].

Similarly, in the case of polymeric NPs, the election strategy is the chemical solvent evaporation method. In instances where the pharmaceutical compound to be encapsulated exhibits hydrophobic characteristics, a dual-phase system is employed. In this configuration, the polymer and the hydrophobic drug are dissolved into the organic phase, and then encounter an emulsifying agent-rich aqueous phase, leading to the constitution of an oil-in-water type emulsion system. Conversely, the creation of water-in-oil-in-water double emulsion for the encapsulation of a hydrophilic molecule is a feasible prospect. In both cases, the reaction ends with the solvent evaporating and polymeric NPs being formed [91]. Thus, the success of this reaction depends on the type of polymer and its concentration, the surfactant, the hydrophobic/hydrophilic nature of the cargo, and stirring method; unfortunately, all these factors hinder batch-to-batch reliability and scaling up production [92, 93].

In the case of inorganic NPs, magnetic NPs, can be achieved by physical processes such as beam lithography or gas deposition, while metal NPs are usually obtained by chemical reduction processes [94, 95]. However, the cited procedures, still face several difficulties; in fact, during the production of inorganic NPs it is impossible to manage directly the particles’ growing, causing significant batch-to-batch fluctuation in size and size distribution [96].

When focusing on the production of siRNA-loaded NPs, it is important to emphasize that there are multiple strategies based on the type of NPs. Due to the need to pay close attention to siRNAs when working with them, the reaction environment requires RNA-free material and a shorter timeline to prevent nucleic acid failure. Polymers including PEI, PEG, polycaprolactone (PCL), PLGA have been thoroughly investigated as siRNA delivery agents via bulk methods [97].

An illustrative example is provided by the research of Mendes and colleagues. where liposomes were prepared and subsequently engineered with PEI–lipid conjugate to co-deliver anticancer drug and MDRs siRNA in an OC model. First, paclitaxel-loaded liposomes were created, and then they were covered with PEI-lipid. Consequently, an electrostatic interaction was established between the surface of the NPs and the siRNAs, thereby complexing the ribonucleic acids onto the NPs [98].

On the other hand, Ghareghomi et al., developed folate-functionalized PLGA NPs that were co-loaded with telomerase reverse transcriptase (hTERT) siRNA, wortmannin, a powerful PI3K inhibitor, and magnetic NPs (MNPs), a theranostic agent, to produce multifunctional NPs for targeted drug administration and molecular targeted therapy. This sophisticated architecture enabled for tiny nanosystems with siRNA encapsulation effectiveness of approximately 75% of the total load. As a result, cell viability assays revealed that wortmannin/siRNA-loaded MNPs-PLGA-F2 NPs exhibited the highest synergistic effect on SKOV-3 °C cells, proving the efficacy of combination therapy and demonstrating that the co-delivery of Wtmn and hTERT siRNA through the MNPs-PLGA-F2 NPs led to an effective targeted molecular inhibition of hTERT [99].

It is a cause for concern that, as a consequence of the aforementioned issues, a poor rate of translation persists between basic research and clinical applications. The primary cause of this is the large batch-to-batch variability that result from the traditional bulk method’s inadequate control over the preparation properties, including particle size and polydispersity index (PDI) [100]. This point should not be underestimated since the physicochemical and surface properties of NPs play a pivotal role as they influence all the phenomena that occur after in vivo administration, including biodistribution, protein corona effect, bio-interactions, and clearance [101, 102]. Thus, it is interesting to note that in vivo fate of NPs mostly depends on their dimensional range and surface properties. According to the available evidence, once administered, the NPs are subjected to the so-called protein corona effect which relies on adsorption of plasma proteins on their surface; this phenomenon is size-dependent and confers unique properties to the NPs influencing their bio-interactions [103]. Moreover, the distinct biodistribution of NPs in organs and tissues is a direct consequence of their size; in fact, NPs bigger than 200 nm are promptly captured by the liver and spleen due to the activation of complement, while smaller NPs experience both prolonged circulation time and increased penetration across all organs, with the exception of the blood-brain barrier [104]. Similar to size, several studies in the literature have demonstrated the critical role that surface properties play in blood circulation and nanocarriers biodistribution, showing that NPs with larger surface charge densities and/or greater hydrophobicity incorporate more proteins compared to those with neutral charges. This results in faster opsonization and removal by the RES [105, 106].

The overwhelming weight of evidence points to the crucial significance of strictly managing the technological properties of NPs; in addition, bench methods require long manufacturing times, the use of non-green solvents, and lack of operational control mechanisms over the chemical-physical processes [107]. As a result, over the last years, the microfluidic technology has gained favour as a viable process for mass-producing NPs with strictly controlled characteristics [108].

Microfluidic approach

Clinical development of siRNA-based treatments for OC is hampered by multiple interrelated linked obstacles encompassing manufacturing, stability, delivery, and translational application. From a biological perspective, siRNAs are particularly sensitive to enzymatic breakdown by circulating ribonucleases and rapid renal clearance, necessitating the use of chemical stabilization techniques or delivery platforms that may influence safety profiles and pharmacokinetic behaviour [109]. NPs operate as a protective barrier for siRNAs, preventing them from being degraded in the circulatory system, increasing their plasma half-life, and improving cellular uptake [110].

When considering siRNA-loaded formulations, the extreme efficiency of microfluidic production plays a critical role in encapsulating the nucleic acid in a finely controlled environment. By virtue of its nature, handling siRNA in a microfluidic facility guarantees that, when compared to conventional production, the contact time between the sensitive material and the organic solvent is drastically reduced, resulting in a lower incidence of chemical degradative phenomena [110, 111]. Achieving batch-to-batch consistency remains a critical concern, as the carrier properties as size distribution, polydispersity, and encapsulation efficacy may significantly influence biological performance and reproducibility [112]. Furthermore, scalability remains a major challenge when evaluating the difficulty of converting preclinical production procedures into cost-effective and GMP-compliant manufacturing pipelines [113].

The aforementioned issues have been largely addressed by the introduction of microfluidic technology in the pharmaceutical industry, which is defined as “the science and technology of systems in which extremely small volumes of solvent are manipulated within microchannels”. In fact, microfluidic technique could yield considerable benefits since during the in-flow processes it is possible to manage the occurring chemical reactions both in space and time [114].

A classical microfluidic system (Fig. 2) is comprised of a software that is coupled to pumping systems which are connected to phase reservoirs (aqueous and organic). These fluids are carried to the microfluidic device; in this instance, the phases are inserted into the chip by keeping fluid rate ratios (FRRs) and total flow rates (TFRs) finely controlled. The outcome from the microfluidic device is the final product, ready to be analyzed and characterized [115].

Fig. 2.

Microfluidic set-up illustration. Reproduced with permission from [116]

The microfluidic device is the core of the entire microfluidic setup since the technology’s high efficiency stems from the channels’ miniaturization, which drives production through diffusive processes rather than convective ones that occur during traditional bulk production [116].

Given that the fluid dynamics of each device differ based on the forces applied and the trajectory the phases follow, it is recommended to choose a specific internal geometry according to the kind of formulation that is required, as defined in literature [100]. This aspect provides precise control of the distribution of fluid inside the compartments, and batch-to-batch variability is removed once optimal conditions are identified, as microfluidic technique guarantees stringent control over all operational parameters [117].

The ability to design and customize NPs properties, e.g., size, shape, functionalization, the narrower size distribution and the automatization of the system facility, the possibility to parallelize the production and scaling-up to industrial application, contribute to identify microfluidics as a viable approach to optimize and produce ready-to-test innovative formulations for a wide range of application [118].

A summary chart of the most widely used geometries for the synthesis of LNPs and liposomes is shown in Fig. 3.

Fig. 3.

Graphical representation of the most widely used geometries of microfluidic devices. A T-junction, B Staggered herringbone micromixer (SHM), C Hydrodynamic flow focusing (HFF)

Many studies looked at the very important benefits of the in-flow production method; in a pivotal work, Belliveau et al. demonstrated the ability to produce reliable nanosized siRNA-loaded LNPs using a simple chip with staggered herringbone micromixer (SHM) internal geometry, showing improved silencing capabilities over those produced via traditional methods [119].

Jürgens et al. recently evaluated several microfluidic platforms for the production of siRNA- and mRNA-loaded LNPs, including T-junction mixers, staggered herringbone mixers (SHM), and hydrodynamic flow focusing (HFF). From a technological standpoint, HFF allowed to produce siRNA-loaded LNPs with the most favorable particle size (approximately 114 nm) and superior performance in in vitro efficacy studies [52].

More specifically, concerning anti-tumor therapy, a recent study demonstrated the feasibility of microfluidic-based fused biomimetic hybrid NPs in the potential treatment of metastatic malignant melanoma. Briefly, fused hybrid liposomes were developed using three-dimensional printed microfluidic device by combining cell membrane fragments from a BRAF wild type metastatic melanoma cell line (MGS). Two targeted agents were utilised: the MEK inhibitor cobimetinib and the multi-target receptor tyrosine kinase inhibitor lenvatinib. As a result, it has been established that the produced biomimetic NPs have physico-chemical properties suitable for administration, and showed substantially stronger homotypic targeting for the MGS cell line, under both 2D and 3D growth condition, when compared to non-hybridized NPs. Furthermore, the delivered drugs demonstrated higher cytotoxic activity than free ones, highlighting the promising application of homotypic targeting in cancer therapy [120].

NP systems are able to act as a shelter for siRNAs, protecting them from the degradative processes that would otherwise occur in the circulatory stream. When considering siRNA-loaded formulations, the extreme efficiency of microfluidic production plays a critical role in encapsulating the nucleic acid in a finely controlled environment. By virtue of its nature, the handling of siRNA in a microfluidic facility ensures that the contact time between the sensitive material and the organic solvent is dramatically reduced if compared to conventional production, leading to a lower incidence of degradation phenomena. In a very recent study, VandenHeuvel and colleagues exploited siRNA SIRPα-loaded LNPs to inhibit malignant development in a 3D OC model. Here, LNPs were generated using the NanoAssemblr Benchtop microfluidic equipment, dissolving the siRNA inside the aqueous phase while maintaining the cationic lipid into the ethanolic solution. Three batches were examined, and characterisation results showed that all formulations had a size of approximately 60 nm and a low PDI (< 0.1). Furthermore, siRNA encapsulation measurements demonstrated encapsulation efficacy greater than 95% across all batches, showing the great proficiency of the in-flow production [111].

Although exploratory and comparative in nature, the cited studies have brought attention to the enormous potential that microfluidics application may have in the nucleic acid delivery and cancer therapy. Indeed, the adoption of fast, scalable, and non-destructive approach might be critical to the application of highly innovative formulations in nanomedicine.

Advancements in 3D cell culture models for ovarian cancer

Over the past decade, it has become clear that conventional cell monolayer cultures and animal models are only in part adequate for investigating human diseases and treatment responses. A key reason is the low translational rate of research findings obtained with such experimental models for which the predictive value is limited.

The available evidence suggests that the response of tumour cells to cytotoxic agents dramatically differs between 2D and 3D cell culture models. It has been demonstrated that the latter is an optimal platform for the purpose of drug screening and toxicity testing [121], due to its capacity of reflecting more accurately the inherent complexity of a tumour in vivo.

Indeed, immortalized human cell culture models fail to incorporate the donor’s heterogeneous genetic background, tissue structure (spatial organization), diverse cell composition, and interactions with the extracellular matrix [122].

Therefore, 3D cell culture models, including spheroids and patients-derived organoids (PDOs), could become the most reliable tools in preclinical research as they combine two key advantages: the ability of the system to reproduce tumor architecture, in conjunction with its capacity for efficient high-throughput drug screening [121].

The limited progress in improving survival outcomes for OC can be largely attributed to the use of ‘one-size-fits-all’ therapies and the lack of clinically relevant experimental models that accurately reflect the advanced stages of such disease [123]. The generation of reliable pre-clinical models for studying OC that fully recapitulate the biological and morphological characteristics of neoplasms of this nature are of great significance for improving our understanding of cancer for guiding the clinical decision making processes.

Kopper et al. developed an organoid platform that enables the long-term in vitro expansion, manipulation, and analysis of multiple OC subtypes, and their findings showed that OC organoids not only retain nuclear and cellular atypia as well as biomarker expression, but also accurately model recurrent mutations and the tumor heterogeneity characteristic of OC [124]. Recent studies demonstrate that OC organoids faithfully mirror patient responses to chemotherapy and exhibit heterogeneous sensitivity to PARP inhibitors (PARPi). Beyond this, they function as powerful experimental models to elucidate determinants of PARPi sensitivity, investigate mechanisms of chemoresistance, and evaluate novel drug combinations aimed at overcoming resistance [125].

The interaction between tumor cells and their surrounding niche, which constitutes the TME, plays a key role in the spread of OC cells within the peritoneum facilitating metastasis, and in their response to treatments [126]. Therefore, in order to design or screen novel therapies targeting OC cells, it is essential that drug testing models incorporate the TME.

Recently, significant advancements have been made in the fields of 3D in vitro cancer models and tumor tissue/microenvironment bioengineering, driven by the development of 3D models that integrate key elements of the TME in a spatially and biomechanically relevant manner. These 3D models enable the study of cancer cell behaviour and drug response under conditions that more closely mimic the physiological environment, proving to be more efficient for drug testing and drug discovery in the perspective of precision medicine [123].

Pilot studies have demonstrated promising results using co-culture systems of OC organoids with diverse components of the TME, including cancer-associated fibroblasts, endothelial cells and immune cells. This approach enhances spatio-temporal resolution and reduces intra-organoid heterogeneity [127, 128].

In this context, the application of dynamic 3D cell culture approaches could potentiate the pre-clinical tools for disease modeling, drug screening and cytotoxicity testing. The organoid-on-a-chip is an experimental tool based on a microfluidic device with potential applications to overcome the limitations of conventional organoids by integrating OC cells with cells of the TME in a controlled environment. The main feature of such device is the controlled perfusion of growth factors which allows cell to cell and cell to stroma interactions [129].

Although numerous studies have been published on the use of nanodrugs in cancer treatment, only a limited number of systems have advanced to clinical application, also due to the poor predictive outcomes achieved with standard 2D cell culture methods in in vitro experiments [130].

While the use of simple 2D monocultures may be suitable for initial screening of nanodrug safety during the early stages of NPs development, they should be replaced in later stages by multicellular systems. Furthermore, many of the currently used in vivo models fail to accurately replicate the TME, immune environment, and the tumor permeability and penetration, typical of the human body [130]. Therefore, the field of nanomedicine calls for the adoption of standardized 3D in vitro models and protocols to efficiently and reproducibly assess the key features that influence NPs behavior in the biological environment and to gain a deeper understanding of bio-nano interactions [131].

Of note, multicellular spheroids and scaffold-based co-culture systems, have emerged as valuable tools for the evaluation of nanoparticle-based siRNA delivery, as they better reproduce key features of the in vivo microenvironment, such as cell–cell and cell–extracellular matrix interactions, tissue-like architecture, and diffusion-limited gradients. In particular, spheroids develop gradients of oxygen, nutrients, and metabolites, leading to heterogeneous cellular states and gene expression profiles that markedly differ from those observed in 2D cultures. Such microenvironmental cues critically influence nanoparticle penetration, intracellular trafficking, and the efficiency of siRNA-mediated gene silencing, thereby providing a more physiologically relevant platform to assess delivery performance and biological response [132, 133].

Regarding DDSs for siRNA delivery, Joshi and colleagues developed a hypoxia-sensitive PEG-azobenzene-PEI-DOPE (PAPD) construct to co-deliver doxorubicin (Dox) and anti-P-gp siRNA (siPgp) which was tested in 2D and 3D human OC cell lines. This DDS contained the enzyme-sensitive PEG-cleavable moiety, which conferred it the ability to induce a de-PEGylation, facilitating the uptake into tumor cells. The results obtained demonstrated that the uptake of PAPD NPs was found to be more efficient in the 3D model than in the cell monolayer, highlighting that, while PEGylated NPs can still be internalized by 2D monolayers, dePEGylation is essential for efficient NP uptake by 3D tumor models. Additionally, they showed that the administration of siRNA versus P-glycoprotein in combination with doxorubicin delivered using PAPD NPs resulted in 80% cytotoxicity in cell monolayers and 20% cytotoxicity in spheroids under hypoxic conditions, suggesting the importance of using 3D models to confirm results generated on 2D monolayers, especially before starting animal studies [134].

Nanoparticles in siRNA-based therapy: critical challenges and translational limitations

RNA technology has the potential to be a groundbreaking therapeutic method for treating a variety of diseases; however, before its application becomes a concrete reality, it is necessary to find an adaptable and robust drug delivery technology capable of overcoming all the issues hampering its clinical application [135].

Although siRNAs are a good option for suppressing target gene expression, they face multiple barriers to their systemic delivery, including possible immunogenicity, the onset of off-target effects, poor intracellular uptake, endosomal escape, and renal clearance [136]. Once naked and unmodified siRNA is administered, serum ribonucleases rapidly degrade it, and the resulting fragments may trigger undesirable immune responses. Conversely, if exogenous siRNAs escape rapid degradation, they are promptly detected by pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and retinoic acid–inducible gene I (RIG-I)-like receptors (RLRs). This recognition activates an immune signalling cascade, leading to the production of inflammatory cytokines and type I interferons [137, 138]. Additionally, exogenous siRNAs may be taken up by non-target cells via phagocytosis and endocytosis, triggering an unintended process that may cause unexpected side effects and gene silencing in healthy cells [139].

Scientific attention has increasingly focused, particularly in recent years, on targeted non-viral vectors. As previously described, the delivery of siRNA via NPs should address problems with chemical–physical instability, cargo loading, and overcoming the intracellular barriers that siRNA must face in order to exert its biological activity in the cytosol [140]. Most significantly, however, off-target effects are minimized by employing novel targeted formulations that limit the therapeutic activity mostly to the site of interest by exploiting the active targeting approach [141].

siRNA uptake and endosomal escape are critical points strictly depending on nucleic acids’ chemical properties; in fact, due to their size and negative charge, RNA and endo molecules require energy-dependent processes, as well as endocytosis, to move through the cell membrane. Issues with cellular uptake and proper subcellular targeting have long limited the clinical use of nucleic acid-based treatments [142].

When siRNA-loaded NPs are internalized within the target cell, an endosome is created leading to a progressive acidification of the internalized material before the vesicle can release its contents into the cytosol. Under physiological conditions, this process results in the complete degradation of unencapsulated siRNA, thereby severely compromising its biological activity [143]. On the other hand, numerous excipients for siRNA-carrying nanoparticle synthesis have been particularly designed to have the ability to escape the endosome, allowing free and unmodified siRNA to be released and perform its biological activity [144].

Despite the numerous advantages, such as the extremely tunable properties of inorganic NPs and the great versatility of polymeric ones, it is necessary to carefully select the type of nanosystem to be used according to the intended outcome. In fact, it is crucial to take into account that non-lipid nanoparticulate systems present greater challenges related to biocompatibility and biodegradability, as well as accumulation toxicity, which significantly limits their clinical application [145]. To this end, research in pharmaceutical technology continues to progress, with increasing attention devoted to materials suitable for gene delivery applications with reduced toxicity. In particular, novel gemini surfactants have emerged as a promising approach to enhance the complexation of genetic material into nanoscale lipoplexes, while simultaneously overcoming limitations related to the cellular uptake of large, charged molecules [146]. Moreover, exploiting the functionalization of anticancer NPs with targeting ligands is essential to maximize site-specific accumulation and precise delivery of the therapeutic payload, thereby substantially limiting off-target distribution and minimizing adverse effects on healthy tissues [147].

Recently, studies with significant translational promise have concentrated on lipid-based NPs, especially LNPs. In these systems, the inclusion of ionizable lipids not only enhances the binding and encapsulation of genetic material but also facilitates endosomal escape by inducing perturbations in the vesicular membrane [144].

Indeed, the translatability of innovative formulations remains challenging. In fact, once the limitations related to scaling up production systems in flow have been overcome, rigorous testing and several phases of clinical trials are necessary to guarantee safety and efficacy. Furthermore, it is crucial to overcome the issues related to the stability, sterilization, and storage of the resulting formulations [148].

Lastly, in addition to research and development challenges, regulatory approval poses a further obstacle for LNP-based technologies, especially given the variety of compounds they can deliver. The FDA and EMA require LNPs to meet quality, safety, and efficacy standards. During preclinical development, cell and animal studies assess the formulation’s toxicity; only if deemed safe can approval be sought to initiate phase 1 clinical trials [149]. These factors make it more challenging to carry out long-term research initiatives and restrict access to these therapies once they are approved [150].

Conclusion

OC remains one of the most lethal gynaecological malignancies, largely due to its asymptomatic progression, late diagnosis, and resistance to conventional therapies. These limitations highlight the urgent need for innovative therapeutic strategies, among which nanotechnology has emerged as a particularly promising approach.

Future research efforts should focus on refining NPs designs, improving production scalability through microfluidic systems, and developing reliable tumor models to better predict clinical outcomes. By overcoming these challenges, nanotechnology has the potential to revolutionize OC treatment, offering more effective and personalized therapeutic solutions that could significantly improve patient outcomes.

Abbreviations

OC

Ovarian carcinoma

NPs

Nanoparticles

DDSs

Drug delivery systems

RES

Reticuloendothelial system

MPS

M phagocyte system

TME

Tumor microenvironment

EPR

Enhanced permeability and retention

TfR1

Transferrin receptor

siRNA

Small interfering RNA

SLNs

Solid lipid nanoparticles

PLK1

Polo-like kinase 1

RRM2

Ribonucleotide reductase

LNPs

Lipid nanoparticles

SUVs

Small unilamellar vesicles

LUVs

Large unilamellar vesicles

GUVs

Giant unilamellar vesicles

MLVs

Multilamellar vesicles

PEG

Polyethylene glycol

DOPC

2-dioleoyl-sn-glycero-3-phosphocholine

NLCs

Nanostructured lipid nanoparticles

GEM

Gemcitabine

OXA

Oxaliplatin

HA

Hyaluronic acid

PLGA

poly(lactic-co-glycolic) acid

CS

Chitosan

SeNPs

Selenium-based NPs

AuNPs

Gold NPs

PTX

Paclitaxel

PEI

Polyethyleneimine

PEG-FA

Polyethylene glycol-folic acid

PEI

Polyethyleneimine

XIAP

X-linked inhibitor of apoptosis protein gene

ATO

Arsenic trioxide

RBC-M

Red blood cell membrane

FCM

Fusion cell membrane

DCs

Dendritic cells

CpG-ODN

CpG-oligodeoxynucleotide

DOR

Diminished Ovarian Reserve

PCL

Polycaprolactone

MNPs

Magnetic NPs

PDI

Polydispersity index

FRRs

Fluid rate ratios

TFRs

Total flow rates

PDMS

Polydimethylsiloxane

PMMA

Polymethyl methacrylate

SHM

Staggered herringbone micromixer

HFF

Hydrodynamic flow focusing

PARPi

PARP inhibitors

PAPD

PEG-azobenzene-PEI-DOPE

Dox

Doxorubicin

siPgp

Anti-P-gp siRNA

Author contributions

Roberta Di Fonte: Data curation, Writing—original draft. Isabella Bolognino: Data curation, Writing—original draft. Federica Sommonte: Data curation, Writing- original draft. Simona Serratì: Writing—review and editing. Rossella Fasano: Writing—review and editing. Ilaria Arduino: Writing—review and editing. Rosa Maria Iacobazzi: Writing—review and editing. Nunzio Denora: Writing—review and editing. Paola Perego: Conceptualization, Writing—review and editing. Giacomina Rossi: Writing—review and editing. Diego Tosi: Writing—review and editing. Letizia Porcelli: Conceptualization, Supervision. Amalia Azzariti: Data curation, Writing- original draft, Conceptualization, Supervision.

Funding

This work was supported by the European Union - Next Generation EU - PNRR M6C2 - Investment 2.1 Enhancement and strengthening of biomedical research in the NHS - project: PNRR-MAD-2022-12376508 (CUP MASTER: B43C22001030006 – CUP Istituto: F73C22002150006).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Authors′ information

The authors affiliated to the IRCCS Istituto Tumori “Giovanni Paolo II”, Bari are responsible for the views expressed in this article, which do not necessarily represent the Institute.

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors give their consent for publication.

Clinical trial

Not applicable.

Competing interests

The authors declare that they have no competing interests.