A superior method for antitumor therapy and application: dual-ligand nanomedicines

Abstract

Currently, nanomedicines have been widely applied in the treatment of various types of tumors. However, due to the complexity of the tumor microenvironment, conventional nanomedicines often exhibit poor efficacy, insufficient site specificity, and susceptibility to off-target effects. In contrast, dual-ligand nanomedicines demonstrate superior targeting ability and drug penetration in tumor therapy. These nanomedicines are equipped with two ligands on their surface, enabling targeting of specific receptors on the same or different cells. The specific binding between ligands and receptors significantly enhances the selectivity and targeting of dual-ligand nanomedicines towards tumors. This review systematically describes the preparation of dual-ligand nanomedicines, the influencing factors, and the types of delivered drugs, focusing on the application of dual-ligand nanomedicines in targeting the treatment of various tumors. We highlight the comprehensiveness of dual-ligand nanomedicines for the treatment of tumors, including glioblastoma, lung cancer, breast cancer, gastric cancer, and many other types of tumors. Finally, the possible challenges for the future development of dual-ligand nanomedicines in terms of preparation, clinic, and safety are further analyzed. We look forward to exploring dual-ligand nanomedicines in greater depth to provide references for their future development and clinical applications.

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Ailing Wang

Ailing Wang is currently pursuing her master's degree at Shandong University of Traditional Chinese Medicine under the supervision of Prof. Jiyong Liu. Her main research interests focus on nanoparticle-based drug delivery systems for anti-tumor applications.

Jiyong Liu

Jiyong Liu received his PhD degree in Pharmacology from Changhai Hospital of Shanghai, Second Military Medical University in 2005. After that, he completed a postdoctoral training in Shanghai University of Traditional Chinese Medicine, and was a visiting scholar of Weill Cornell Medical College of Cornell University from 2013–2014. He is currently a doctoral advisor of Clinical Pharmacy in Fudan University and serves as the director of Department of Pharmacy, Fudan University Shanghai Cancer Center. He focuses on the study of innovative drugs and novel anti-tumor drug delivery systems based on clinical demand orientation.

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1 Introduction

Current medical methods still face difficulties in treating cancer. With the advancement of nanotechnology, physicians can now treat tumors with drugs laden with nanoparticles (NPs) through the enhanced permeability and retention (EPR) effect, where nanomedicines passively cross the peritumoral vascular system to reach tumors, alternatively, ligands on the surface of nanomedicine can bind to receptors overexpressed by tumor cells or vascular endothelial cells enabling nanomedicines to actively target tumors.¹

Nevertheless, emerging research elucidates that although the EPR effect plays a pivotal role in murine models, its translational relevance in humans is limited due to tumor heterogeneity or deficient fenestrations in the tumor endothelium.² Moreover, considerable heterogeneity exists in the EPR effect across preclinical models and even among patients with identical tumor profiles.³ A review of the literature over the past decade demonstrated that only 0.7% of NP doses effectively reached solid tumors.⁴ This suggests that active tumor targeting using ligands is increasingly becoming the focal point of research, as passive tumor targeting with nanomedicines proves to be less efficacious. Nanomedicines can be functionalized with one, two, or multiple ligands on their surface to actively target various cellular receptors. The fundamental mechanism of active targeting relies on the specific binding between ligands and receptors. Following cellular internalization, primarily via receptor-mediated endocytosis, these nanocarriers must efficiently escape from lysosomes to release their therapeutic cargo into the cytoplasm. Several strategies have been developed to facilitate lysosomal escape, such as increasing the osmotic pressure within lysosomes to disrupt their membrane integrity, thereby enabling the nanocarriers to reach the cytosolic space and exert their therapeutic effects.⁵ Ligand binding can be inhibited by receptor alterations, which can impact targeting efficacy. Moreover the receptors on tumor cells are dynamically changing, some of them are also expressed on healthy cells, which may enable healthy cells to absorb nanomedicines.^6,7 For these reasons, single ligand nanomedicines may exhibit off-target effects as a result of poor targeting efficiency.

In contrast, dual-ligand nanomedicines for tumor therapy exhibit heightened cellular selectivity and enhanced cellular uptake. Qiu et al.⁸ developed a dual-ligand modified nanomedicine (cGA/cRGD-LP-DOX) incorporating glycyrrhetinic acid (GA) and cyclic Arg-Gly-Asp peptide (cRGD), targeting the GA receptor and α_vβ₃ integrin, respectively, in hepatic tumor tissues. In vivo studies revealed that the clearance rate of GA- and cRGD-modified nanoparticles from plasma was faster than that of non-targeted nanoparticles (Fig. 1(A)). Biodistribution analysis indicated that the accumulation of nanoparticles in tumor tissues increased over time (from 4 to 8 hours), with GA/cRGD-LP-DOX (adriamycin) exhibiting significantly higher tumor uptake at 8 hours compared to GA-LP-DOX, cRGD-LP-DOX, and LP-DOX. Meanwhile, drug accumulation in the heart gradually decreased (Fig. 1(B)). Furthermore, ex vivo fluorescence imaging performed 4 hours post-injection demonstrated that GA/cRGD-LP-DOX showed the strongest fluorescence signal in tumor tissues, indicating superior tumor-targeting efficiency (Fig. 1(C)). Ligand-modified nanomedicines not only significantly extend the drug's half-life in vivo but also enable precise targeted delivery to tumor tissues. Compared to single-ligand-modified and unmodified nanomedicines, dual-ligand-modified nanomedicines exhibit superior antitumor efficacy (Table 1).

Fig. 1 Schematic diagram of pharmacokinetic correlation. (A) Time profile of DOX concentration in plasma after intravenous injection of different DOX formulations. (B) Doxorubicin concentration in heart, liver, and tumor acquired from nude mice xenografted HepG2 tumor at different times after intravenous injection of liposomal DOX formulations. (C) Biodistribution of DOX liposomal formulations after intravenous injection. (*P < 0.05, **P < 0.01, ***P < 0.001).⁸ Copyright 2023, ACS Publications.

Table 1 A meta-comparison of dual vs. single vs. no-ligand efficacy

Formulation	Drug release rate	Tumor drug accumulation	Tumor volume	Body weight	Liver and kidney toxicity	Survival time extension
Abbreviations: experimental conditions: each formulation was injected four times at 4 day intervals by tail vein at a dose of 6 mg kg^−1. (10 mice per group); statistical analysis: (*P < 0.05, **P < 0.01, ***P < 0.001); survival time extension calculation formula:
No ligand	55%	10%	Moderate	Increased	No significant toxicity	25%
GA single ligand	64%	15%	Small	Increased	No significant toxicity	50%
cRGD single ligand	60%	25%	Small	Increased	No significant toxicity	32%
GA + cRGD dual ligand	70%	40%	Small	Increased	No significant toxicity	86%

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Dual-ligand nanomedicines bind two ligands targeting different or the same receptor site. Through receptor-mediated endocytosis, the nanomedicine enters the tumor cell releasing the drug and exert a better therapeutic effect, demonstrating significant application potential (Fig. 2). This review analyzes the preparation, influencing factors, and types of delivered drugs of dual-ligand nanomedicines are analyzed in detail. As dual-ligand nanomedicines in tumor therapy have not been comprehensively described before, this review focuses on their potential application in targeted tumor therapy. Various combinations of ligands are designed according to different tumor types to achieve better therapeutic effects, emphasizing the wide-ranging nature of the treatment. Finally, we offer our insights and summarize the challenges encountered in the current development of dual-ligand nanomedicines.

Fig. 2 Schematic illustration of dual-ligand nanomedicines navigating the tumor microenvironment and selectively targeting tumor cells through enhanced binding affinity and cellular uptake. The dual-ligand nanomedicines are circulated into the tumor microenvironment, where their surface dual ligands bind to receptors on the surface of the tumor or vascular endothelium and enter the cells. Then the nanocarriers are cleaved by the lysosomes in the tumor cells to release the anti-tumor drugs therein, which play a role in the death of the tumor cells.

2 Preparation of dual-ligand nanomedicines

2.1 Dual-ligand components

In contrast to single-ligand nanomedicines, dual-ligand nanomedicines are generally more effective, utilizing two ligands to target distinct receptors expressed either on the same cell or on different cells. The ligands can be distributed on the surface of the NPs or combined into a single molecule. Common ligands include antibodies, proteins, peptides, small molecules, glycans, aptamers, etc. (Fig. 3).

Fig. 3 Types of nanocarriers/ligands used for nanomedicines: ABC for Liposomes (where B represents multicompartment liposomes, while A and C are unilamellar liposomes, with drugs encapsulated in their respective internal compartments and structures), DEF for Micelles (where D and E represent micelles viewed from different angles, and F refers to polymeric micelles), GHI for Nanohydrogels (where G depicts the internal structure of hydrogels, H and I represent hydrogels with different properties and shapes), JKL for metal/inorganic nanoparticles (where J refers to iron oxide nanoparticle carriers, K represents graphene or other planar nanostructures, and L stands for gold nanoparticles). For ligands, RGD, HA, and TF bind to specific receptors, biotin is transported into cells via the sodium-dependent multivitamin transporter (SMVT), and aptamers specifically bind to target molecules.

2.1.1 Antibodies. Antibodies are among the most widely used natural ligands for modifying the surface of nanomedicines. Although the preparation of antibodies is complex and associated with high production costs, their ability to bind to specific antigens with high affinity allows for highly specific targeting, providing significant clinical translation potential. In particular, bispecific antibodies can bind two overexpressed receptors simultaneously, and this dual receptor targeting strategy improve tissue penetration and selectivity for tumor-targeted delivery. Emami et al.⁹ utilized functionalized nanoparticles modified with two different antibodies: anti-epidermal growth factor receptors (EGFR) antibody (Panitumumab) and anti-PD-L1 antibody, and encapsulated with docetaxel (DTX) to target tumor cells. This strategy leads to tumor cell death through a synergistic signaling mechanism and is more selective than single ligand-targeted nanoparticles. Wu et al.¹⁰ designed a dual-ligand magnetic nanoparticle, functionalized with two highly specific antibodies—anti-Lyve-1 and anti-podoplanin antibodies. This design enabled the nanoparticles to more precisely target lymphatic endothelial cells within tumors, facilitating in vivo fluorescence/magnetic resonance (MR) molecular imaging. This approach significantly improved the accuracy and effectiveness of cancer diagnosis.

2.1.2 Proteins. When proteins are used as ligands to functionalize the surface of nanoparticle carriers, the potential expression of protein receptors in normal tissues may lead to nonspecific uptake, Thereby reducing the targeted delivery of nanoparticle-based drugs to tumor cells. However, proteins are naturally present in the human body and exhibit lower immunogenicity compared to antibodies, making protein ligands more suitable for long-term or repeated administration, with enhanced safety and applicability. Transferrin (TF) is the most commonly used protein ligand, binding to the TF receptor (TfR) and enters the cell through receptor-mediated endocytosis. This process facilitates the transport of iron absorbed by the alimentary canal and freed from erythrocyte degradation into the cell from the bloodstream. TfR expression in cancer cells can be up to 100 times higher than that of normal cells in average. TfR is an attractive target for cancer treatment due to its extracellular accessibility, internalization capacity, and crucial function in the cytopathology of human cancer. Wang et al.¹¹ designed a dual-ligand nanodrug functionalized with TF and folic acid (FA). This drug enters tumor cells through the specific affinity of TF with TfR and folic acid (FA) with folate receptor (FR). Compared to non-targeted and single-targeted nanoparticles, this dual-ligand nanodrug exhibits higher cytotoxicity and stronger cellular uptake, showing superior anti-tumor efficacy. Zhang et al.¹² functionalized the surface of nanogels with lactoferrin (Lf) and phenylboronic acid (PBA) for dual-targeted drug delivery in glioma treatment. The system specifically targets low-density lipoprotein receptor-related protein-1 (LRP-1) and sialic acid (SA), achieving precise tumor-targeted delivery across the blood–brain barrier.

2.1.3 Peptides. Compared to antibodies, peptides exhibit smaller steric hindrance, making them more readily able to bind to targets, and they also have lower immunogenicity. As intermediate products of protein hydrolysis, peptides are composed of α-amino acids linked by peptide bonds. Due to their unique structural characteristics, peptides, when used as ligands, can undergo modifications such as cyclization or amino acid substitution to prevent degradation from by protein hydrolysis, thereby enhancing their stability and functionality within drug delivery systems. Commonly used peptides are RGD and cell penetrating peptide (CPP). The RGD cyclic peptide can activate signal transduction pathways by competitively binding to integrin receptors to control a variety of cellular processes required for the genesis, development, and metastasis of solid tumors, preventing tumor cells from attaching to the extracellular matrix or directly causing tumor cells to undergo apoptosis, consequently achieving the goal of inhibiting tumor cell growth, invasion, and metastasis.¹³ The CPP structure contains cations and amphiphilic amino acids, which can cross cell membranes to deliver medications or other cell-impermeable compounds to cells and enhance uptake by tumor cells. Amin et al.¹⁴ modified liposomal nanoparticles with RGD cyclic peptide and TAT (trans-activator of transcription) cell-penetrating peptide to target the tumor neovasculature and tumor cells. As a member of the CPP family, TAT peptide significantly enhances the cellular uptake of nanodrugs through receptor-mediated endocytosis. This ligand combination significantly improved nanoparticle–cell interactions under normal conditions, showing more pronounced effects compared to single-ligand modifications. Zhang et al.¹⁵ coated iron oxide (Fe₃O₄) nanoparticles with a layer of poly(lactic-co-glycolic acid) (PLGA) and further functionalized them with cRGD and GA-EWVDV (a Glu-Trp-Val-Asp-Val pentapeptide modified with gallic acid), thereby constructing a dual-ligand nanoparticle system. These nanoparticles targeted glycoproteins and P-selectin on platelet membranes. The results indicated that the nanoparticles exhibited specific targeting ability and stable binding to thrombi. This study represents the first application of dual-ligand-modified nanoparticles for dual-modality molecular imaging of thrombosis, providing a novel approach for the diagnosis and treatment of thrombosis.

2.1.4 Small molecules. Small molecules are advantageous in the modification of nanomedicines because of their low immunogenicity and inexpensive manufacturing cost. Compared to proteins and peptides, small molecules exhibit better chemical stability and are more resistant to variations in pH, temperature, and proteolytic enzymes. However, their target expression is limited, typically being highly expressed only in certain tumors. This limitation can be addressed by combining small molecules with other ligands to enhance targeting efficacy. Due to the high expression of FR and biotin receptor (BR) on the surface of tumor cells, small molecules like FA and biotin are often modified as ligands on the surface of nanomedicines. FA is a natural anti-tumor small-molecule substance that binds to FR on the surface of tumor cells to affect their gene expression and trigger apoptosis. Compared to other nanomedicines, tumor cells primarily absorb biotin via sodium-dependent multivitamin (SMVT) transporters, which are use a transmembrane sodium gradient for transport, and its overexpression on tumor cell surfaces makes it a target for biotin functionalization.¹⁶ For instance, Wang et al.¹⁷ enhanced the targeting capability of their delivery system by functionalizing its surface with both biotin and FA ligands. The results demonstrated that the dual-targeted delivery system exhibited better targeting efficiency and more significant anti-tumor effects compared to single-targeted and non-targeted systems. Zang et al.¹⁸ developed a dual-targeted drug delivery system aimed at inhibiting the tumor microenvironment, functionalized with biotin and mannose ligands. This system can bind to biotin receptors overexpressed on tumor cells and mannose receptors on macrophages, leading to internalization and suppression of their survival. It significantly reduces tumor angiogenesis and reprograms the tumor microenvironment, thereby exerting a therapeutic effect.

2.1.5 Glycans. Glycans are complexes generated by covalently combining polypeptide chains on the surface of polysaccharides as the main body, which are low-cost and non-toxic; hyaluronic acid (HA) is the most common ligand modified in nanomedicines that can bind to CD44 receptors that are overexpressed on the surface of various kinds of tumor cells. However, HA sometimes may something shield another ligand. For example, when HA is conjugated to a ligand, the ligand's effective surface density is decreased due to the random coil conformation of HA in solution, which covers the ligand; alternatively, due to its high molecular weight, HA may potentially have a shielding effect when it is encapsulated in NPs.¹⁹ When constructing dual-ligand nanomedicines with HA and other ligands to enhance targeting, these problems should be taken into consideration. For instance, Liu et al.²⁰ proposed a dual-targeted drug formulation targeting both tumor cells and M2 tumor-associated macrophages. This formulation utilizes Fe³⁺ and tannic acid to immobilize HA, forming a metal-polyphenol network (MPNs) coating on the nanoparticle surface, termed SAMMH. HA targets the CD44 receptor on M2 tumor-associated macrophages, while SAMMH also targets M2 macrophages and promotes their transformation into M1 macrophages, achieving a combined anti-tumor effect. This approach provides a novel strategy for the development of efficient anti-tumor therapeutics. Kumar et al.²¹ modified chitosan nanoparticles with sialic acid and cetuximab to target the overexpressed SA-binding receptors and EGFR on A-549 lung cancer cells. The results showed that the modified nanoparticles exhibited significantly better anti-proliferative activity against A-549 cells compared to non-targeted nanoparticles, demonstrating synergistic cancer cell targeting ability with reduced systemic toxicity.

2.1.6 Other ligands. In addition to the ligands mentioned above, such as antibodies, proteins, peptides, small molecules, and glycans, many other ligands continue to be widely utilized. For instance, aptamers are a class of single-stranded functional oligonucleotides capable of folding into specific tertiary structures, which enable them to target a variety of compounds. Compared to antibodies, aptamers exhibit smaller molecular sizes (ranging from 12 to 30 kDa) and lower production costs. Moreover, aptamers possess high specificity and affinity for their targets, allowing them to effectively differentiate between target proteins that share similar structural epitopes.²² Camorani et al.²³ functionalized the surface of gold–core/silica–shell nanoparticles with two RNA aptamers—CL4 2′-fluoropyrimidine (2′F-Py) RNA aptamer and 2′F-Py RNA Gint4.T aptamer. These aptamers efficiently targeted the extracellular domains of the EGFR and platelet-derived growth factor receptor β (PDGFRβ), respectively. This study is the first to demonstrate the significant potential of a dual EGFR and PDGFRβ aptamer-functionalized nanoparticle system to simultaneously target both breast cancer cells and stromal cells within the tumor microenvironment. Li et al.²⁴ functionalized magnetic Fe₃O₄ nanoparticles with epithelial cell adhesion molecule (EpCAM) aptamers and protein tyrosine kinase-7 (PTK7) aptamers. EpCAM is a major epithelial tumor antigen overexpressed on most tumor cells and can be utilized for targeting circulating tumor cells (CTCs). However, the targeting efficacy of EpCAM significantly decreases when tumor cells undergo phenotypic heterogeneity and epithelial–mesenchymal transition (EMT). To address this challenge, the researchers introduced another aptamer—PTK7—to recognize and target EMT-positive CTCs. The results demonstrated that this functionalized magnetic nanoparticle system exhibited high sensitivity and targeting capability toward CTCs. Additionally, organic compounds such as boronic acids play an indispensable role as ligands in nanoparticle-based drug delivery systems.²⁵

2.1.7 The correlation between ligand selection. The selection of ligands in anti-tumor dual-ligand nanodrugs is interrelated. Dual-ligand nanodrug delivery systems typically involve two distinct ligands, which can target either different or the same biological targets. Through synergistic effects, these ligands enhance the targeting capability and therapeutic efficacy of the drug. The rationality and complementarity of ligand selection are key factors in achieving precise drug delivery and improving anti-tumor efficacy.
2.1.7.1 Enhanced targeting. The dual-ligand system significantly improves the targeting of tumor cells by binding to two distinct receptors on the same cell surface. FA is introduced as a second ligand into galactosylated nanocarriers, enabling targeting of both FRs on tumor cells and asialoglycoprotein receptors (ASGPRs). This strategy effectively enhances the targeting efficiency of nanodrugs toward tumor cells while minimizing non-specific uptake by normal cells, thereby further optimizing the therapeutic efficacy and selective delivery of the nanodrugs.²⁶
2.1.7.2 Complementary interactions between ligands. The complementary interaction between ligands plays a critical role in the targeted delivery of nanodrugs. During the process of nanodrugs reaching their targeted sites, they often encounter biological barriers, such as the blood–brain barrier. Therefore, the rational selection of ligands is essential to ensure that the nanodrugs can efficiently cross these barriers and reach the designated target site. Lf can specifically bind to the low-density LRP-1 on the endothelial cells of the blood–brain barrier, facilitating the trans-barrier delivery of nanodrugs. Meanwhile, PBA exhibits high affinity and specificity for SA on the surface of cancer cells. By functionalizing the nanodrug surface with both Lf and PBA as dual ligands, the system can effectively overcome the blood–brain barrier, enabling precise drug delivery to tumor tissues and thereby significantly enhancing therapeutic efficacy.¹²
2.1.7.3 Complementarity of molecular mechanisms. Ligand selection can also be optimized based on the molecular mechanisms inside and outside tumor cells. For instance, nanoparticles can specifically target cell membrane receptors by binding to chondroitin sulfate (CS) ligands, followed by receptor-mediated endocytosis. Once inside the cell, the nanoparticles release triphenylphosphine (TPP) ligands in response to the acidic environment, further targeting the mitochondria. This process leads to a significant decrease in mitochondrial membrane potential, resulting in the generation of excessive reactive oxygen species (ROS) and ultimately inducing cell apoptosis.²⁷
2.1.7.4 Tumor microenvironment responsiveness. The selection of dual ligands often takes into account the specific roles of proteins in the tumor microenvironment, optimizing ligand function to achieve the best outcomes. For instance, lysosomal cysteine protease (legumain) expressed on the cell surface exhibits high specificity for cleaving certain protein substrates. The tetrapeptide tuftsin binds to the neuropilin-1 (NRP-1) receptor and promotes its internalization; however, tuftsin is readily internalized by the mononuclear phagocyte system, limiting its application in cancer therapy and tumor-associated macrophage (TAM) targeted drug delivery. To overcome this challenge, researchers proposed fusing the Legumain substrate peptide—alanine–alanine–aspartic acid (AAN)—to tuftsin, enhancing its specificity. Using this strategy, a dual-targeted nanocarrier (ATpep-NPs) was developed, with AAN acting as a targeting ligand, significantly enhancing selective delivery to tumor cells and TAMs. After recognition and specific cleavage by overexpressed Legumain on tumor cells and TAMs, ATpep-NPs expose tuftsin, thereby promoting internalization in tumor cells and phagocytosis in TAMs, ultimately achieving efficient dual-targeted drug delivery and combined therapy for tumors and TAMs.²⁸

In conclusion, the selection of ligands in anti-tumor dual-ligand nanodrugs is closely interrelated. By optimizing the selection and combination of ligands, the therapeutic efficacy and targeting capability of the drug can be significantly enhanced, while minimizing side effects on normal cells. In practical applications, the selection of ligands should consider multiple factors, including tumor type, receptor characteristics, tumor microenvironment, and the physicochemical properties of the drug.

2.2 Classification of nanocarriers

The selection of nanocarriers stands as a paramount aspect in the design of nanomedicine, with exemplary nanocarriers necessitating superior safety, stability, and straightforward preparatory procedures. Choosing appropriate nanocarriers for dual-ligand nanomedicines can maximize active targeting of ligands and improve the antitumor effects of the drugs. For the effective delivery of dual-ligand nanomedicines, researchers have constructed a range of carriers with distinct morphologies and functionalities, among which the most commonly used are liposomes, micelles, nanohydrogels, and inorganic materials (Fig. 3), Additionally, a number of polymeric carriers that play an important role (Table 2).

Table 2 Polymer-based dual-ligand nanomedicines as drug delivery carriers

Carrier type	Preparation method	Dual-ligand	Targeting cell	Tumor	Related literature
Abbreviations: MLS peptides: mitochondrial localizing sequence peptides; DEX: dexamethasone; GE11-HA-ss-Chol: GE11 peptide-conjugated hyaluronic acid derivatives grafted with hydrophobic cholesteryl moiety; 5-FU: 5-fluorouracil.
Nanosized metal organic frameworks	Thermal synthesis method	GA	HepG2 cells	HCC	29
		LA
Chitosan nanoparticles	Ionic cross-linking	FR	A549 cells	Lung cancer	30
		EGFR
Polyamidoamine	Coupling	RGD	MCF-7 cells	BC	31
		TAT
Low molecular weight heparin	Coupling	GA	HepG2 cells	HCC	32
		LA
Photostable nanodiamonds	—	MLS peptides	MCF-7 cells	BC	33
		FA
Mesoporous Silica nanoparticles	—	FA	HeLa cells	CCa	34
		DEX
GE11-HA-ss-Chol NPs	Self-assembled	GE11	MDA-MB-231 cell	BC	35
		HA	MCF-7 cells
Chitosan nanoparticles	Ionic crosslinking technique	GA	SMMC-7721 cells	Liver cancer	36
		5-FU	LO2 cells

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2.2.1 Liposomes. Liposomes are both hydrophilic and lipophilic due to their structure, which places hydrophilic hydroxyl endings along the surface of the bilayer, hydrophobic ends toward the middle layer, and hydrophilic ends inside the inner core. Given their amphiphilic characteristics, liposomes emerge as optimal drug vectors for compounds exhibiting diverse polarities.³⁷ Moreover, the predominant constituents of liposomes comprise phospholipids and cholesterol, exhibiting notable cell affinity and complete biodegradability within the human system. Consequently, liposomes exhibit commendable biocompatibility, safety, and immunogenicity profiles. Tf and TAT, a peptide that penetrates cells, were co-modified on liposomes by Yuan et al.³⁸ which showed that the dual-ligand-modified liposome had a more potent anti-tumor effect than the single-ligand modifications. Kang et al.³⁹ demonstrated the capability of this nanomedicine delivery system, using liposomes as carriers and FA along with penetratin peptide-1(Pep-1) peptide as ligands, to selectively deliver chemotherapeutic agents to cancer cells.

2.2.2 Micelles. In aqueous media at low concentrations, both components are present as monomers in solution, however, the hydrophilic head groups and hydrophobic tail groups combine and self-assemble to form micelles when the concentration increases to a critical micelle concentration.⁴⁰ The thermodynamic stability of micelles is enhanced, as indicated by lower values of the critical micelle concentration. Micelles predominantly encapsulate drugs through chemical conjugation, physical entrapment, or a hybrid of both methods. Drugs encapsulated in micelles are protected from metabolization and prolong circulation time in the body, resulting in protection and a slow release. Zhou et al.⁴¹ utilized polymeric micelles modified with FA and fibroblast activation protein α (FAPα), Cao et al.⁴² constructed dual-ligand micelles by combining functionalized galactose (Gal) and PBA, both approaches enhance drug uptake by tumor cells and facilitate drug penetration into tumors. As research progresses, many micellar nanomedicines are currently undergoing clinical trials. For example, NK105 is a nanoparticle drug delivery system in which paclitaxel (PTX) is encapsulated within polymer micelles. In 2019, Fujiwara et al. conducted an open-label Phase III non-inferiority clinical trial (NCT01644890) in Japan, comparing the efficacy and safety of NK105 and PTX in patients with metastatic or recurrent breast cancer. The results indicated that the incidence of peripheral sensory neuropathy (PSN) was 1.4% in the NK105 and 7.5% for PTX. Patient-reported PSN data demonstrated that NK105 significantly outperformed PTX in terms of PSN incidence (P < 0.0001). These findings provide strong clinical evidence supporting the use of NK105 in the treatment of breast cancer. In 2022, researchers initiated a clinical trial titled “A Phase I Trial of Cancer-targeting Micelles for Non-muscle-invasive Bladder Cancer” (NCT05519241), aimed at developing a bladder cancer-specific targeting ligand, PLZ4, in combination with a PTX-loaded nanoparticle micelle (PPM) platform. This platform has the capability to specifically deliver the drug to bladder cancer cells both in vitro and in vivo. This clinical trial marks the first human trial with PPM treatment for non-muscle-invasive bladder cancer (NMIBC), currently in the phase I clinical trial stage, focusing on assessing the safety and efficacy of the platform.

Although targeted nanomedicines have gradually progressed into clinical trials, the clinical studies involving dual-targeted nanomicelles remain relatively scarce. Compared to single-target or non-targeted nanomicelles, the preparation of dual-targeted nanomicelles is more complex, entail higher production costs, and carries greater clinical risks. However, dual-targeted nanomicelles can precisely deliver drugs to target sites, significantly enhancing drug bioavailability and thereby improving their anticancer efficacy. This makes dual-targeted nanomicelles a promising approach in cancer therapy. With advancements in technology and clinical research, it is anticipated that future clinical trials will validate their therapeutic potential.

2.2.3 Nanohydrogels. Nanohydrogels exhibit strong water molecule adsorption, form a three-dimensional mesh structure in water, and possess inherent biocompatibility, hydrophilicity, and high porosity, etc. These properties make nanohydrogels ideal for encapsulating large-molecule biotherapeutics, ensuring their superior stability in biofluids.⁴³ For instance, Xie et al.⁴⁴ developed a redox-responsive IL-2/Fc nanohydrogel system for delivering interleukin-2 (IL-2) and co-cytokines, enhancing the safety and efficacy of overdose T-cell therapy in immunotherapy for related tumors. Additionally, T cells express various antigens on their surface, such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4), which regulate key pathways to inhibit tumor progression.⁴⁵ Zhang et al.⁴⁶ synthesized a dual-ligand DNA nanohydrogel carrying both PD-1 and a CTLA-4 aptamer. PD-1 aptamers can specifically bind to PD-1 receptors, while CTLA-4 aptamers bind to CTLA-4 receptors on T cell membranes. The combination of both aptamers more effectively blocks tumor immune escape, reshapes the immune system, and enhances the anti-tumor effect and the overall effectiveness of tumor immunotherapy. Moreover, the reduced size and hydrophilicity of the nanohydrogels facilitate systemic delivery and extended blood circulation.⁴⁷ Nanohydrogels are now widely applied in fluorescence imaging, drug delivery, and wound treatment.^48–50

2.2.4 Metallic/inorganic materials. Metallic elements or other non-biodegradable substances, such as silicon dioxide, carbon, iron oxide, gold, and quantum dots are frequently used to construct inorganic material nanocarriers, which can be designed in a variety of sizes, structures, and geometries. Inorganic NPs have a wide range of applications in photothermal therapy, drug imaging, and diagnostics due to their magnetic, radioactive, or plasma properties.^51,52 For instance, gold NPs are particularly effective in cancer thermal destruction due to their ease of surface functionalization and photothermal heating capabilities. They are used in medical applications where cancer cells are selectively destroyed by localized photothermal heating, as well as in photothermal imaging and therapy.⁵³ Xu et al.⁵⁴ developed a multidimensional sensor array using gold NPs functionalized with six near-infrared fluorescent dual ligands (attached via distinct amino acids) as sensing receptors, enabling the analysis of various proteins and identification of patients at different stages of breast cancer (BC). Tao et al.⁵⁵ designed an accessible functional sensor to detect cancer cell targets using seven dual-ligand-functionalized gold NPs as effective cell recognition elements and signaling sensors. Yuan et al.⁵⁶ developed a mannose/HA dual-ligand-modified superparamagnetic iron oxide nanocomposite. This nanocomposite induces the slow release of iron ions, activating the caspase-3 pathway, promoting the distribution of TH1-type cytokines, enhances the phagocytic ability of TAMs towards BC cells, and induces the M1 polarization of TAMs. Additionally, the iron content on nanoparticles surface imparts superparamagnetic properties and dual-ligand modification significantly enhances the anti-tumor effect, demonstrating its considerable potential in targeted therapy. However, the therapeutic utility of inorganic NPs is limited by factors such as inadequate solubility, prolonged toxicity, carcinogenic potential, immunogenicity, inflammatory responses, and tissue damage. To successfully translate inorganic NPs into clinical practice, it is essential to develop a simple, safe, cost-effective, and environmentally friendly production approach. Furthermore, more data on the pharmacokinetics, biodistribution, and safety profiles of NPs are needed.⁵⁷

2.3 Ligand functionalization strategies

For dual-ligand nanomedicines, the introduction of the ligand onto the nanocarrier is crucial. The targeting efficiency is influenced by the strength of ligand-nanocarrier binding, the arrangement of ligands on the nanocarrier, and the positioning of different ligands. To ensure stability and minimize off-target effects, ligands can be attached to the nanocarrier through either non-covalent or covalent interactions (Fig. 4).

Fig. 4 Comparison of noncovalent and covalently bound ligand-activated nanocarriers. Ligands that can be used to achieve active targeting can be installed on nanocarriers via either noncovalent binding or covalent binding. Both strategies have their own pros(black) and cons (red).⁵⁸ Copyright 2022, Elsevier.

2.3.1 Non-covalent binding. Non-covalent binding refers to the attachment of targeted ligands to the NP surface, including physical adsorption, ionic binding, and biotin-affinity interactions. Physical adsorption occurs through weak interactions such as van der Waals forces, hydrophobic interactions, and hydrogen bonding.^59,60 Ionic binding involves the attraction between oppositely charged ligands and NPs. Non-covalent binding does not alter the ligand's structure or its interaction with biological targets, making it a straightforward process. For example, Zang et al.¹⁸ modified biotin and FA onto the surface of nanoparticles through hydrophobic interactions to achieve targeted drug delivery. However, ionic binding and physical adsorption are easily influenced by pH and temperature. This can be mitigated by biotin–affinity interactions, which offer better ligand stability and resistance to denaturants, proteolytic enzymes, and varying pH and temperature conditions.⁶¹

2.3.2 Covalent binding. Covalent binding refers to the creation of covalent bonds between the ligand and the functional groups present on the nanocarrier surface. Common binding methods include carbodiimide chemistry, maleimide chemistry, and click chemistry.^58,62 The carbodiimide chemistry involves the binding of carboxyl and primary amines to the ligand and the nanocarrier. Generally, chemical modification of the ligand and carrier is not required. Optimal efficiency is achieved when the reaction is performed in an acidic buffer free of extraneous carboxyl and amine groups. To improve binding efficiency, N-hydroxysuccinimide (NHS) can alternatively be added to the buffer.⁶³ Sulfhydryl groups on the ligand surface and maleimide groups on the nanocarrier form stable thioether bonds through maleimide chemistry, which occurs rapidly at neutral pH and can be applied to protein ligands, GE11 peptides, and anti-EGFR aptamers.⁶² Click chemistry involves the formation of a stable triazole bond through a chemo-selective reaction between an azide and an alkyne group. This reaction produces a single reaction product, exhibits high specificity, and can be carried out under mild conditions without requiring complex purification.⁶³ Emami et al.⁹ employed the EDC/NHS coupling chemistry method to conjugate anti-EGFR and anti-PD-L1 antibodies onto the surface of nanoparticles. Initially, they activated the nanoparticle surface with an EDC/NHS solution, followed by the addition of antibodies. The –COOH groups on the nanoparticle surface reacted with the –NH₂ groups on the antibodies, successfully achieving the conjugation of the antibodies onto the nanoparticle surface (Fig. 5).

Fig. 5 DRT-DTX-PLGA were developed by conjugating anti-PD-L1 and anti-EGFR antibodies (Panitumumab) on surface of DTX-PLGA via EDC/NHS coupling chemistry. Notes: DRT is dual receptor targeting.⁹ Copyright 2023, Elsevier.

3 Factors influencing the action of dual-ligand nanomedicines

Dual-ligand nanomedicines are functionalized with two different targeting ligands, enabling selective binding to two distinct receptors and releasing the drug into tumor cells for therapeutic effect. Dual-ligand nanomedicines target receptors on the same cell as well as on different cells or organelles. The two ligands can be utilized together as a single molecule bound to the nanomedicine's surface or independently.¹⁹ The ligand combinations commonly used in the preparation of dual-ligand nanomedicines are RGD cyclic peptides, HA, TF, FA, and biotin in combination with a second ligand.^19,64 Targeting efficiency is significantly affected by ligand conformation and surface density, which are crucial in determining the binding affinity of particle receptors. To enhance nanomedicine targeting and minimize non-specific interactions, optimizing ligand density is necessary.⁶⁵ The therapeutic efficacy of nanomedicines is influenced by factors, such as ligand combinations, length, density, and electrostatic interactions (Fig. 6).⁶⁶

Fig. 6 Schematic illustration of dual-ligand nanomedicine is influenced by ligand. (A) the interaction between the cell membrane and NPs with different ligand decorations. (B) Targeting mechanism of dual-ligand NPs with different ligand lengths.⁶⁶ Copyright 2018, Wiley.

3.1 Ligand length

Xia et al.⁶ proposed that the length of the ligand also influences cellular binding and uptake of nanomedicines, and they performed dissipative particle dynamics (DPD) simulations to compare the binding of single- and dual-ligand nanomedicines on the cell surface. The study revealed that the cell surface energy for both nanomedicines changed negligibly with a shorter ligand. This phenomenon arises because the enhancement in ligand–receptor binding is minimal during the binding of dual-ligand nanomedicines to cells. This is due to the difficulty in pairing the second ligand with its receptor after one ligand–receptor interaction, which leads to mismatches caused by volume effects. However, with longer ligands, the cell surface energy changes significantly because the ligands rearrange, aggregate, and bind to their respective receptors without interference, which greatly increases the binding rate. However, excessive ligand length may hinder cellular phagocytosis and uptake. For this reason, the ligand must be the proper length—neither too long nor too short.⁶⁶

3.2 Ligand density

Elevated ligand densities on the surface of nanomedicines typically lead to enhanced ligand–receptor binding and heightened cellular absorption.⁶⁷ A study by Moradi et al.⁶⁸ showed that cell targeting increases progressively and stabilizes once a critical ligand density is reached. Ligand density does not directly correlate with cellular uptake because (1) as ligands bind to receptors and enter the cell, the number of available receptors on the cell surface decreases, leaving fewer ligands to bind and reducing endocytosis and uptake. (2) The accumulation of ligands in a densely packed configuration on the nanomedicine's surface can cause site-blocking, hindering ligand–receptor recognition, impairing binding, and reducing cellular uptake.⁶⁹ Thus, an optimal ligand density on the nanomedicine's surface, which is not maximized. Sultana et al.⁷⁰ constructed a dual-ligand nano-delivery system modified with FA and HA to evaluate the effect of ligand density on the targeting ability. They found that cellular targeting increase with ligand density up to a certain point, after which it decreased. Furthermore, compared to single-targeted NPs, dual-ligand NPs demonstrated better cellular targeting in expressing both receptors, with the receptor expression level determining the ideal ligand density for nanomedicine. Further research on ligand density showed that it alters cellular uptake mechanisms and intracellular transport, in addition to impacting the overall cellular uptake of nanomedicines. At low ligand densities, nanomedicines enter cells via lattice protein-mediated endocytosis and are subsequently degraded in lysosomes. Conversely, at high ligand densities, nanomedicines are phagocytosed by macrophages and exhibit limited lysosomal solubilization. Moreover, higher ligand density promotes protein adsorption on the surface, triggering immune-mediated NP elimination.⁷¹

Recent studies have demonstrated that the one-step microfluidic method enables precise regulation of ligand density on the surface of nanoparticles by adjusting the composition of the solvent phase. This approach effectively avoids the issues associated with excessively high or low ligand presentation, which can compromise targeting efficiency.⁷² For instance, in the synthesis of dual-ligand-modified PLGA-PEG nanoparticles (5F2H NPs, containing 5 mol% FA and 2 mol% HA), a solvent mixture comprising 93 mol% PLGA-PEG5K, 5 mol% PLGA-PEG5K-FA, and 2 mol% PLGA-HA was employed. By fine-tuning the molar ratio of polymer precursors, the density of both the polymer backbone and the targeting ligands (FA/HA) can be accurately controlled, offering a practical strategy for the fabrication of highly targeted drug delivery systems.⁶⁶

3.3 Electrostatic interaction

Non-specific interactions between the two ligands, primarily electrostatic and hydrophobic, can occur. However, as the ligands binding to nanomedicines are typically hydrophilic, only electrostatic interactions are considered. Electrostatic interactions attract the heads and tails of adjacent ligands, affecting ligand–receptor binding and reducing cellular uptake efficacy.⁶ Furthermore, electrostatic interactions happen not only between nearby ligands but also between blood proteins and nanomedicines. As a result, various proteins adhere to the nanomedicine's surface, shortening its circulation time, reducing cellular uptake, and weakening ligand–receptor binding.⁷³ The probability of protein adsorption is typically decreased when nanomedicines have a hydrophilic or amphiphilic polymer coating on their surface.^74,75 Using two typical coating polymers—hydrophilic and amphiphilic polymers—as examples, Ding et al.⁷⁶ used two typical coating polymers—hydrophilic and amphiphilic—as examples and systematically examined their impact on the delivery efficiency of ligand NPs in the presence of serum proteins through DPD simulations. Their findings show that although both polymers are electrically neutral, they both reduce protein adsorption and enhance NP uptake by cells. Amphiphilic polymer-coated NPs are more effective in treating tumors because cancer cells can better absorb pH-responsive polymer-coated particles than non-responsive, charge-neutral polymer-coated NPs.

4 Dual-ligand nanotechnology delivers different types of tumor drugs

4.1 Cytotoxic drugs

Cytotoxic drugs primarily hinder cell growth and replication by disrupting cell structure and function, thus reducing tumor proliferation. However, the term ‘cytotoxic drug’ is sometimes used interchangeably with ‘antitumor drugs’.^77,78 To assess safety risks, the pharmaceutical industry defines cytotoxic drugs as those that directly destroy cancerous cells and prevent tumor spread. A drug is deemed to exhibit a cytotoxic mechanism if it meets the following criteria: (1) inducing direct damage to DNA structure or disrupting mitotic function, leading to cellular demise; (2) cytotoxic drugs often lack sufficient specificity for malignant cells, resulting in damage to healthy, rapidly dividing cells alongside cancer cells.⁷⁸ Commonly employed cytotoxic drugs in dual-ligand nanotechnology are DOX,^33,34,79 PTX,^80,81 DTX,^9,82 and others.

4.2 Targeted therapeutic drugs

Drugs or preparations known as “targeted therapeutics” possess the ability to selectively target lesions, accumulate and release active components at the target site, and inhibit tumor cell growth and proliferation so as to achieve the goals of therapy. Trastuzumab is a recombinant humanized monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2), and serves as a cornerstone in current targeted cancer therapies. By specifically binding to the overexpressed HER2 receptors on the surface of tumor cells, trastuzumab effectively disrupts downstream signaling pathways, thereby inhibiting cellular proliferation and tumor progression.⁸³ Studies have shown that HER2 overexpression can be detected in approximately 74% of breast cancer cases.⁸⁴ Notably, beyond its therapeutic role, trastuzumab can also function as a targeting ligand by being conjugated to the surface of nanocarriers. This modification significantly enhances the targeting efficiency of nanoparticle-based drug delivery systems, offering a promising strategy for precision nanomedicine in HER2-positive cancer treatment.⁸⁵

4.3 Immunotherapeutic drugs

Unlike conventional antitumor drugs, immunotherapeutic drugs that restore anti-tumor immunity in the tumor microenvironment, produce a sustained immunosurveillance effect, and enhance immune system function to combat the disease. Common immunotherapeutic drugs include, but are not limited to, immune checkpoint blockers (e.g., CTLA-4, PD-1 or its primary ligand PD-L1⁸⁶), and autologous T cells targeting the chimeric antigen receptor (CAR) of CD19,⁸⁷etc. However, immune system stimulation during immunotherapy usually results in an adverse auto-reactive immune response with related side effects,⁸⁸ Some researchers have made use of dual-ligand nanocarriers containing small interfering RNAs (siRNAs), which promote the activation and proliferation of immune cells, interfere with tumor gene expression, and reduce the side effects of immunotherapy.^89–91

5 Application of dual-ligand nanomedicines in different tumors

Cancer is generally categorized into early, intermediate, and advanced stages based on its progression. In early and intermediate stages, treatment primarily involves surgical resection, aiming for curative removal of the tumor. However, in advanced stages where surgical options are often limited, targeted therapy has emerged as a principal therapeutic approach. This modality enables precise intervention by acting on specific molecular targets associated with tumorigenesis (Table 3).

Table 3 An overview of dual-ligand nanomedicines for drug delivery in various tumors

Tumor type	Dual-ligand	Targeting receptor	Carrier type	Animal model	Related literature
Abbreviations: PEG–PCL: poly(ethylene glycol)–poly(ε-caprolactone); MET: mesenchymal-epithelial transition factor; cMBP: a MET targeting peptide (KSLSRHDHIHHH); MPC: 2-methacryloyloxyethyl phosphorylcholine; dNP2 peptide: a blood–brain barrier-permeable peptide; TPE@Zn: zinc-tetraphenylethene; mPEG-PE: polyethylene glycol–phosphatidylethanolamine; PEOz-PLA: poly(2-ethyl-2-oxazoline)-poly(D,L-lactide).
GBM	TGN peptide	—	PEG-PCL NPs	Orthotopic transplantation	92
	AS1411 DNA	Necleolin protein
	IL-13p	IL-13Rα2	Polymeric NPs		93
	RGD cyclic peptide	αvβ3 integrin
	Inherbin3	EGFR	MPC-NPs		94
	cMBP	MET
	Pep-1	IL-13Rα2	PEG-PLGA NPs		95
	CREKA peptide	Fibrin-fibronectin complexes
NSCLC	PD-L1 antibody	PD-1	ARAC	Pulmonary metastasis	96
	PKL1 inhibitor
	PD-L1 antibody	PD-1	PLGA NPs	Patient-derived NSCLC cells	9
	Panitumumab	EGFR
TNBC	P-selectin peptide	P-selectin	Liposomal NPs	BC metastasis	97
	αvβ3 integrin peptide	αvβ3 integrin
	HA	CD44	Saporin-loaded nanogels		98
	GE11 peptide	EGFR
	FA	FR	Liposomal NPs	BC and brain metastasis	99
	dDP2 peptide	—
	Peptide K237	KDR/Flk-1 tyrosine kinase	Polymeric NPs	Lung metastasis	100
	Ep23 aptamer	EpCAM
GC	AS1411 DNA	—	Gold nanoprisms	Subcutaneous transplantation	101
	TPE@Zn	—
	—	—	pH-responsive liposome NPs	Orthotopic transplantation	102
	—	—
HCC	GA	GA-R/PKCα	Liposomal NPs	Orthotopic transplantation	103
	PNA	Mucin 1
	RGD cyclic peptide	αVβ3 intergrin	Liposomal NPs		104
	TAT peptide	Cell-surface glycoproteins
	TF	TFR	Liposomal NPs	Subcutaneous transplantation	105
	TAT peptide	Cell-surface glycoproteins
	TF	TFR	Solid lipid nanoparticles		106
	M	MR
PC	MCC-555	PPARs	—	Patient-derived PC cells	107
		KLF4
OC	TF	TFR	Liposomal NPs	Subcutaneous transplantation	108
	R8	—
	TF	TFR	mPEG-PE micelle		109
	mAb 2C5	—
PCa	HA	CD44	PLGA-PEG NPs	Patient-derived PCa cells and subcutaneous transplantation	82
	GE11	EGFR
	YPSMA-1	PSMA	Polymeric micelles	Patient-derived PCa cells	81
	RGD cyclic peptide	αvβ3 intergrin
MM	TRAIL	EGFR	Lumazine synthase protein cage NP	Subcutaneous transplantation mice	110
	EGFRAfb
	TF	TFR	Liposomal NPs		105
	TAT peptide	—
CC	HA	CD44	Silica NPs	Patient-derived CC cells	70
	FA	FAR
RCC	NGR motif peptide	CD13	PEG-LP	Subcutaneous transplantation	111
	R4	—
OSCC	FAPα ligand	FAPα	PEOz-PLA polymeric micelles	Subcutaneous transplantation	41
	FA	FR
CCa	Pep-1	—	Liposomal nanocarrier	Patient-derived CCa cells	39
	FA	FR

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5.1 Glioblastoma

Glioblastoma (GBM), originating from glial and neuronal cells of the nervous system, includes GBMs and grade II–III astrocytomas, constituting 81% of malignant brain tumors. The age-standardized incidence rate is 4.7 cases per 100 000 person-years.¹¹² Due to their nature as brain tumors, GBMs necessitate drug therapy to traverse the blood–brain barrier (BBB). Nanomedicines offer a promising approach for delivering drugs to the central nervous system and facilitating their preferential accumulation in the brain (Fig. 7). Additionally, the bond of ligands to the surface of nanomedicines enhances drug transport to certain diseased areas in the brain and improves drug targeting.¹¹³

Fig. 7 Dual-ligand nanomedicines cross the BBB to target tumor cells. To simultaneously mitigate EGFR and MET activation, a dual functionalized brain-targeting nanoinhibitor. BIP-MPC-NP, is developed by conjugating Inherbin3 and cMBP on the surface of NHS-PEG8-Mal modified MPC nanoparticles. Permitted for use from an open access article;⁹⁴ copyright 2020, Springer Nature.

The dual-ligand configuration of nanomedicines enhances their capability to cross the BBB and target GBM cells. Gao et al.⁹² developed a cascade delivery system for GBM treatment, termed ASTNP, which integrates Tat-Glu-Tyr-NH2 (TGN) peptides derived from phages and AS1411 aptamers as dual ligands on nanomedicines. The TGN peptide demonstrates selectively binds to endothelial cells in the BBB, facilitating nanomedicine transport across the barrier. Meanwhile, the AS1411 aptamer binds specifically to nucleolin, a protein highly expressed on tumor cells. As a result, ASTNP cross the BBB and selectively target GBM cells after administration. Another therapeutic strategy for GBM treatment involves the inhibition of tumor-associated neovascularization the higher the H1F1α content, the better the tumor invasion and resistance to chemotherapy. Whereas anti-tumor neovascularization combined with anti-tumor cells significantly reduced H1F1α expression.¹¹⁴ To effectively treat GBM, Gao et al.⁹³ developed a combinatorial strategy incorporating interleukin-13 (IL-13) peptide and cRGD into nanoparticles to selectively target tumor cells and endothelial cells. This dual-targeting approach suppressed HIF1α expression, reducing tumor invasiveness and chemotherapy resistance, and enhanced tumor cell apoptosis. Moreover, GBMs can be successfully treated by building a dual-ligand nano-delivery system that decreases drug resistance⁹⁴ and shortens the period that nanomedicines remain in the tumor microenvironment.⁹⁵

5.2 Lung cancer

Lung cancer is the most prevalent and leading cause of cancer-related mortality, with exceedingly high and rising incidence and mortality rates.¹¹⁵ Lung cancer is classified into subtypes, including adenocarcinoma, squamous carcinoma, non-small-cell lung cancer (NSCLC), and small-cell lung cancer, which generally originate in the trachea, bronchial mucosa, and glands.¹¹⁶

PD-1 is expressed in 24%–60% of NSCLC cases.^117,118 Therefore, PD-1/PD-L1 inhibitors are more commonly studied in lung cancer immunotherapy. PD-1, a member of the CD28/CTLA-4 family of T-cell regulatory proteins, is mainly expressed on mature T cells, while its ligand, PD-L1, is predominantly expressed on tumor cells. When PD-1 and PD-L1 bind, they reduce the activity and proliferation of CD4⁺ and CD8⁺ T cells, which in turn suppresses the immune response to surrounding tissues. However, in tumors, this combination reduces T cell ability to eliminate immune agents, promoting immune escape and tumor growth.¹¹⁹ Inhibiting PD-1/PD-L1 binding enhances T cell proliferation and killing. For example, Reda et al.⁹⁶ developed a dual-ligand nano-delivery system (ARAC) by modifying a nanomedicine with a PD-L1 antibody and the PLK1 inhibitor Volasertib. Volasertib not only inhibits excessive proliferation of lung cancer cells but also upregulates PD-L1 expression (Fig. 8). They enhance the binding of PD-L1 and PD-1, enabling T lymphocytes to perform their immune killing function. The nano-system not only reduced the effective dose of the drug but also alleviated the toxicity of the drug, which had a better synergistic effect. In addition, Emami et al.⁹ designed a dual-ligand-modified nanomedicine delivery system using anti-EGFR and anti-PD-L1 antibodies. The study's findings demonstrated increased binding affinity and cytotoxicity compared to the single anti-PD-L1 antibody-modified nanodrug delivery system.

Fig. 8 Proposed mechanism of action of ARAC nanoconstruct. (Left cell) ARAC binds to PD-L1 on the surface of cancer cells and is internalized via receptor-mediated endocytosis. Following endosomal escape, Volasertib is released to inhibit PLK1 activity, resulting in G2/M cell cycle arrest and apoptotic cell death. However, G2/M arrest induced by Volasertib also upregulates PD-L1 expression in surviving cancer cells, thereby rendering them less responsive to immune-mediated effects due to PD-L1-mediated immunosuppression. (Right cell) This property is exploited by using the elevated PD-L1 expression in surviving cancer cells as a homing target for subsequent ARAC delivery, enabling a feedforward targeting mechanism—i.e., enhanced tumor targeting with successive treatment cycles.⁹⁶ copyright 2022, Springer Nature.

5.3 Breast cancer

The health of women is seriously threatened by female BC, which has a high incidence and death rate.¹²⁰ BC is categorized into the triple-negative, HER2-positive, luminal A, and luminal B subtypes. Triple-negative breast cancer (TNBC) is aggressive and frequently spreads, accounting for 15–25% of cases.

Triple-negative breast cancer is a highly aggressive and heterogeneous subtype of breast cancer. CTCs can adhere to the vasculature of metastatic sites through mechanisms similar to leukocyte adhesion cascades. After initial endothelial adhesion, the cellular behavior of CTCs at the metastatic site undergoes a transition from P-selectin-dependent endothelial rolling to firm adhesion mediated by α_vβ₃ integrin. Based on this mechanism, Doolittle et al.⁹⁷ proposed a nanoparticle vascular targeting strategy based on P-selectin and α_vβ₃ integrin. This strategy specifically targets CTCs at different stages of metastasis, thereby enabling complementary targeting of different tumor sites. Studies have shown that the dual-ligand strategy significantly enhances the high-precision targeting ability in early metastasis, whereas single-ligand nanoparticles may fail to recognize heterogeneous changes in the tumor microenvironment, reducing targeting efficiency (Fig. 9). One further method for treating BC metastases is to target and release therapeutic drugs to the sites of metastasis.^121,122 EGFR and CD44 receptors are commonly overexpressed in metastatic cancer cells.^123–126 Chen et al.⁹⁸ utilized GE11 peptide and HA dual ligands to target EGFR and CD44 receptors, respectively, with superior drug delivery and high cellular uptake compared to CD44 mono-ligand. Li et al.⁹⁹ developed cascade-targeted nanomedicines using FA and dNP2 peptides to treat brain metastases secondary to breast cancer. As a BBB permeable peptide, the dNP2 peptide facilitates the nanomedicine's passage across the barrier and enhances its uptake by tumor cells.^127,128 The function of dNP2 peptide is affected by FA's spatial site resistance; therefore, in order to fully utilize the function of dNP2 peptide and optimize drug penetration, the lower pH was utilized to cleave the stilbene bond that connects FA to nanomedicine after it has entered the tumor microenvironment. Furthermore, in an attempt to prevent tumor spread by causing harm to the primary BC tumor, Yao et al.¹⁰⁰ targeted tumor neovascularization and circulating tumor cells in the circulation using the K237 peptide and Ep23 aptamer, respectively, in an effort to destroy the original tumor and prevent tumor metastasis.

Fig. 9 Schematic illustration of dual-ligand nanoparticle target at different stages of metastasis. (A) the dual-ligand nanoparticle, and (B) targeting of the nanoparticles to metastatic sites using vascular targeting and a dual-ligand strategy. Inset: Interactions of circulating tumor cells and vascular bed.⁹⁷ copyright 2015, ACS Publications.

5.4 Gastric cancer

Annually, more than 990 000 individuals worldwide are diagnosed with gastric cancer (GC), resulting in approximately 738 000 deaths. GC typically manifests asymptomatically and carries a grim prognosis upon initial detection. The stomach mucosa is the source of GC, which is usually detected at an advanced stage. The Lauren classification categorizes GC into three histologic subtypes: intestinal, diffuse, and indeterminate.¹²⁹

Two major targets for GC nanomedicine delivery systems have been explored: HER2 and the vascular endothelial growth factor (VEGF) receptor.^130–132 Among them, HER2 protein abnormal overexpression is related to GC and is a key target for treatment.¹³¹ Zhang et al.¹⁰¹ constructed a gold-based nanomedicine targeting HER2 and immunoadjuvant CPG sequences, modified with zinc tetraphenylvinyl and AS1411 DNA aptamer on gold nanoparticles. Zinc tetraphenylethylene specifically targets the cell membrane of early apoptotic cells, with no effect on normal cell membranes, while the AS1411 DNA aptamer has nucleus-targeting capabilities. With its enhanced gene transduction capacity and synergistic therapeutic effect, this nano-delivery method has promise as a potential treatment for GC. In addition to stimulating angiogenesis and improving vascular permeability, VEGF also binds to receptors on the surface of tumor cells, activates downstream signaling pathways, and has a direct role in the development, genesis, and migration of tumor stem cells.¹³³ Long et al.¹⁰² used a pH-responsive liposome loaded with apatinib and cinobufagin, which was hybridized membrane-coated, for the combined treatment of GC. The results indicated that the LP-R/C@AC nanocomplex effectively inhibits tumor invasion and metastasis through the VEGFR2 and STAT3 signaling pathways. Additionally, it induces autophagy and apoptosis in tumor cells, effectively killing them in vitro.

5.5 Liver cancer

Liver cancer carries a mortality rate of 8.2% and is categorized into two primary types: primary and secondary.¹¹⁵ Primary liver cancer comprises hepatocellular Carcinoma (HCC), and mixed liver cancer. More than 90% of primary liver cancer are due to HCC.^134,135 Surgical resection and liver transplantation are common treatments for early-stage liver cancer. Nevertheless, patient death occurs frequently before to liver donor acquisition due to donor shortages and high costs. In the realm of liver cancer therapeutics research, there is currently a significant focus on liver-targeted medication delivery.

In actively targeted therapy for liver cancer, protein kinase Cα (PKCα) is frequently used as a target because liver cancer tissues overexpress PKCα in comparison to normal liver tissues.¹³⁶ Its corresponding ligand is GA,¹³⁷ a triterpenoid saponin, that is one of the main active ingredients extracted from licorice. GA binds specifically to PKCα, enabling the carrier to distinguish between tumor liver tissue and normal liver tissue.^136,138 Numerous researchers have developed a dual-ligand nano-delivery system for liver cancer research using GA and other ligands. For example, Li et al.¹⁰³ modified DOX-containing liposomes with GA and peanut agglutinin (PNA) as dual ligands to create a dual-ligand nano-delivery system. PNA can bind to mucin 1, which is overexpressed on tumor cells, thereby precisely target liver tumor cells. This prevents GA from binding to normal liver tissue cells, increases DOX uptake by tumor cells, and inhibits the growth of tumor cells. Moreover, some researchers have created modified nanomedicines that containing both lactic acid (LA) and GA as dual ligands. These ligands can bind to the Asialoglycoprotein receptor, which is overexpressed on the surface of tumor cells, and they simultaneously target tumor liver tissues, improving targeting accuracy.^{36,79,139,140} although specific ligand-modified nano-delivery systems have improved cellular selectivity, their less effective tumor penetration still has an impact on the therapeutic efficiency of nanomedicines. Due to their non-specificity, cell-penetrating peptides can only enhance carrier uptake to a limited extent,¹⁰⁴ and combining the two can enhance the effectiveness of ligand nano-delivery systems. For example, TF¹⁰⁵ or RGD cyclic peptides,¹⁰⁴ along with TAT peptides, are used to modify nanomedicine. TAT is a cell-penetrating peptide that enables the intracellular transport of molecules with various sizes and physicochemical characteristics by interacting electrostatically between positively charged amino acids and negatively charged glycoproteins on the cell surface. The absorption of nanomedicines is increased by the combination of the receptor-targeting characteristics of TF or RGD cyclic peptides with the enhanced cellular uptake impact of TAT in nano-delivery systems. In addition, it can also be used for the treatment of liver cancer by targeting two different types of cells, Jing et al.¹⁰⁶ modified nanomedicines with mannan (M) and TF to specifically target tumor hepatocytes and macrophages, respectively, and macrophages are crucial elements that stimulate inflammatory responses in cancer and could be therapeutic targets.¹⁴¹ The TF and M dual ligands are designed to target two different cells in the liver, deliver the drugs at multiple points. This approach improves the drugs’ therapeutic impact, minimizes their adverse effects, and increases their uptake in the target cells.

5.6 Pancreatic cancer

With a five-year overall survival rate of less than 10 percent, pancreatic cancer (PC) stands as the malignancy with the highest mortality rate among major tumors and represents the primary cause of cancer-related deaths in developed nations.¹⁴² Precancerous lesions, genetic alterations, and inherited factors are the primary causes of PC.

Targeting the two isoforms of peroxisome proliferator-activated receptors (PPARs), PPARα and PPARγ, thiazolidinediones exhibit antitumor action in various cancer cells,¹⁴³ and are more potent than traditional ligands specific to PPARs. A novel category of thiazolidinediones, MCC-555, has both PPARγ-dependent and PPARγ-independent antitumor effects against prostate and colorectal cancer, influencing the KLF4 transcription factor in PC cells. KLF4 overexpression inhibits tumor growth in PC cells.¹⁴⁴ Furthermore, research suggests that KLF4 can inhibit the spread of several malignancies, including PC. Min et al.¹⁰⁷ used MCC-555 as a PPAR dual ligand to investigate the expression changes of p21, NAG1, cyclin D1, and KLF4 in PC cells. They found that MCC-555 binding to PPARα and PPARγ decreased cyclin D1 expression, while MCC-555 regulated KLF4 expression independently of PPARs, increasing the expression of p21 and NAG-1, thereby supporting KLF4's antiproliferative effect. This result consistent with studies showing that KLF4 targets p21 and NAG-1 in cancer cells,¹⁴⁵ MCC-555 induces a decline in cyclin D1 expression and an increase in p21 and NAG-1 expression, leading to growth inhibition and apoptosis in PC cells.

5.7 Ovarian cancer

Ovarian cancer (OC) originates in the fallopian tubes.¹⁴⁶ Approximately 70% of patients are diagnosed only after the disease has progressed to stages III or IV. Studies have shown that the 5-year survival rate for OC is 47.4%.¹⁴⁷

Polyarginine (R8) cell-penetrating peptide rich in arginine, and it is believed that the optimal chain length for effective translocation is 8 arginine units. R8 shares similarities with the translocation domains of TAT.¹⁴⁸ Numerous studies have convincingly demonstrated R8's efficacy in enhancing intracellular drug delivery within nano-delivery systems.^149,150 Deshpande et al.¹⁰⁸ designed R8- and TF-modified dual-ligand nanomedicines, which improved drug delivery and penetration into OC cells, enhancing anticancer effects in vitro and in vivo by targeting the TfR on cancer cells. These findings highlight the substantial benefits of combining R8 and TF to enhance the therapeutic efficacy of DOX. Sawant et al.¹⁰⁹ constructed dual-ligand nanomedicines by modifying TF and monoclonal antibody 2C5 (mAb 2C5), which targets multiple tumor cells by recognizing nucleosomes presented on the surface of tumor cells near apoptotic and dying cells. Experimental evidence has shown that mAb 2C5 exhibits superior anti-tumor effects in tumor-bearing mice, both in vivo and in vitro. Scientists predicted that nanomedicines modified with both TF and mAb 2C5 would have superior therapeutic effects compared to those modified with a single ligand. The results demonstrated that although dual-ligand nanomedicines significantly increased activity in vitro, single-ligand (TF) nanomedicines showed stronger therapeutic effects in vivo. This suggests that the relationships among targeting ligands in vivo are more intricate than in simplified in vitro models, highlighting the need for superior dual-ligand nanomedicines to demonstrate consistent therapeutic efficacy both in vivo and in vitro.

5.8 Prostate cancer

Prostate cancer (PCa), one of the most common malignancies in males, accounts for 20% of newly diagnosed cases and 9.8% of cancer-related deaths in men.^115,151 It is classified into prostate adenocarcinoma and various other subtypes. PCa is diagnosed through prostate-specific antigen-based early screening, magnetic resonance imaging, rectal examination, and prostate biopsy.¹⁵²

Research indicates that most patients with PCa exhibit detectable levels of EGFR.¹⁵³ Dong et al.⁸² constructed binary nanomedicines targeting CD44 and EGFR through HA and GE11 dual ligands modifications. The HA-modified nanomedicine is combined with the GE11-modified nanomedicine to form a binary system, with the encapsulated drugs in the two nanocarriers either being the same or different (Fig. 10). If the drugs encapsulated in both carriers synergistically interact, the dosage can be reduced while maintaining potent anti-tumor efficacy. Compared to dual-ligand co-modified nanoparticles, the strategy of mixing two separately single-ligand-modified nanoparticles offers improved ligand stability and simplifies quality control during formulation. This approach enables independent optimization of each ligand, thereby minimizing steric hindrance and competitive binding between ligands on the same nanoparticle surface, ultimately enhancing the efficiency and specificity of target recognition. A classically glycosylated transmembrane protein called prostate-specific membrane antigen (PSMA) is overexpressed in some primary and metastatic PCa cells (such as 22Rv1 cells) as well as in tumor neovascularization.¹⁵⁴ Its expression is not androgen-dependent, and it increases as the disease progresses and following chemotherapy or surgical ablation treatment. It has been demonstrated that the PSMA antibody YPSMA-1-modified carriers enhances therapeutic efficacy and has a positive homing impact on tumor cells and xenografts that express PSMA. Gao et al.⁸¹ created dual-ligand nanomedicines that target PSMA and the tumor angiogenesis biomarker integrin α_vβ₃, respectively, using YPSMA-1 and RGD cyclic peptide modifications. Prior studies have shown in the literature that RGD cyclic peptide-conjugated nanocarriers, via α_vβ₃-mediated endocytosis that facilitates their internalization into tumor cells, demonstrate increased cytotoxicity and antiproliferative activity against tumor cells.^155–157 The study's findings indicated that dual ligand-modified nanomedicines could efficiently and synergistically deliver drugs into PCa cells that expressed PSMA and were rich in integrin α_vβ₃. Which resulted in a significant improvement in the recognition of PCa cells and facilitated cellular uptake and intracellular drug release.

Fig. 10 HA and GE11 dual ligand-modified binary nanoparticles (C) were constructed by the self-assembling of GE-NPs (A) and HA-NPs (B). FMN is flavin mononucleotide.⁸² Copyright 2022, Dove Medical Press https://www.dovepress.com/drug-design-development-and-therapy-journal.

5.9 Melanoma

Melanoma (MM), recognized as one of the most formidable malignancies in humans, often defies complete eradication through conventional surgical or chemotherapeutic interventions.

The member of the TNF superfamily of ligands, TNF-related apoptosis-inducing ligand (TRAIL), frequently triggers cell apoptosis by binding to pro-apoptotic death receptors (DR4/DR5). Consequently, TRAIL is a promising biological anticancer medication that can specifically cause apoptosis in cancer cells that overexpress death receptors.¹⁵⁸ Jun et al.¹¹⁰ utilized adhesion molecules that target both EGFR and TRAIL for dual-ligand targeting. Their findings demonstrated that nanomedicines modified with dual ligands significantly increased their cytotoxicity against MM cells. Additionally, by preventing EGF/EGFR binding, the nanomedicine efficiently breaks the EGF-mediated EGFR survival signaling pathway and dramatically stimulates both endogenous and exogenous apoptotic pathways, leading to maximum accelerated cancer cell death. Additionally, other researchers have developed liposomal nano-delivery systems modified with TF and the cell-penetrating peptide TAT.^38,105 Among them, TF ligand specifically binds to the TF receptor overexpressed on the surface of MM cells, while TAT ligand, as a classical cell-penetrating peptide, helps the dual-ligand liposomes to be internalized by MM cells, which improves the targeting efficiency and therapeutic efficacy of the nano-delivery system.

5.10 Other tumors

Apart from the aforementioned extensively studied cancers, various other tumor types can derive benefits from the utilization of dual-ligand nanomedicines. For example, in colon cancer (CC) studies, Sultana et al.⁷⁰ targeted the FR and the CD44 receptor on CC cells using HA- and FA-modified NPs. Cellular assays exhibited the drug delivery system's efficacy in precisely targeting cancer cells, thereby holding promise for advancing CC therapeutics in the future. In renal cell carcinoma (RCC) studies, Takara et al.¹¹¹ prepared dual-ligand-modified liposomes of the NGR peptide and the cell-penetrating peptide tetraarginine (R4). R4 helps liposomal medications penetrate cells, while NGR targets CD13, which is overexpressed in the RCC vasculature. Experimental findings corroborated that the liposomal drug exerted a more potent anti-tumor effect. In oral squamous cell carcinomas (OSCC) studies, Zhou et al.⁴¹ synthesized dual-ligand polymeric micelles that specifically target tumor cells and cancer-associated fibroblast cells, respectively, in order to encapsulate medications with improved anticancer effects and fewer side effects; in CCa studies, Kang et al.³⁹ prepared a liposome nano-delivery system modified with dual ligands of FA and the cell-penetrating peptide Pep-1 and successfully demonstrated its antitumor potential for selective delivery of chemotherapeutic agents to FR-positive cancer cells.

6 Challenges for dual-ligand nanomedicines

Dual-ligand nanomedicines that provide enhanced drug loading, accurate tumor targeting, and selectivity. However, some research has also demonstrated that there are no appreciable benefits to dual-ligand nanomedicines over single-ligand nanomedicines.^6,109,159 In certain cases, dual-ligand nanomedicines may be less effective than single-ligand nanomedicines, particularly when receptor competition or significant steric hindrance is present. When there is a considerable difference in the expression levels of the two targeted receptors on the target cells, the receptor with lower expression may become a bottleneck for dual-ligand binding. Furthermore, steric hindrance could prevent both ligands from effectively binding to their respective receptors simultaneously. Additionally, the structural complexity of dual-ligand systems may make them more susceptible to recognition and clearance by the immune system, thereby potentially reducing their therapeutic efficacy.

The three primary concerns with dual-ligand nanomedicines are discussed in this chapter, including ligand distribution arrangements, clinical translation, and safety.

6.1 Distribution of ligands on dual-ligand nanomedicines

Conventionally, dual-ligand nanomedicines improve NPs targeting, but the absorption of the particles is not invariably improved. Optimal ligand length facilitates enhanced cellular uptake through spontaneous ligand rearrangement; however, nonspecific ligand interactions or length disparities may diminish cellular uptake (as elaborated further in 3).⁶ The targeting efficacy of dual-ligand nanomedicines is hindered by site-blocking effects and both excessive and insufficient ligand density.^6,9 Additionally, the targeting impact is also greatly influenced by the ligand's location, affinity for the receptor, and activity upon coupling. In vitro research may assess and optimize these factors, offering a dependable foundation for in vivo application.¹⁹ Optimal distribution and three-dimensional arrangement of the ligand are imperative for efficient binding to the target molecule and subsequent therapeutic efficacy.¹¹³

6.2 Clinical applications of dual-ligand nanomedicines

Despite considerable investigation, few dual-ligand nanomedicines have been applied in clinical settings to treat tumor patients.⁵⁸ Currently, the majority of researchers only concentrate on new drugs by enhancing their mechanism of action and increasing the effectiveness of tumor therapy; they are not taking into account the actual conditions needed for fabrication. Due to the number and variety of ligands in dual-ligand nanomedicines, there are no reliable methods to quantify ligand distribution on nanocarrier surfaces. The pharmaceutical business finds it challenging to adjust to mass production due to the complicated procedures and high preparatory needs. Even when manufacturing on a large scale, it is difficult to control the quality of the drugs produced and the variations between batches.^58,113 Nowadays, switching dual-ligand nanomedicines from preclinical to clinical use remains a great challenge.

6.3 Safety of dual-ligand nanomedicines

Safety considerations for dual-ligand nanomedicines predominantly revolve around the nanocarrier, with approximately 20% of clinically rejected NPs attributed to safety concerns.¹⁶⁰ The physical characteristics of NPs, such as size, surface area, surface charge, and aggregation state, determine their toxicity.¹⁶¹ These characteristics have been demonstrated to impact NP distribution and deposition in various organ systems, as well as change their molecular interactions with different proteins and other macromolecules. Additionally, the degree of exposure and the method of administration—ingesting, injecting, inhaling, or coming into contact with the skin—are also related to the toxicity of NPs.¹⁶² For example, transpulmonary exposure to NPs can cause an inflammatory response, fibrosis, and necrosis of lung tissues. However, strategies exist to mitigate or circumvent NP toxicity. For instance, employing NPs with a charged surface promotes their enhanced accumulation in target organs compared to their uncharged counterparts, while employing specific coating materials to modify NP surfaces can mitigate the risk of NP toxicity.

7 Summarize

Nowadays, although dual-ligand nanomedicines still have difficulties with ligand distribution, clinical translation, and safety, they still have an indispensable place in tumor therapy and are a superior therapeutic pathway following surgery, radiotherapy, and chemotherapy. Dual-ligand nanomedicine surface ligands can interact with each other to enhance therapeutic efficacy (Fig. 11) or can be modified to have multiple ligands acting together, and likewise they can bind to other targeting forces (e.g., magnetic targeting forces), which can enhance targeting and minimize potential off-target effects.^42,163 As time passes, dual-ligand nanomedicines have also demonstrated a non-negligible role in diagnosis and prognosis; we believe that with the collaborative efforts of researchers in the fields of molecular biology, pharmacy, oncology, immunology, clinical medicine, and other areas of medicine, dual-ligand nanomedicines will show the greatest therapeutic results, work better in clinical settings, and pave new ground in the treatment of human illnesses.

Fig. 11 Schematic illustration of the reversible tumor-targeting system based on a pH-responsive dual-ligand mutual-shielding strategy. PBA and Gal residues could form a complex at pH 7.4 and mutually shield their targeting function. At pH 6.8, the binding affinity between PBA and Gal weakened, and PBA and Gal bind to their receptors respectively.⁴² Copyright 2019, ACS Publications.

Author contributions

Ailing Wang: conceptualization, writing – original draft, formal analysis, investigation. Xuejun Wang: data curation, formal analysis, writing – review & editing. Dan Li: formal analysis, writing – review & editing. Aixue Li: investigation, writing – review & editing. Mengyuan He: methodology. Yingying Yuan: resources, software. Li Ye: supervision, validation, bisualization. Jiyong Liu: supervision, validation, visualization.

Data availability

No data was used for the research described in the article.

Conflicts of interest

The authors have no conflicts of interest to declare.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (grant numbers 82274103, 82305039), the National Key R&D Program of China (grant number 2023YFC3404103), the Science and Technology Commission of Shanghai Municipality (grant number 24S1190150, 23S21900200), the State Key Laboratory of Neurology and Oncology Drug Development (grant number SKLSIM-F-202418), and the Cooperation Program of the Xuhui District Medical College with Enterprises (grant number 23XHYD-24). Additionally, we were also supported by the drawing tools provided by Figdraw.

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Footnote

† These authors made equal contributions to this work.

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