Dual-Ligand Liposomes Nano carrier with Cisplatin and Anti-PD-L1 siRNA in Head and Neck Squamous Cell Carcinoma: A Review

INTRODUCTION  
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer globally, with over 890,000 new cases annually [1]. It arises from mucosal epithelia in the oral cavity, pharynx, and larynx, often linked to tobacco use, alcohol consumption, and human papillomavirus (HPV) infection [2]. Despite advances in surgery, radiotherapy, and chemotherapy, the 5-year survival rate remains below 65% due to late diagnosis, metastasis, and recurrence [3]. New therapies for HNSCC are limited by systemic toxicity, chemoresistance, and an immunosuppressive tumor microenvironment (TME) [4]. Cisplatin, a cornerstone of HNSCC chemotherapy, often fails in advanced cases due to dose-limiting nephrotoxicity, neurotoxicity, and acquired resistance [4]. Additionally, tumor cells evade immune surveillance by upregulating PD-L1, which binds PD-1 on T cells to inhibit their activity [5]. Monotherapies targeting PD-1/PD-L1 achieve only modest response rates (15–20%), underscoring the need for combinatorial strategies [6].  Immune checkpoint inhibitors (ICIs) have revolutionized oncology, but their efficacy in HNSCC is hindered by poor TME penetration and adaptive resistance [7]. Silencing PD-L1 via siRNA offers a gene-editing approach to block immune evasion at its source, while cisplatin promotes immunogenic cell death (ICD), releasing tumor antigens that prime T-cell responses [8]. Combining these agents could synergistically enhance antitumor immunity and cytotoxicity. Co-delivering these agents ensures spatial-temporal coordination, maximizing therapeutic synergy while minimizing systemic immunosuppression [9].  
Nanocarriers, particularly liposomes, improve drug solubility, prolong circulation, and enhance tumor targeting [10]. Dual-ligand liposomes functionalized with two distinct targeting moieties (e.g., folate and transferrin receptors) exploit overexpression of multiple receptors on HNSCC cells, enabling precise delivery to both tumor and immune cells. This design overcomes the heterogeneity of HNSCC and improves penetration into the dense TME [11].  
The novelty of this approach lies in the integration of dual-ligand liposomes for simultaneous delivery of cisplatin and anti-PD-L1 siRNA. This review aims to evaluate the mechanistic basis, preclinical efficacy, and translational potential of this combinatorial nanotherapy, offering insights into its role in redefining immune checkpoint modulation for HNSCC treatment.

DUAL-LIGAND LIPOSOME DESIGN AND FORMULATION
Liposome Engineering for Dual-Targeting
Dual-ligand liposomes represent an advanced nanoscale drug delivery system designed to facilitate the co-administration of cisplatin and anti-PD-L1 siRNA. This formulation aims to enhance tumor-specific targeting, improve drug stability, and increase therapeutic efficacy, addressing challenges associated with tumor heterogeneity and immunosuppressive tumor microenvironments[12]. Cisplatin exerts cytotoxic effects by inducing DNA crosslinking and promoting immunogenic cell death, whereas anti-PD-L1 siRNA modulates immune responses by downregulating PD-L1 expression, thereby restoring T-cell function. The simultaneous delivery of these therapeutic agents is intended to optimize antitumor immune responses and inhibit mechanisms of immune evasion [13]. Traditional liposomal formulations primarily rely on passive accumulation; however, variability in tumor characteristics in head and neck squamous cell carcinoma can limit treatment efficacy [14]. The dual-ligand approach incorporates two receptor-targeting moieties, increasing binding affinity for both tumor cells and immune cells, thereby improving therapeutic delivery precision [15]. In head and neck squamous cell carcinoma, folate receptors and transferrin receptors are frequently overexpressed, facilitating selective tumor uptake [16]. Folate functions as an essential cofactor in DNA synthesis, with cancer cells exhibiting increased folate uptake due to heightened metabolic activity [17]. Similarly, transferrin plays a crucial role in iron homeostasis and is highly expressed in proliferative tumor cells, making it a viable targeting ligand [18]. The combination of folate and transferrin ligands in the dual-functionalized liposomal system ensures precise tumor accumulation, optimizing drug delivery efficiency [19]. Achieving effective drug penetration within the tumor microenvironment is critical for PD-L1 silencing and immune modulation [20]. 

siRNA Loading: Challenges and Solutions
The delivery of anti-PD-L1 siRNA presents several inherent challenges, primarily related to its stability, cellular uptake, and susceptibility to nucleic acid degradation. Free siRNA molecules are highly vulnerable to enzymatic degradation by nucleases in circulation, resulting in a short half-life and limited therapeutic efficacy [21]. Additionally, the negative charge and hydrophilic nature of siRNA molecules hinder their ability to traverse lipid membranes, leading to poor intracellular penetration and inefficient gene silencing [22]. Overcoming these obstacles necessitates the development of optimized delivery systems that enhance siRNA stability, facilitate cellular entry, and ensure effective gene silencing without premature degradation. Several key challenges must be addressed to achieve successful siRNA delivery. One major limitation is rapid clearance from circulation, which significantly reduces bioavailability and necessitates stabilization strategies to extend siRNA half-life [23]. Low transfection efficiency further complicates therapeutic application, as siRNA must enter tumor cells efficiently and suppress PD-L1 expression without being neutralized prior to intracellular processing [24]. Another critical barrier is the endosomal escape limitation, wherein siRNA molecules are internalized via endocytosis but frequently undergo degradation within lysosomal compartments before reaching the cytoplasm, thereby reducing their functional availability for gene silencing [25]. To improve the stability and delivery efficiency of siRNA therapeutics, several formulation strategies have been explored. Cationic liposomes serve as an effective delivery platform by incorporating positively charged lipids that interact electrostatically with negatively charged siRNA molecules [26]. This electrostatic association enhances siRNA encapsulation, improves retention within lipid-based carriers, and facilitates cellular uptake [27]. Surface modifications such as polyethylene glycol functionalization further contribute to siRNA stabilization by shielding the therapeutic cargo from enzymatic degradation and immune recognition, thereby prolonging systemic circulation and enhancing biodistribution [23]. Additionally, endosomal escape strategies are critical for maximizing siRNA bioavailability within the cytoplasm [28]. Lipid-based carriers often integrate pH-sensitive compounds such as histidine-modified lipids, which induce endosomal membrane destabilization in response to acidic intracellular environments, facilitating the release of siRNA into the cytoplasm and ensuring effective gene silencing [29]. These optimization strategies collectively enhance the efficacy of PD-L1 suppression, thereby potentiating the immunomodulatory effects required for antitumor activity [30]. When combined with cisplatin-induced immunogenic cell death, siRNA-mediated PD-L1 downregulation contributes to sustained immune activation and improved therapeutic outcomes in head and neck squamous cell carcinoma [31]. 

Ligand Selection for Tumor-Specific Targeting
The selection of ligands for tumor-specific targeting is a critical factor in optimizing drug delivery, ensuring precise accumulation within tumor cells and immune compartments. While passive targeting strategies rely on the enhanced permeability and retention effect, active targeting mechanisms leverage ligand-receptor interactions to achieve selective drug localization [32]. By incorporating ligands that are overexpressed on malignant cells, it is possible to enhance therapeutic specificity while minimizing off-target effects. The strategic selection of ligands is particularly relevant in the context of head and neck squamous cell carcinoma, where tumor heterogeneity and an immunosuppressive microenvironment pose significant challenges to conventional drug delivery approaches [33]. Among the various targeting ligands investigated, folate has emerged as a highly effective molecule for tumor-specific drug accumulation. Folate receptors are frequently overexpressed in head and neck squamous cell carcinoma, facilitating ligand-mediated endocytosis and enhancing the intracellular delivery of therapeutic agents [34]. The high-affinity interaction between folate and its receptor allows selective uptake, reducing systemic exposure and minimizing off-target effects. This ligand is particularly advantageous in circumventing normal tissue accumulation, thereby improving the therapeutic index of liposomal formulations [35]. Transferrin represents another widely utilized ligand for active targeting. The transferrin receptor is highly expressed in proliferative tumor cells, playing a pivotal role in iron homeostasis and cellular metabolism [36]. The overexpression of transferrin receptors in malignant tissues makes transferrin an ideal targeting moiety for liposomal drug delivery systems. By facilitating receptor-mediated endocytosis, transferrin improves drug uptake efficiency and enhances tumor-specific accumulation. This ligand is especially beneficial for nanoparticle-mediated cisplatin delivery, as iron metabolism influences tumor progression and therapeutic sensitivity [37]. Epidermal growth factor receptor inhibitors offer a targeted approach for addressing tumors with high EGFR expression. Certain