Vinh Phuoc

Katel Nguyen

Igor Hervé-Aubert

Emilie Chourpa

Allard-Vannier

Active targeting strategy in nanomedicines using anti-EGFR ligands -a promising approach for cancer therapy and diagnosis

Keywords: EGFR, nanomedicine, active targeting, cancer therapy, imaging, theranostics Graphical

As active targeting using nanomedicines establishes itself as a strategy of choice in cancer therapy, several target receptors or ligands overexpressed in cancer cells have been identified and exploited. Among them, the epidermal growth factor receptor (EGFR) has emerged as one of the most promising oncomarkers for active targeting nanomedicines due to its overexpression and its active involvement in a wide range of cancer types. Henceforth, many novel EGFR-targeted nanomedicines for cancer therapy have been developed, giving encouraging results both in vitro and in vivo. This review focuses on different applications of such medicines in oncotherapy. On an important note, the contribution of EGFR-targeting ligands to final therapy efficacy along with current challenges and possible solutions or alternatives are emphasized.

Introduction

Cancer is one of the most prevalent, predominant and fatal diseases in the world with an increasing number of new cases each year. In the United States, it is estimated that around 1,898,160 new cases will be detected and 608,570 Americans will die from cancer in 2021 [START_REF] Siegel | Cancer Statistics[END_REF]. With the advent and the development of new modalities used in diagnosis and/or early treatment, the cancer death rate has continuously fallen since 1991, reaching a total decline percentage of 31% in 2018. Despite this positive sign, the overall survival rate has yet to be improved, especially in the era of new public health threats posed by the novel coronavirus, also known as SARS-Cov-2, during which many studies have reached a consensus on the increasing vulnerability of cancer patients to COVID-19 complications [START_REF] Moujaess | Cancer patients and research during COVID-19 pandemic: A systematic review of current evidence[END_REF].

Currently, several types of cancer treatment are available including surgery, radiotherapy, and chemotherapy that can be applied either alone or in combination with each other [START_REF] Huang | A review on the effects of current chemotherapy drugs and natural agents in treating non-small cell lung cancer[END_REF]. Chemotherapy remains the standard modality for cancer therapy, but confronts several issues such as quick clearance, low stability, low drug tolerance due to non-specific biodistribution, and the emergence of drug resistance. One of the most promising strategies to overcome these problems is to use nanoscale drug delivery systems (Pawar and Prabhu, 2019a). These nanosystems are developed not only to improve drug delivery but also to cover a broad spectrum of cancer therapy including gene delivery, phototherapy, diagnosis, and theranostic purposes. Compared to conventional adjuvant agents, nanoparticles (NPs) offer remarkable benefits such as: i) improving the delivery of highly hydrophobic drug, ii) facilitating the passage of drugs across biological barriers, iii) protecting and improving the delivery of therapeutic molecules to their action sites, iv) combining imaging and therapy, and v) specifically reducing drug toxicity [START_REF] Hirsjarvi | Passive and Active Tumour Targeting with Nanocarriers[END_REF].

Tumor targeting with NPs can be achieved by two strategies namely passive or active targeting. Passive targeting relies on the Enhanced Permeation and Retention (EPR) effect or abnormalities of the tumor microenvironment to better deliver therapeutic agents [START_REF] Attia | An overview of active and passive targeting strategies to improve the nanocarriers efficiency to tumour sites[END_REF][START_REF] Byrne | Active targeting schemes for nanoparticle systems in cancer therapeutics[END_REF][START_REF] Hirsjarvi | Passive and Active Tumour Targeting with Nanocarriers[END_REF]. Although passive targeting can enhance drug tumor accumulation, it cannot specifically distinguish between tumors and healthy tissues and may lead to intolerant side effects. Therefore, recent studies are focusing on a new strategy termed "active targeting" to maximize the therapeutic efficiency and minimize toxicity on normal tissues. Active targeting refers to the functionalization of NPs' surface with biological ligands that can bind specifically to the target molecules or receptors present on cancer cells [START_REF] Yoo | Active Targeting Strategies Using Biological Ligands for Nanoparticle Drug Delivery Systems[END_REF]. Among various kinds of target molecules or receptors, Epidermal Growth Factor Receptors (EGFRs) appear among the most interesting targets for active targeting with nanomedicines due to their remarkable overexpression in a broad spectrum of cancer types and their involvement in diverse cancer cell processes [START_REF] Nguyen | Nanomedicines functionalized with anti-EGFR ligands for active targeting in cancer therapy: Biological strategy, design and quality control[END_REF]. EGFR is a 170 kDa transmembrane glycoprotein in the tyrosine kinase family that plays a key role in the control of cell proliferation in normal tissues. In cancer, the EGFR-overexpression is a common phenomenon in various kinds of human cancers such as lung, head and neck, colon, pancreas, breast, ovary, and bladder and kidney cancers. Its overexpression has been proven highly related to multiple cancer-implicated signaling pathways and significantly contributes to cancer angiogenesis as well as cancer resistance. Considering its active involvement in cancer, many researchers are currently working on this field and achieved important advances in the development of active targeted nanomedicines with anti-EGFR ligands. As information on its structure, signaling pathways and available EGFR-targeting ligands has been precisely described in our previous review [START_REF] Nguyen | Nanomedicines functionalized with anti-EGFR ligands for active targeting in cancer therapy: Biological strategy, design and quality control[END_REF], this paper will specifically focus on the application of such nanomedicines in cancer therapy and diagnosis.

Structure of EGFR-targeted nanomedicines

Various kinds of NPs, EGFR-targeting moieties and conjugation methods have been developed for cancer therapy [START_REF] Nguyen | Nanomedicines functionalized with anti-EGFR ligands for active targeting in cancer therapy: Biological strategy, design and quality control[END_REF]. Diverse nanomaterials are available and can be classified into two main groups including organic and inorganic NPs. Table 1 provides information on the structures, advantages, and drawbacks of different kinds of EGFR-targeted NPs and different strategies to conjugate anti-EGFR ligands onto these NPs.

In general, these NPs can be used for various kinds of applications. However, according to a specific objective, some kinds of NPs can be preferred over others. For example, in EGFR-targeted NPs, to deliver a hydrophobic drug, organic NPs such as liposomes, solid lipid NPs, polymeric NPs or micelles are more frequently used than inorganic NPs. In contrast, for phototherapy or imaging agents delivery, inorganic NPs such as gold nanoparticles (AuNPs), superparamagnetic NPs (SPIONs) or quantum dots (QDs) are usually employed. In EGFR-targeted NPs, these inorganic NPs are normally attached to organic components such as polymer layers or EGFR active targeting ligands in order to improve their stability and functionality, and therefore, belong to the family of hybrid NPs [START_REF] Yao | Nanoparticle-Based Drug Delivery in Cancer Therapy and Its Role in Overcoming Drug Resistance[END_REF]. For gene therapy with EGFR-targeted NPs, dendrimers are employed in many studies.

Since their discovery in 1961, liposomes have been widely used as drug delivery systems in cancer treatment. These NPs comprise a bilayer lipid membrane made of nontoxic phospholipids and cholesterol surrounding an aqueous core. Thanks to their small size ranging from 50 nm to 100 nm, their hydrophilic and hydrophobic structural properties, liposomes can be used to deliver both hydrophilic and hydrophobic therapeutic agents and are considered one of the most widely used nanosystems in drug delivery [START_REF] Aghebati-Maleki | Nanoparticles and cancer therapy: Perspectives for application of nanoparticles in the treatment of cancers[END_REF][START_REF] Akbarzadeh Khiavi | Multifunctional nanomedicines for targeting epidermal growth factor receptor in colorectal cancer[END_REF]. Doxorubicin encapsulated in liposomes (Doxil®) is the first approved nanoformulation in cancer treatment [START_REF] Barenholz | Doxil® -The first FDA-approved nano-drug: Lessons learned[END_REF]. In EGFR-targeted NPs, their application is highly regarded in drug and gene delivery. As an example, Wang et al conjugated anti-EGFR scFv fragments onto their liposomes to improve the therapeutic index of chemotherapy (doxorubicin and vinorelbine) in head and neck cancer (HNC). On the one hand, with a size inferior to 150 nm (100 nm for non-targeted liposomes and 120 nm for targeted ones), both targeted and non-targeted liposomes demonstrated an efficient EPR effect, as reflected by better inhibition on tumor growth compared to free drugs. On the other hand, the advantage of EGFR-active targeting was clearly revealed by a significant reduction in the IC50 values of various cell lines of HNC (FaDu and Ca9-22 cells), and a prolonged median survival time of animals treated with targeted NPs compared to non-targeted counterparts or free drugs (94 days versus 60 days and 56 days, respectively) [START_REF] Wang | Novel anti-EGFR scFv human antibodyconjugated immunoliposomes enhance chemotherapeutic efficacy in squamous cell carcinoma of head and neck[END_REF].

Polymeric NPs are the most common kind of NPs used for drug delivery, with Abraxane® (approved in 2005) being the first polymeric nanodrug in the market. The choice of polymers is made according to the type of application and the encapsulated agents. However, these polymers, either natural or synthesized, have to be biocompatible, nontoxic and have a suitable half-life time [START_REF] Aghebati-Maleki | Nanoparticles and cancer therapy: Perspectives for application of nanoparticles in the treatment of cancers[END_REF]. Among different kinds of polymers used in nanomedicines, poly(lactic-co-glycolic acid) or PLGA and poly(lactic acid) or PLA are the most successful nanomaterials due to their capability of being hydrolyzed inside the body into metabolite monomers with minimal toxicity [START_REF] Aftab | Nanomedicine: An effective tool in cancer therapy[END_REF]. Not surprisingly, many EGFR-targeted polymeric NPs have been developed and cover a large spectrum of cancer therapy from therapeutic to diagnostic applications. This aspect will be further illustrated in each section of this review.

Micelles are colloidal carriers made of amphiphilic molecules that can self-assemble into nanosized drug delivery systems. Most micelles are made of polymers (polymeric micelles) but amphiphilic molecules such as amphiphilic peptides can also be used. As regards EGFR-targeting application with non-polymeric micelles, anti-EGFR peptides such as GE11 peptides have been extensively applied as a component to construct micelles while preserving their EGFR-targeting properties. In this strategy, the GE11 serves as the hydrophilic head and a hydrophobic tail is introduced, as shown in a study of Liang et al [START_REF] Liang | Development of self-assembling peptide nanovesicle with bilayers for enhanced EGFRtargeted drug and gene delivery[END_REF]. In another study, Du et al have demonstrated the potency of selfassembly amphiphilic peptide NPs with GE11 in the delivery of gemcitabine and olaparib to treat pancreatic cancer with breast cancer 2 mutation. An extra glycine and two glutamic-acid containing carboxyl groups were added into GE11 to increase its hydrophilicity. Furthermore, to have sufficient hydrophobicity, a C18 tail was added into the monomer. The targeted micelles were stable, exhibited high encapsulation efficiency for both drugs (up to 98%), had their half-life prolonged (>3h vs 1h for the free drugs), and provided better tumor suppression compared to non-targeted ones with minimal side effects [START_REF] Du | Epidermal Growth Factor Receptor-Targeting Peptide Nanoparticles Simultaneously Deliver Gemcitabine and Olaparib To Treat Pancreatic Cancer with Breast Cancer 2 ( BRCA2 ) Mutation[END_REF].

Dendrimers are highly branched polymers that comprise three main components: a central core, repetitive branching units, and terminal groups. There are three types of dendrimers. The most widely used in research included polyamidoamine (PAMAM), poly(propylene imine) (PPI), and poly-Llysine (PLL) dendrimers. Compared to conventional linear and branched polymeric nanocarriers, dendrimers present several advantages such as: i) higher control in terms of physico-chemical properties with good solubility, ii) good ability to pass through cell barriers, and iii) high possibility of surface functionalization. Therefore, dendrimers are getting increasing attention, and their great potency as a drug or gene delivery system has been confirmed [START_REF] Abedi-Gaballu | PAMAM dendrimers as efficient drug and gene delivery nanosystems for cancer therapy[END_REF][START_REF] Mendes | Dendrimers as Nanocarriers for Nucleic Acid and Drug Delivery in Cancer Therapy[END_REF]. In EGFR-targeted nanomedicines, the application of dendrimers in gene delivery is very promising. To be precise, dendrimers are able to protect nucleic acids from nuclease degradation by forming stable dendrimer-nucleic acid complexes via electrostatistic forces. Furthermore, the possibility of modifying their surface with different kinds of anti-EGFR ligands can contribute effectively to enhancing the efficacy of the finalized nanocarriers [START_REF] Abedi-Gaballu | PAMAM dendrimers as efficient drug and gene delivery nanosystems for cancer therapy[END_REF]. Therefore, the application of EGFR-targeted dendrimers in gene therapy are focused in this paper.

In addition to the previous "organic" NPs, inorganic nanomaterials have also been extensively applied in EGFR-targeted nanomedicines. These NPs include gold NPs (AuNPs), mesoporous silica NPs (MSNPs), superparamagnetic iron oxides NPs (SPIONs) and quantum dots (QDs). Compared to organic NPs, inorganic NPs can offer unique advantages including high stability, easy synthesis, easy modification and inertness. Their application can cover a broad spectrum in cancer therapy, e.g. the delivery of radiotherapy, phototherapy or imaging agents thanks to their unique intrinsic properties. For example, AuNPs are of great interest owing to their unique properties in size and shapedependent optical and electronic characteristics. MSNPs are extensively used in the medical field due to several advantageous properties such as their large surface area, well-defined morphology, and biocompatibility. SPIONs can be served as excellent contrast agents in magnetic resonance imaging (MRI). QDs are outstanding in biomedical imaging for cancer compared to traditional organic dyes due to their unique optical and electronic characteristics. In addition to their unique intrinsic properties, these NPs can also be served as promising drug nanocarriers due to the possibility of easily modifying their surface [START_REF] Bayda | Inorganic Nanoparticles for Cancer Therapy: A Transition from Lab to Clinic[END_REF]. In EGFR-targeted nanomedicines, these inorganic NPs can be easily functionalized with various kinds of anti-EGFR ligands via different conjugation methods and have been exploited in many studies [START_REF] Nguyen | Nanomedicines functionalized with anti-EGFR ligands for active targeting in cancer therapy: Biological strategy, design and quality control[END_REF]. The accurate applications of such nanomedicines will be discussed in the following sections.

Mesoporous silica NPs

NPs made of silica with welldefined porosity and morphology

Via chemical conjugation

Biocompatibility, easy surface modification, large surface area, welldefined morphology Short circulation time, low stability [START_REF] Attia | An overview of active and passive targeting strategies to improve the nanocarriers efficiency to tumour sites[END_REF][START_REF] Pasqua | Mesoporous Silica Nanoparticles in Cancer Therapy: Relevance of the Targeting Function[END_REF][START_REF] Reda | PLK1 and EGFR targeted nanoparticle as a radiation sensitizer for non-small cell lung cancer[END_REF]

Magnetic NPs

NPs made of superparamagnetic iron oxides

Via chemical conjugation

Biocompatibility, easy surface modification, easy synthesis, possibility to exploit magnetic properties No internal loading capacity [START_REF] Chu | Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy[END_REF][START_REF] Revia | Magnetite nanoparticles for cancer diagnosis, treatment, and treatment monitoring: recent advances[END_REF][START_REF] Vinh Nguyen | Targeted nanomedicine with anti-EGFR scFv for siRNA delivery into triple negative breast cancer cells[END_REF][START_REF] Xiao | Superparamagnetic nanoparticles for biomedical applications[END_REF]

Quantum dots

Semiconductor nanocrystals with cores made of inorganic elements (e.g. Cd and Se) enclosed within a metallic shell (e.g. ZnS, Ag2S)

Via chemical conjugation

Innate optical properties, easy surface modification

No internal loading capacity, potential toxicity [START_REF] Lee | Characterization and Cancer Cell Specific Binding Properties of Anti-EGFR Antibody Conjugated Quantum Dots[END_REF][START_REF] Richards | Antibody fragments as nanoparticle targeting ligands: a step in the right direction[END_REF][START_REF] Zhang | Affibodyfunctionalized Ag 2 S quantum dots for photoacoustic imaging of epidermal growth factor receptor overexpressed tumors[END_REF] 3. Applications of EGFR-targeted nanomedicines in the context of cancer therapy

Targeted delivery of chemotherapeutics or therapeutic radionuclides

As EGFR expression is strictly related to a variety of cancer processes, anti-EGFR moieties on nanomedicines can serve, at the same time, as active targeting ligands and therapeutic agents for cancers with a high EGFR expression level. The introduction of anti-EGFR moieties onto nanomedicines can offer several advantages as follows.

Higher drug accumulation in cancer cells and tumors overexpressing EGFR

At the cellular level, EGFR-targeted NPs can improve drug cellular uptake via specific EGFR-mediated endocytosis. While non-targeted NPs internalize into cancer cells via non-specific pathways (diffusion or membrane fusion), the targeted NPs can enter directly into cancer cells via both non-specific and specific pathways. As regards the specific pathway, the targeted NPs firstly bind to cancer cells via the EGFR ligand-receptor complex, and subsequently activate the corresponding endocytosis, which involves endosomal or lysosomal uptake of these NPs. Due to this specific pathway, a better and faster cellular internalization process can be achieved with EGFR-targeted NMs [START_REF] Clemons | Distinction Between Active and Passive Targeting of Nanoparticles Dictate Their Overall Therapeutic Efficacy[END_REF][START_REF] Singh | EGFR-targeted gelatin nanoparticles for systemic administration of gemcitabine in an orthotopic pancreatic cancer model[END_REF]. Following the endocytosis, NPs are usually disassembled and release the entrapped drugs, directly delivering them into the site of cell activation, which is not observed in the case of non-targeted NPs [START_REF] Clemons | Distinction Between Active and Passive Targeting of Nanoparticles Dictate Their Overall Therapeutic Efficacy[END_REF]. On another aspect, the functionalization with EGFRtargeting ligands can help distinguish tumor cells from healthy ones, this may be useful to reduce the side effects related to encapsulated drugs [START_REF] Du | Epidermal Growth Factor Receptor-Targeting Peptide Nanoparticles Simultaneously Deliver Gemcitabine and Olaparib To Treat Pancreatic Cancer with Breast Cancer 2 ( BRCA2 ) Mutation[END_REF]. This advantage is based on the relatively low expression of EGFR in normal cells (4.0-10.0 x10 4 EGFRs/cell) compared to the high EGFR expression level in cancer cells (up to over 10 6 EGFR/cell) [START_REF] Akbarzadeh Khiavi | Multifunctional nanomedicines for targeting epidermal growth factor receptor in colorectal cancer[END_REF]. Finally, as EGFR-overexpression is a common phenomenon in a large spectrum of cancers, the possibility to develop a nanosystem that can cover several kinds or cancers or highly heterogeneous cancers can be achieved [START_REF] Reda | PLK1 and EGFR targeted nanoparticle as a radiation sensitizer for non-small cell lung cancer[END_REF].

In vivo, EGFR-targeting provides substantial benefits in enhancing anti-tumor activities of NPs and reducing severe side effects compared to non-targeted NPs or free drugs. Many studies have developed EGFR-targeted NMs for various kinds of cancers, which are resumed in Table 2. Herein, a standard example was carried out by Sandoval et al. The authors developed gemcitabine-loaded liposomes functionalized with EGF proteins for breast cancer treatment. While the cellular uptake of non-targeted nanoparticles exhibited no difference among different breast cancer cell lines, EGFRtargeted nanoparticles exhibited a better cellular internalization capacity according to an increasing level of EGFR density in cancer cells (MDA-MB-468> MDA-MB-231> MCF-7, respectively). As a result, a 2-fold higher tumor accumulation level in MDA-MB-468 tumors of gemcitabine was obtained and a significant slower tumor growth rate of MDA-MB-468 tumors treated with EGFR-targeted NPs was achieved compared to that treated with non-targeted ones [START_REF] Sandoval | EGFR-targeted stearoyl gemcitabine nanoparticles show enhanced anti-tumor activity[END_REF].

As the advantages of EGFR-targeted NMs compared to conventional NMs have clearly been observed both in vitro and in vivo for a wide range of cancers, it is reasonable to state that EGFRs are among the most appealing oncomarkers for the development of targeted nanotherapies and worthy of further clinical trials.

Overcoming multidrug resistance

In addition to severe secondary effects, multidrug resistance (MDR) is another challenge imposed to chemotherapy application in cancer treatment. In MDR-related processes, drugs are pumped out of cancer cells before they can exert any therapeutic function. It has been reported that EGFR-overexpression plays an important role in the survival of MDR-associated phenotypes (hypoxic MDR tumors). To be more precise, EGFR-overexpression upregulates the hypoxia inducible factor (HIF-1α), which activates several genes related to MDR such as mdr1 and mrp1. These genes subsequently cause the overexpression of two essential MDR-related proteins including P-glycoprotein (P-gp) and multidrug resistant protein 1 (MRP-1). The upregulation of these transmembrane drug efflux pumps induces the MDR phenomenon by pumping out anticancer drugs, resulting in little or no therapeutic action [START_REF] Huang | Demethoxycurcumin-Carrying Chitosan-Antibody Core-Shell Nanoparticles with Multitherapeutic Efficacy toward Malignant A549 Lung Tumor: From in Vitro Characterization to in Vivo Evaluation[END_REF]. Therefore, EGFR-targeted NPs may help to overcome this problem by significantly increasing drug accumulation inside MDR tumors with EGFR overexpression. On the other side, the therapeutic activities of conjugated EGFR ligands on the NPs may also be helpful to inhibit the production of factors such as HIF-1α linked to MDR and provide a potential solution to overcome MDR.

As an example, Milane et al used GE11-conjugated polymeric NPs (PCL, PLGA-PEG NPs) for paclitaxel and lonidamine co-delivery in MDR human breast and ovarian tumor cells. The authors showed that hypoxia promoted the MDR characteristics in EGFR-positive ovarian cancer (SK-OV-3 cells) and breast cancer cells (MDA-MB-231 cells), as reflected by a clear increase in the IC50 of paclitaxel compared to those obtained from corresponding wild types. As expected, the expression of EGFRs in the hypoxic SK-OV-3 and MDA-MB-231 cells was found to be significantly upregulated compared to normoxic ones. As a result, a higher endocytosis level of the targeted NPs in normoxic cells (SK-OV-3 and MDA-MB-231 cells) was recorded even after only 30 minutes of treatment. This phenomenon reconfirmed the interest of using EGFR as a target in case of MDR. In addition, the targeted NPs loaded with the two drugs demonstrated a superior pharmacokinetic profile in comparison to free drugs or nontargeted NPs, resulting in 5-10% cell viability for normoxic and hypoxic cell lines, respectively. All these results proved that relying on EGFR-targeted NPs is a promising strategy to actively target and overcome MDR (Milane et al., 2011a(Milane et al., , 2011b)). Using a similar strategy, Huang et al coated their curcumin-loaded chitosan NPs with cetuximab and used them to treat normoxic and multidrug resistant hypoxic lung cancer cells overexpressing EGFR (A549 cells). In vitro, free drugs were ineffective against hypoxic A549 cells and lightly effective against normoxic A549 cells with an IC50 of 40 µg/mL, whereas cetuximab-coated NPs achieved high cytotoxicity in both hypoxic and normoxic A549 cells with IC50 of 10 µg/mL and 5 µg/mL, respectively. In addition, the authors showed that the improvement in cytotoxicity of the targeted NPs was clearly noticeable in the case of hypoxic A549 cells, highlighting the efficacy of EGFR targeting in MDR hypoxic tumors [START_REF] Huang | Demethoxycurcumin-Carrying Chitosan-Antibody Core-Shell Nanoparticles with Multitherapeutic Efficacy toward Malignant A549 Lung Tumor: From in Vitro Characterization to in Vivo Evaluation[END_REF].

In a word, targeted nanomedicines with anti-EGFR ligands can be used as a promising strategy to improve the therapeutic index of conventional chemotherapy in hypoxic MDR tumors. To better exploit EGFRs, their involvement in other kinds of MDR needs to be better clarified. Besides, the therapeutic activity of anti-EGFR ligands on nanomedicines in MDR cancers is another interesting aspect to investigate.

Change in the downstream signaling pathway

Anti-EGFR ligands on NPs can bind to EGFRs present on cancer cells, block the binding of EGFRs on cells with other EGFR ligands, suppress or disrupt the downstream signaling pathway, and lead to a better therapeutic activity. As a proof of concept, a double advantage of anti-EGFR ligands on NPs was highlighted in the study of Reda et al. On the one hand, the authors demonstrated that targeted mesoporous silica NPs (MSNPs) better internalized into EGFR positive cancer cells (A549 and H460 cells) by a factor of eight compared to the low-expressed EGFR normal lung cell line (NL20 cells). On the other hand, the grafted cetuximab on NPs could also provide a therapeutic effect by binding to EGFRs on cancer cells and changing the signaling pathway. In fact, following X-ray irradiation, activated EGFRs on cancer cells are normally phosphorylated and translocated to the nucleus, where they play a role in mediating DNA repair. This process can reduce the efficiency of radiotherapy. Therefore, if it is blocked, cancer cells will be more sensitive to radiotherapy, improving treatment efficiency. On this point, it is interesting to note that conjugated cetuximab NPs were more effective than free mAbs in reducing cell surface EGFR receptors despite their lower concentration. This phenomenon was probably due to the higher density of grafted antibodies on NPs and resulted in higher interaction mAbs-EGFRs. As a result, by analyzing the level of DNA damage, the authors found that cetuximab grafted on NPs could prevent cancer cells from repairing DNA damage induced by irradiation [START_REF] Reda | PLK1 and EGFR targeted nanoparticle as a radiation sensitizer for non-small cell lung cancer[END_REF].

In another study, Chen et al developed EGFR-targeted albumin-cisplatin NPs functionalized with anti-EGFR aptamers to improve the efficacy and tolerability of cisplatin in EGFR-positive cervical cancer.

The results indicated that both free aptamers in solution and aptamers conjugated with functionalized NPs could specifically bind to EGFR-positive cancer cells (Hela). Thanks to this specific binding, targeted NPs achieved higher tumor inhibition effects with the highest cytotoxicity level (IC50 = 26 µM) that is twice better than that of free cisplatin (IC50 = 55 µM) and that of non-targeted NPs (IC50 = 56 µM). Moreover, cisplatin encapsulated with targeted NPs accumulated significantly better than that with non-targeted NPs and free cisplatin in tumors (1.4 and 1.75 times, respectively), and accumulated less in healthy organs (1.1 and 3.9 times in liver, respectively). For example, ex vivo data of drug accumulation in liver showed that free cisplatin accumulated more in liver by a factor of 3.5 and 3.9 than cisplatin conjugated with non-targeted and targeted NPs, respectively. This selective tumor accumulation not only helped to enhance drug therapeutic index but also reduced systemic toxicity. As cisplatin application in clinical trials is usually limited due to its high toxicity, aptamerfunctionalized NPs are a promising strategy to address this problem. In addition to active targeting properties, the authors also insisted that EGFR-aptamers conjugated on these NPs played a vital role in the antitumor effects of the finalized NPs. The conjugated anti-EGFR aptamers effectively blocked EGF, induced EGFR phosphorylation, changed the signaling downstream pathway, and contributed significantly to the final antitumor activities of the targeted NPs [START_REF] Chen | Aptamer Functionalized Cisplatin-Albumin Nanoparticles for Targeted Delivery to Epidermal Growth Factor Receptor Positive Cervical Cancer[END_REF].

Although the therapeutic activity of anti-EGFR ligands on nanomedicines has been proven, only several kinds of ligands such as aptamers or mAbs can be applied. Moreover, their activity is highly dependent on their quantity and their conformation on the NPs. Therefore, appropriate conjugation strategies need to be considered in the design step [START_REF] Nguyen | Nanomedicines functionalized with anti-EGFR ligands for active targeting in cancer therapy: Biological strategy, design and quality control[END_REF]. Moreover, as the yield of ligand conjugation is relatively low, the quantity of ligands present on NPs is far from that required to have any therapeutic activity. In addition, the introduction of so many ligands onto NPs may have a negative impact on colloidal properties of the finalized NPs. As a result, an equilibrium between costbenefit and efficacy-stability is a relevant issue to take into consideration.

Gene delivery

Recently, gene therapy increasingly arouses interest in cancer therapy. Thanks to their potency and their ability to silence specific oncogenes in a selective manner, interfering nucleic acids (iNAs) have been widely applied to ensure targeted molecular intervention and achieve a higher level of specific actions than conventional cytotoxic chemotherapy. Nevertheless, the clinical application of naked iNAs is limited due to their high charge, hydrophilicity, big size, and nuclease degradation. To overcome these problems, a suitable delivery system is needed to deliver these potent agents into target sites. One promising approach is to nanovectorize these iNAs. Among different available nanosystems, EGFR-targeted nanomedicines have demonstrated their potency in cancer treatment.

Targeted intracellular iNAs delivery

Similar to drug delivery with nanomedicines, EGFR-targeted nanomedicines have been shown to be effective in protecting siRNA and are better delivered into cancer cells. Among various kinds of NPs, the choice of targeting NPs with anti-EGFR ligands for gene delivery is quite limited and only certain kinds of NPs have been applied including cationic polymers or liposomes, cationic polymers NPs, dendriplexes and extracellular vesicles.

The association of iNAs with cationic polymers is normally achieved via electrostatic forces between the negative charges of iNAs and the cationic charges on NPs. Several cationic polymers can be applied for this purpose such as chitosan, poly-L-arginine, polyethylenimine (PEI), poly-lysine [START_REF] Ben Djemaa | Versatile electrostatically assembled polymeric siRNA nanovectors: Can they overcome the limits of siRNA tumor delivery?[END_REF]. In a previous study, our team developed for the first time a novel EGFRtargeted nanomedicine based on the complexation between EGFR-scFv fragment-decorated SPIONs, siRNA and cationic polymers (chitosan and poly-L-arginine) for better gene delivery into triple negative cancer cells (MDA-MB-231 cells). The results showed that by increasing the number of EGFR fragments on the NPs surface, a better cellular uptake level by a factor of two can be obtained. Moreover, the conjugation of EGFR scFv fragments helped increase gene silencing effects from 25.3% to 69.4% in TNBC (triple negative breast cancer) cell lines (MDA-MB231 cells) (Vinh [START_REF] Vinh Nguyen | Targeted nanomedicine with anti-EGFR scFv for siRNA delivery into triple negative breast cancer cells[END_REF].

Recently, extracellular vesicles are considered emerging therapeutic carriers, especially in gene delivery due to their role in intercellular communication and their ability to enter cells through multiple routes that result in a long half-life time (Table 1). However, the lack of specificity remains one of the biggest challenges for their application. To address this problem, Pi et al incorporated EGFR-specific 2' F-RNA aptamers to extracellular vesicles to enhance targeting gene delivery into EGFR-positive breast cancer cells. This targeted nanosystem exhibited successful targeted delivery of surviving siRNA to breast tumor cells and inhibited the expression of the target gene at both protein and mRNA levels. Moreover, targeted NPs were able to accumulate more in tumors than nontargeted NPs. Consequently, with a dose of 0.5 mg of siRNA per kg of mice body weight (six doses weekly), the targeted NPs significantly suppressed in vivo tumor growth, as monitored by the tumor volume with and without NPs (tests versus controls) [START_REF] Pi | Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression[END_REF].

Reduced toxicity of iNAs-loaded dendrimers

Compared to conventional cationic polymers, dendrimers are interesting materials and are considered a revolution in gene delivery with nanocarriers [START_REF] Dufes | Dendrimers in gene delivery[END_REF]. However, their application is still limited due to their cytotoxicity and hemolytic properties (Table 1). Numerous studies have revealed that this toxicity is highly related to the strong cationic charges of these nanocarriers. In fact, the highly positive charge interacts strongly with the negatively charged cell membranes and results in cell destabilization, cytoplasmic proteins leakage and eventual lysis (Palmerston [START_REF] Mendes | Dendrimers as Nanocarriers for Nucleic Acid and Drug Delivery in Cancer Therapy[END_REF]. Hence, recent studies with dendrimers are focusing on strategies to reduce or mask their charge, and surface modification with EGFR-targeting ligands may help solve this problem.

The first example is a novel EGFR-targeted complex of EGF proteins, plasmid DNAs and PAMAM dendrimers developed by Zhang et al. The authors employed electrostatic absorption to conjugate negatively charged EGF proteins and DNAs onto their cationic dendrimers. This strategy was shown to be effective in reducing the positive charge of the finalized dendrimers (from +16.2 ± 0.2 mV to +2.4 ± 0.8 mV, respectively), which may attenuate the dendrimers toxicity. Using Renilla luciferase DNAs as models, the targeted NPs exhibited higher gene transfection efficiency both in vitro in MCF-7/EGFR and MDA-MB-231 cells and in vivo in MDA-MB-231 tumors, as reflected by a higher in vivo luciferase expression level by a factor of around two. Furthermore, when non-targeted dendriplexes exhibited remarkable toxicity, the cellular toxicity of the targeted dendrimers was significantly decreased according to an increasing amount of EGF proteins on their surface. Firstly, this result may be explained by the decrease in the charge of the targeted dendrimers. Secondly, while the only possible way for non-targeted NPs to enter the cell is the non-specific pathway that may damage the lipid bilayer structure and cause cell death, targeted NPs can internalize through both non-specific and EGFR-specific endocytosis, which is a normal physiological process and causes less damage to the cell [START_REF] Zhang | A novel EGFR-targeted gene delivery system based on complexes self-assembled by EGF, DNA, and activated PAMAM dendrimers[END_REF].

In another study of Kim and colleagues, a polyanionic peptide was genetically fused to the EGFR repebody that subsequently enabled the complexation of DNA, dendrimers and repebodies through electrostatic interactions. In this study, the authors compared the gene delivery efficiency of the resulting targeted dendrimer into cell lines with different levels of EGFR expression including MDA-MB-231 (high expression), HeLa (moderate expression) and HepG2 (low expression). The gene delivery efficiency of the targeted NPs in cells was correlated with the EGFR expression level, revealing their selective binding activity. Furthermore, a reduced cytotoxicity level related to an increasing amount of EGFR repebodies was observed for the targeted dendriplexes. It is likely that the EGFR repebody may play a dual role as a targeting moiety and a cytotoxicity-reducing agent for gene delivery with dendrimers [START_REF] Kim | Electrostatically assembled dendrimer complex with a highaffinity protein binder for targeted gene delivery[END_REF].

For gene delivery with viral vectors, EGFR-targeted NPs have been proven beneficial to improving the specificity and therapeutic efficacy of gene therapy. As an example, Yoon et al. showed that by conjugating cetuximabs onto their PEGylated dendrimers and complexing the latter with an oncolytic adenovirus (Ad), the finalized formulation was efficient to enhance the delivery of this oncolytic Ad into EGFR-positive tumor cells with attenuated toxicity compared to non-targeted dendrimers or naked oncolytic adenovirus. Adenovirus (Ad) is a mediated cancer gene therapy that contains a linear double-stranded 34-36 kb DNA genome within an icosahedral capsid, and is considered a promising alternative to conventional therapy for cancer [START_REF] Uusi-Kerttula | Oncolytic Adenovirus: Strategies and Insights for Vector Design and Immuno-Oncolytic Applications[END_REF]. However, its application in clinical trials is limited due to its highly immunogenicity and its weakness in cellular internalization.

To solve this problem, the authors complexed their oncolytic Ad with cetuximab-conjugated dendrimers to better deliver Ad into the tumor sites. Among three cell lines with different levels of EGFR expression including EGFR-overexpressed lung cancer (A549), EGFR low-expressed breast cancer (MCF-7) and normal fibroblast (HDF) cells, the EGFR-targeted complex showed a selective binding capacity with the highest cellular uptake into A549 cells. Moreover, in these cancer cells, the finalized formulation exhibited a cellular uptake level that was 8.6 and 6.5 times higher than those of the naked Ad and the non-targeted dendrimers, respectively. The immunogenicity and hepatotoxicity of the EGFR-targeted formulation were also reduced in comparison to the naked Ad [START_REF] Yoon | Antitumor effect and safety profile of systemically delivered oncolytic adenovirus complexed with EGFR-targeted PAMAM-based dendrimer in orthotopic lung tumor model[END_REF].

Combination of gene therapy and chemotherapy

Drug resistance is currently a challenge to cancer treatment with chemotherapy. To address this issue, higher doses of adjuvant agents are normally used, but result in more severe side effects. The combination between gene therapy mediated by EGFR-targeted NMs and chemotherapy may allow the use of lower but more efficient doses of anticancer drugs and effectively address this issue. To this end, EGFR-targeted NMs can be used to deliver interfering nucleic acids (iNAs) in combination with free drugs or to co-deliver iNAs along with drugs. As an example, Nascimento and colleagues developed GE11 peptide-conjugated chitosan NPs as a versatile delivery system for silencing the essential mitotic checkpoint gene Mad2 that induces cell death. The GE11-decorated NPs showed efficient and targeted delivery of Mad2-siRNA in the EGFR positive A549 cell lines, resulting in a better gene silencing effect than non-targeted NPs. These results were also confirmed in tumorbearing mice, as the targeted NPs were shown to have higher targeting efficiency and accumulate better in tumor cells. Importantly, in cisplatin-resistant tumors, monotherapy using EGFR-targeted NPs loading siRNA presented an efficient inhibitory activity on tumor growth better than that involving non-targeted ones, as reflected by a delayed tumor growth rate of 51.2% vs 43%, respectively, in comparison to non-treated groups. In combination with cisplatin (DDP), either EGFRtargeted NPs + DDP or non-targeted NPs + DDP demonstrated a greater antitumor efficiency level than that of cisplatin alone (70.6% and 58.9% in comparison to that of DDP alone, respectively). Besides, the co-therapy also helped to reduce dramatically the effective dosage of cisplatin with negligible toxicity in comparison with free ciplastin [START_REF] Nascimento | Overcoming cisplatin resistance in non-small cell lung cancer with Mad2 silencing siRNA delivered systemically using EGFR-targeted chitosan nanoparticles[END_REF][START_REF] Nascimento | Biodistribution and pharmacokinetics of Mad2 siRNA-loaded EGFR-targeted chitosan nanoparticles in cisplatin sensitive and resistant lung cancer models[END_REF]. In another effort to combine drug and gene delivery, Steinborn et al co-delivered methotrexate (MTX) and siRNA using cationic liposomes functionalized with GE11 peptides. As expected, with the help of EGFR-targeting properties from GE11 peptides, in EGFR-positive and drug-resistant cancer cell lines (KB cells), an enhanced cellular internalization level and improved antitumor activities were observed for GE11modified NPs compared to free drugs or non-targeted NPs [START_REF] Steinborn | Epidermal growth factor receptor targeted methotrexate and small interfering RNA co-delivery[END_REF]. Liang et al used GE11-binding amphiphilic peptides to construct their ultra-stable self-assembling peptide nanovesicles. Anti-luciferase siRNA transfection efficiency was twice and five times higher in EGFRpositive SMMC-7721 and Eca-109 cells, respectively, when GE11-conjugated nanovesicles were used instead of GE11-free NPs. In contrast, no observable difference between these nanovesicles was recorded for EGFR-negative cells (SGC-7901 cells). These results demonstrated that GE11 conjugation helped selectively deliver siRNA into cells with high EGFR expression. Furthermore, this system can be used to co-deliver doxorubicin and siRNA to EGFR-overexpressed cancer cells. Indeed, the rate of tumor growth in nude mice treated with drugs/genes co-delivered with targeted NPs dramatically decreased compared with that of mice treated with non-targeted NPs, whereas the free drugs or genes exhibited a negligible antitumor activity [START_REF] Liang | Development of self-assembling peptide nanovesicle with bilayers for enhanced EGFRtargeted drug and gene delivery[END_REF].

All aforementioned studies have demonstrated the great potential of NPs functionalized with EGFR ligands in gene delivery. However, their application remains limited to a few kinds of NPs and a few types of nucleic acids. Hence, there remains so much room for improvement as regards the development of gene delivery with EGFR-targeted nanomedicines. Moreover, as the mechanism of gene therapy with nanomedicines is not completely clarified, the intracellular trafficking of such NPs also needs to be studied in depth to provide a new and efficient method for cancer treatment.

Phototherapy

In addition to traditional therapies, phototherapy has recently drawn increasing attention in cancer treatment due to its remarkable benefits, especially in terms of safety compared to other methods. Phototherapy application can be classified into two main branches including photodynamic and photothermal therapies (Figure 1). EGFR-targeted nanomedicines have been applied to phototherapy and demonstrated excellent results in cancer treatment.

Figure 1. Photodynamic therapy (PDT) and photothermal therapy (PTT) with EGFR-targeted nanomedicines in cancer treatment

Photodynamic therapy with EGFR-targeted nanoparticles

Photodynamic therapy (PDT) employs light sensitive molecules, which once exposed to a specific wavelength, will produce cytotoxic Reactive Oxygen Species (ROS) [START_REF] Lim | Nanophotosensitizers toward advanced photodynamic therapy of Cancer[END_REF]. PDT is a minimally invasive therapy approved for the treatment of carcinoma because photosensitizers (PS) are only cytotoxic when they are activated in tumor regions. This property is its key advantage compared to conventional chemotherapy. PDT efficacy has been clinically tested in various kinds of cancer and was found to have limited side effects and a low risk of resistance [START_REF] Master | EGFR-mediated intracellular delivery of Pc 4 nanoformulation for targeted photodynamic therapy of cancer: in vitro studies[END_REF]Pawar and Prabhu, 2019a). However, the poor solubility and low tumor specificity of PS entail many issues in PDT application such as risks of phototoxicity, photosensitivity and an undesirable pharmacokinetic profile [START_REF] Master | EGFR-mediated intracellular delivery of Pc 4 nanoformulation for targeted photodynamic therapy of cancer: in vitro studies[END_REF]Pawar and Prabhu, 2019a). One of the most promising ways to overcome these challenges is targeting nanoparticles with biological ligands such as anti-EGFR moieties. On the one hand, the presence of anti-EGFR ligands on NPs can help PS to better internalize into cancer cells, which ensures a sufficient quantity of PS for efficient activities and resolves the problem of low photosensitivity. On the other hand, the selective uptake only in cancer cells and not in healthy cells can reduce the risk of phototoxicity for EGFR-targeted NPs. As an example, Tsai et al constructed EGFR-targeted chitosan nanoparticles to deliver curcumin, a photosensitizer, into gastric cancer cells. In this study, the chitosan NPs were decorated with EGF proteins to target EGFR-overexpressed gastric cancer cell lines (MKN45). The cytotoxicity caused by PDT with the targeted NPs was approximately four times higher than that of non-targeted NPs, with IC50 values at 3.4 µM and 11.9 µM, respectively. In contrast, similar IC50 values (13.1 µM and 12.8 µM, respectively) were observed for these NPs in EGFR-negative cells (GES), suggesting a selective uptake of these targeted NPs for EGFR-positive cells. More interestingly, the authors revealed that EGFRtargeted NPs exhibited a better PDT effect than folate-targeted NPs in MKN45 cells by a factor of two. This could be due to different expression levels of EGFR and folate receptors in MKN45 and confirmed the importance of choosing the appropriate targeting ligands to ensure treatment efficiency [START_REF] Tsai | EGFR-targeted photodynamic therapy by curcumin-encapsulated chitosan/TPP nanoparticles[END_REF]. In another study, Chu et al developed multifunctional micelles with GE11 peptides to target EGFR and better deliver the photosensitizer chlorin e6 (Ce6) into colorectal cancer cells. Thanks to GE11 specificity, Ce6 uptake from Ce6/GE11-micelles in EGFR-positive cancer cells (HCT-116 cells) was 1.7 times higher than that from non-targeted micelles, whereas no significant difference in Ce6 uptake was observed for targeted and non-targeted NPs in EGFR-negative cancer cells (SW620 cells). Consequently, Ce6 released from targeted NPs exhibited a better cancer cell elimination activity than its counterpart [START_REF] Chu | pH-Responsive Nanophotosensitizer for an Enhanced Photodynamic Therapy of Colorectal Cancer Overexpressing EGFR[END_REF]. Liu et al also developed EGFR-targeted polymeric micelles with anti-EGFR nanobodies as selective photodynamic therapy in head and neck cancer. The targeted NPs presented not only their selectivity as their binding affinity with EGFRoverexpressing A431 cells was higher than that with low-EGFR expressing HeLa cells, but also higher photocytotoxicity (four times) on A431 cells compared to non-targeted micelles [START_REF] Liu | EGFR-Targeted Nanobody Functionalized Polymeric Micelles Loaded with mTHPC for Selective Photodynamic Therapy[END_REF].

For non-targeted NPs, the non-specific pathway is the only way for them to enter cancer cells. This process depends on time-resolved cell membrane-mediated processes. In vivo, accumulated NPs via the passive pathway (EPR phenomenon) can re-enter the blood stream via the diffusion mechanism over time, resulting in decreased drug concentrations at tumor tissues. The active targeting that highly upregulates internalization processes via specific receptor-mediated pathways can either prevent the re-diffusion of drug in vivo or promote rapid intracellular uptake. For instance, Master and colleagues utilized GE11-decorated polymeric NPs (PEG-PCL NPs) to encapsulate a PDT agent (Pc 4) for a better delivery, rapid intracellular uptake and enhanced PDT activity in EGFR-positive cancer cells (A431). In comparison with non-targeted ones, the targeted NPs presented not only a higher but also a faster intracellular uptake of drugs into cancer cells. As a result, the targeted NPs enhanced the PDT-induced cytotoxicity in cancer cells within a shorter time after PDT application (10 minutes vs 5 hours) [START_REF] Master | EGFR-mediated intracellular delivery of Pc 4 nanoformulation for targeted photodynamic therapy of cancer: in vitro studies[END_REF]. This result is promising especially for further application in vivo and in clinical trials. Currently, the interval for various current clinical formulations of PS such as Cremophor is unpredictable and varies between 1-4 days. This long waiting time can hinder the treatment process and reduce its efficacy especially in the cases of certain aggressive types of tumors that require repeated locoregional therapy after primary treatment such as head-and-neck cancer. At this point, targeted nanomedicines with anti-EGFR ligands offer an additional advantage.

Despite its potency and safety, PDT clinical applications are limited to the treatment of superficial cancers due to the low tissue penetration ability of visible light. To address this issue, the application of deep tissue penetrating near infrared (NIR) excitable upconversion NPs loaded with suitable PDT agent has shown great potential in improving the penetration depth of conventional PDT. The upconversion NPs are able to convert low-energy NIR light to high-energy visible or ultra-violet (UV) light. The up-converted light excites subsequently the PS attached on the surface of the NPs and exhibits the PDT activity. In addition, the treatment efficacy of these NPs in PDT therapy can be promoted if they are decorated with targeting ligands such as anti-EGFR moieties. Lucky et al employed this strategy for better PDT application against a non-superficial cancer (head and neck cancer) by functionalizing their upconversion NPs with EGFR targeting ligands. The authors chose EGFR-affibodies as targeting agents due to their small size and low immunogenicity. As a result, the affibody-decorated NPs were internalized much more rapidly and efficiently (3.8 times) than the unmodified NPs in EGFR-overexpressed cancer cells (A431 and H596 cells), whereas no significant difference between the targeted and the non-targeted NPs was observed in EGFR-negative cells (MCF-7 and HepG2 cells). In in vivo model, the targeted NPs exhibited higher PDT toxicity in EGFRpositive cells than in EGFR-negative cells (35% vs 0%), a significant delay in tumor growth, an enhanced survival rate, and long-term safety (up to 120 days) [START_REF] Lucky | In vivo Biocompatibility, Biodistribution and Therapeutic Efficiency of Titania Coated Upconversion Nanoparticles for Photodynamic Therapy of Solid Oral Cancers[END_REF].

All these studies demonstrated that targeting nanomedicines with EGFR-targeting ligands could provide efficient strategies to promote PDT application in EGFR-overexpressing cancers. Compared to conventional PDT, this strategy offers outstanding advantages such as enhancement in drug cellular uptake in cancer cells for a sufficient drug dose, and faster internalization resulting in a shorter druglight interval. Further studies on this application in other EGFR-positive cancers, especially nonsuperficial ones, may offer promising methods for better treatment of these so-called "incurable" diseases.

Photothermal therapy with EGFR-targeted nanoparticles

In addition to PDT, photothermal therapy (PTT) is the second type of phototherapy, which is based on the use of biocompatible and special materials (PTT agents) to convert light energy into heat energy, resulting in cancer cells destruction. Non-invasive and localized treatments are PTT advantages compared to traditional chemotherapy. Due to minimal absorption by the human body, near infrared (NIR) light (700-900 nm) is normally employed in PTT. As one of the key parameters in PTT is the choice of the PTT agent, such agents need to have a good photostability region, photothermal efficiency in NIR and a desirable safety profile. A variety of PTT agents are currently available including carbon-nanotubes, quantum dots, graphene oxides, and metal NPs [START_REF] Kwon | Proteinticle/Gold Core/Shell Nanoparticles for Targeted Cancer Therapy without Nanotoxicity[END_REF]Pawar and Prabhu, 2019b).

One of the most important challenges that hinders PTT clinical applications is its lack of specificity. This phenomenon may cause severe side effects and reduce drug tumor accumulation, leading to insufficient PTT efficiency, especially in the case of AuNPs. In fact, PTT using AuNPs is normally limited due to its toxicity that originates from the long retention time of AuNPs in the body (Table 1).

AuNPs with a size superior to 40 nm can cause severe tissue damages in the liver, spleen, and kidney after 28 days post intravenous injection. On the one side, the active targeting of AuNPs can attenuate this phenomenon by selective delivery into tumor tissues and their reduced accumulation in healthy ones. On the other side, the amount of AuNPs required to achieve efficient PTT therapeutic effects can be reduced with the help of these EGFR-targeted nanomedicines. Several studies have proven that this strategy may be the right solution to overcome these issues. As an example, Kwon et al developed proteinicle/gold core/shell nanoparticles for targeted and nanotoxicity-free cancer therapy. The core of NPs is made of the hepatitis B virus capsid decorated with EGFR-affibodies for active targeting and dotted with many small gold NPs for PTT activity. The anti-EGFR affibodies helped the NPs to be effectively delivered into EGFR-expressing breast cancer cells (MDA-MB-468 cells) and exhibited increased tumor cell necrosis and remarkable tumor size reduction after NIR irradiation compared to AuNPs. In addition, while non-targeted AuNPs caused extensive and histological damages to the liver and kidney of healthy mice, no visible macroscopic and histological change was recorded in major organs in healthy mice for 3 weeks post-injection in the case of targeted NPs [START_REF] Kwon | Proteinticle/Gold Core/Shell Nanoparticles for Targeted Cancer Therapy without Nanotoxicity[END_REF]. In another effort to make use of targeted PTT in glioma, Lu et al functionalized their Fe3O4@Au NPs with cetuximab. In EGFR-overexpressed (U251 tumor) animal cancer models, targeted NPs showed higher tumor inhibitory effects than non-targeted NPs. In addition, targeted NPs did not affect the viability of cancer cells when hyperthermia was not applied. As soon as hyperthermia was applied, enlarging zones of necrosis following the diffusion of targeted NPs were observed, resulting in tumor growth suppression [START_REF] Lu | Fe3O4@Au composite magnetic nanoparticles modified with cetuximab for targeted magneto-photothermal therapy of glioma cells[END_REF]. In another effort to enhance the effectiveness of PTT in superficial bladder cancer, Chen et al functionalized their AuNPs with anti-EGFR scFv. The targeted NPs exhibited sufficient thermal energy to kill urothelial carcinomas (MBT-2 cells) both in vitro and in vivo. Under the same conditions for MBT-2 cells, the NPs conjugated with EGFR-scFv were able to damage cancer cells at a 3-fold lower energy level than that required for non-targeted NPs. As a result, the lower energy required by the NPs helped to reduce possible toxicity related to PTT on healthy tissues in vivo [START_REF] Chen | Photo-thermal therapy of bladder cancer with Anti-EGFR antibody conjugated gold nanoparticles[END_REF].

Interestingly, a combination of PTT, chemotherapy and EGFR targeting can be achieved within a single nanocarrier. He et al developed PLGA/polydopamine core-shell NPs for cancer photothermal therapy and chemotherapy delivery. Compared to other conventional NPs such as AuNPs, the first advantage of this nanosystem is its biodegradability. In addition, with the help of cetuximab as the active targeting ligand, the targeted NPs could effectively internalize into head and neck cancer cells, convert NIR light to heat, and trigger drug release from the PLGA core. As expected, in combination with doxorubicin, targeted NPs presented a better tumor suppression rate without drug-associated side effects than that of non-targeted NPs. On the other hand, the EGFR targeted NPs could quickly elevate the temperature of tissues to 55 °C within 2 min and maintain this temperature during the whole course of NIR irradiation [START_REF] He | Mussel-inspired PLGA/polydopamine coreshell nanoparticle for light induced cancer thermochemotherapy[END_REF].

Although the potential of targeting nanomedicines with anti-EGFR ligands in PTT has clearly been confirmed for a broad range of cancers, several critical parameters need to be carefully considered in the design steps. The first problem is the photochemistry from UV wavelengths that may be detrimental to the stability and recognition properties of the conjugated biomolecules. Thus, protecting strategies are required to preserve the biological functions of these ligands. As an example, Silva and colleagues protected the functionality of EGF proteins conjugated on their AuNPs from UV wavelengths using oleic acid (OA) and hyaluronic acid (HA). The results showed that the presence of polymeric-coated AuNPs helped reduce or even avoid the formation of photoproducts when EGF was exposed to UV light, protecting the structure and function of EGF proteins. In addition to EGF protection capacity, the HA present on NPs surface was shown to effectively provide other internalization pathways than EGFR-mediated endocytosis due to its binding ability to the CD44 receptor [START_REF] Silva | EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy[END_REF]. Besides, the high temperature emitted by PTT can also have a negative impact on the biological functions of anti-EGFR ligands. Especially, if NPs are not accumulated enough in tumor tissues and photo-irradiation is applied, such a high temperature can deactivate the targeting properties of NPs in blood stream and lead to both insufficient therapeutic activity and offsite effects. Consequently, the interval time between NPs administration and photo-irradiation needs to be evaluated with more accuracy.

EGFR-nanomedicines for diagnostic and theranostic applications

Diagnostic applications

In addition to perspective applications in the delivery of therapeutic agents, diagnostic tools developed with EGFR-targeted NPs also provide a myriad of benefits. Thanks to its targeting delivery and high loading capacity of different molecules, this strategy can enable efficient accumulation of imaging agents in tumors, increase the signal-to-noise ratio and/or the intrinsic imaging functionality of NPs as in the case of SPIONs, AuNPs or Quantum dots (QDs). Many studies have indicated that EGFR-targeted NPs are useful not only for early detection of related cancers but also for therapy response monitoring during treatment, contributing actively to the final treatment efficacy, allowing better drug monitoring. Therefore, a large variety of EGFR-targeted nanomedicines have been developed for Magnetic Resonance Imaging (MRI), nuclear imaging, and optical imaging used in cancer (Fig. 2).

Fig. 2. EGFR-targeted nanomedicines used for cancer diagnosis

Targeted MRI agents

Due to its noninvasive and nondestructive nature and multidimensional tomographic capabilities, Magnetic Resonance Imaging (MRI) has emerged as one of the most powerful imaging modalities in the diagnosis and clinical staging of cancer. Due to its noninvasive and nondestructive nature and multidimensional tomographic capabilities, Magnetic Resonance Imaging (MRI) has emerged as one of the most powerful imaging modalities in the diagnosis and clinical staging of cancer. However, one of the biggest limitations of MRI is its low contrast that results in low sensitivity. To enhance the contrast in MRI, several kinds of NPs can be applied. In EGFR-targeted NPs for MRI, thanks to their innate magnetic properties, their biocompatibility and facile synthesis, superparamagnetic iron oxides NPs (SPIONs) have become the most widely used for MRI [START_REF] Leary | Nanoparticles for multimodal in vivo imaging in nanomedicine[END_REF] (Table 1).

Nevertheless, for early tumor detection, MRI application with SPIONs is limited because of its lowsignal sensitivity and its specificity. To address this issue, the use of EGFR-targeted NPs as contrast agents presents potential properties for improving the specificity and sensitivity of related-cancers' imaging. As an example, Tseng et al developed cetuximab-conjugated SPIONs for MRI in EGFRoverexpressing tumor cells. Around 31 antibody molecules were conjugated to each SPION, which helped the latter to specifically bind to EGFR-positive tumor cells (A431 cells) and enhanced the contrast on MRI in comparison to free-antibody SPIONs. In addition, antibody-dependent cellmediated cytotoxicity of cetuximab was observed for antibody-conjugated SPIONs, resulting in a significant increase in apoptosis. These results showed a promising strategy for early detection with MRI and the treatment of EGFR-overexpressing tumor cells [START_REF] Chu | Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy[END_REF]. In another study, to identify patients who could benefit from EGFR-targeted therapies, Chen and colleagues also constructed cetuximab-conjugated SPIONs for their targeted MRI. While Tseng et al used dextran to cover their SPIONs, whose net charge remained and influenced their non-targeted internalization, Chen et al covered their SPIONs with a lipid coating to mask SPION charges and allow further antibody conjugation via maleimide chemistry. However, only 1.5 cetuximab molecules were conjugated per NP, much lower than the number reported in the study of Tseng et al (31 molecules). Despite a lower number of EGFR-targeting ligands, the targeting efficiency of the targeted SPIONs was confirmed and the targeted NPs were able to distinguish EGFR-positive cancer cells (U-251 cells) from U-87MG cells with low EGFR expression [START_REF] Chen | Identification of epidermal growth factor receptor-positive glioblastoma using lipid-encapsulated targeted superparamagnetic iron oxide nanoparticles in vitro[END_REF]. These results have clarified the advantage of the presence of EGFR-targeting ligands even in a small quantity on the active targeting of the finalized NPs. However, the over-presence of such ligands might also have a negative impact on the colloidal properties of the finalized NPs. Therefore, in order to have optimal efficacy, this aspect requires further investigations.

In addition to single imaging and targeting, dual imaging and double targeting are feasible with EGFRtargeted NPs. Wu et al carried out an interesting study in which a bispecific antibody fragment able to target both EGFRs and HER2s was developed. This fragment was constructed by connecting EGFR-scFv (from Erbitux) and HER2-scFv (from Herceptin) via a (Gly4Ser)3 linker. The obtained fragments were subsequently conjugated onto SPIONs, which were labeled beforehand with the NIR fluorescence dye. In vitro results with high HER2 and moderate EGFR expression cancer cells (SK-BR-3) showed that the dual-targeted SPIONs internalized better than the non-targeted or single-HER2 targeted SPIONs. Furthermore, the cellular uptake of the dual-targeted NPs into both HER2+/EGFR-MCF-7 cells and HER2-/EGFR+ MDA-MB-231 cells was higher compared to that of the non-targeted NPs. These results indicated that the dual-targeted NPs possess a high binding activity towards cancer cells with high HER2/EGFR expression. Remarkably, in in vivo MRI, the contrast enhancement of the dual-targeted NPs with SK-BR-3 cells (high HER2 and moderate EGFR expression) was 61.5 times higher than in HER2-/EGFR-cancer cells (Colo-205 cells) at 24 hours after IV injection at a dose of 10 mg/kg [START_REF] Wu | Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu-and EGFR-Expressing Tumors[END_REF].

Targeted nuclear imaging agents

Nuclear medicine is a branch of medicine that uses radiation to provide functional and anatomical data from a specific organ. The application of nuclear medicine in cancer treatment is considered the most powerful technology available for early cancer detection. Several nuclear imaging techniques are available for cancer imaging including Positron Emission Tomography (PET) and Single Photo Emission Computed Tomography (SPECT). Currently, the application of nanotechnology in nuclear medicine is drawing increasing attention and presents remarkable advantages such as its ability to deliver a large quantity of imaging agents and to achieve better sensitivity imaging, along with the possibility to perform multimodality imaging by delivering different kinds of imaging agents [START_REF] Welch | The Advantages of Nanoparticles for PET[END_REF]. In addition to the above advantages, NPs with active targeting ligands such as EGFR can enhance the selective delivery of imaging agents and result in a better diagnosis tool. Two strategies can be applied to deliver nuclear imaging agents into cancer tumors including i) radiolabeling of the NPs' surface and ii) radiolabeling of the active targeting ligand, and its subsequent conjugation onto NPs (Fig. 2B).

Currently, the direct conjugation of imaging agents onto EGFR-targeting ligands is widely used for EGFR-targeting nuclear imaging. Among the available ligands, cetuximab radiolabeled with 99m Tc, 111

In for SPECT imaging or 64 Cu, 89 Zr, 86 Y for PET has been largely utilized [START_REF] Eiblmaier | Correlating EGFR expression with receptor-binding properties and internalization of 64Cu-DOTAcetuximab in 5 cervical cancer cell lines[END_REF][START_REF] Menke-Van Der Houven Van Oordt | 89Zr-cetuximab PET imaging in patients with advanced colorectal cancer[END_REF][START_REF] Nayak | PET imaging of HER1-expressing xenografts in mice with 86Y-CHX-A''-DTPA-cetuximab[END_REF]. However, antibody-based imaging agents confront several limitations due to their slow penetration rate into the tumor, leading to a low tumor-to-blood ratio, low contrast and therefore low-sensitivity imaging. Other targeting ligands such as affibodies, repebodies or GE11 peptides may be alternatives to antibodies. As an example, Burley et al developed a constructed 89 Z-EGFR affibody for targeted PET imaging in head and neck cancer. PET imaging data in this study showed a clear differentiation in tumor accumulation between tumors with varying EGFR expression levels, demonstrating the benefit of using anti-EGFR affibodies [START_REF] Burley | Affibody-Based PET Imaging to Guide EGFR-Targeted Cancer Therapy in Head and Neck Squamous Cell Cancer Models[END_REF]. In another study, Pyo and colleagues used EGFR-repebodies labeled with 64 Cu for PET imaging in EGFR-expressing tumors. The 64 Cu-repebodies exhibited a specific uptake in EGFRpositive cancer cells (H1650 cells), which was over 4 times higher than that in EGFR-negative cells (SW620 cells). Moreover, these targeted complexes demonstrated a rapid uptake in EGFR-positive tumors within 1h and with high tumor-to-background ratios, and therefore, were deemed potential as novel EGFR imaging agents for PET [START_REF] Pyo | 64 Cu-Labeled Repebody Molecules for Imaging of Epidermal Growth Factor Receptor-Expressing Tumors[END_REF]. In another study of Paiva et al, the authors introduced 64 Cu-NOTA (2,2',2''- (1,4,7-triazacyclonane-1,4,7-triyl)triacetic acid) onto the tyrosine residues of GE11 peptides using azo coupling. These labeled-GE11 peptides were then added onto polymeric NPs for EGFR targeting. In vivo analysis of 64 Cu-labeled polymeric NPs with PET imaging demonstrated a higher accumulation level in HCT116 colon tumors for GE11-functionalized NPs than non-targeted NPs. Moreover, increased tumor-to-muscle ratios for both targeted and non-targeted NPs over 48h were also observed in comparison to the case of free radionuclides [START_REF] Paiva | Synthesis and Analysis of 64 Cu-Labeled GE11-Modified Polymeric Micellar Nanoparticles for EGFR-Targeted Molecular Imaging in a Colorectal Cancer Model[END_REF]. These studies have not only presented the interest of using EGFR-targeting for nuclear imaging in related cancer diagnosis but also inspired scientists in the field of nanotechnology to functionalize their NPs with anti-EGFR ligands for EGFR-targeting diagnosis with nuclear medicine, and more importantly, for the development of multifunctional or "theranostic" NPs. As an example, Gill et al conjugated DTPA-modified EGF proteins to their polymeric NPs for a bimodal activity including nuclear imaging with 111 Indium and radiotherapy with a radiosensitizer Ru(phen)2(tpphz) 2+ (Ru1). The targeted NPs accumulated preferentially by EGFR-positive esophageal cancer cells (OE21 cells) and exerted efficient radiotoxicity. Furthermore, this radiotoxicity was enhanced by the combinational activity between 111 Indium and Ru1. Besides, substantially attenuated radiotoxicity of targeted NPs for esophageal cancer cells (OE33 cells) and normal human fibroblast (HFF-1 cells) with a normal EGFR level in comparison to OE21 cells confirmed the advantage of the EGFR-targeting strategy [START_REF] Gill | 111 In-labelled polymeric nanoparticles incorporating a ruthenium-based radiosensitizer for EGFR-targeted combination therapy in oesophageal cancer cells[END_REF].

Targeted optical imaging agents

Optical imaging agents delivery with EGFR-targeted NPs can be achieved via three main strategies including i) direct conjugation of imaging agents such as organic fluorescent dyes onto the targeted NPs surface, ii) utilization of fluorescent-labeled targeting ligands, or iii) utilization of nanomaterials with optical intrinsic properties (Fig. 2C).

For the first strategy, these EGFR-targeted NPs can help to distinguish EGFR-positive tumors from other kinds of cancers or normal tissues and enhance the signal of loaded imaging agents. As an example, in the study of Ryu et al, the authors conjugated a near-infrared (NIR) fluorophore (Cy5.5) and a quencher (BHQ-3) onto EGF proteins. The quencher in these nanoprobes helped to reduce background signals and boost strong fluorescence intensity. The EGFR-targeted and fluorescent nanoprobes exhibited a stronger in vivo fluorescence signal in EGFR-overexpressing tumors (MDA-MB-468 cells) than that in EGFR-negative tumors (MDA-MB-436 cells). Furthermore, in comparison with Cetuximab-Cy5.5, the EGF-decorated nanoprobes showed a much faster tumor accumulation signal that was explained by the better tissue permeability of low-molecular weight EGF proteins [START_REF] Ryu | In vivo fluorescence imaging for cancer diagnosis using receptortargeted epidermal growth factor-based nanoprobe[END_REF]. In another study of Chariou and colleagues, virus-based NPs conjugated with GE11 peptides were used to carry imaging agent-Fluor 647. In different cancer cells with EGFRoverexpression including A431, HT-29 and MDA-MB-231, the presence of GE11 helped to increase the cellular uptake of these NPs in an EGFR-expression level-dependent way. In contrast, no difference in cell uptake between targeted and non-targeted NPs was observed in EGFR-negative ductal breast carcinoma (BT-474 cells) [START_REF] Chariou | Detection and Imaging of Aggressive Cancer Cells Using an Epidermal Growth Factor Receptor (EGFR)-Targeted Filamentous Plant Virus-Based Nanoparticle[END_REF].

Despite their effectiveness, conventional labeling methods using organic fluorophores or fluorescent proteins confront several restrictions such as photobleaching (photochemical degradation of the dye) or low quantum yields. Therefore, NPs made of innately fluorescent materials such as quantum dots (QDs) are preferred. Moreover, the possibility of surface modification allows the introduction of targeting ligands such as anti-EGFR ones onto QD (Table 1). A good example for this phenomenon is illustrated in the study of Lee et al. The authors conjugated cetuximab onto QDs and compared it with Cy5-conjugated cetuximab. As expected, QDs-cetuximab clearly exhibited higher photostability than that of Cy5-cetuximab, whose fluorescent signal decreased about 50% after 30 min. Besides, cetuximab conjugated on QDs presented a high EGFR targeting property, as reflected by an increase by a factor of two in fluorescence intensity in EGFR-positive A549 cells compared to EGFR-negative CT26.WT cells [START_REF] Lee | Characterization and Cancer Cell Specific Binding Properties of Anti-EGFR Antibody Conjugated Quantum Dots[END_REF]. Although QDs are widely used as imaging agents, they suffer from the presence of cytotoxicity linked to heavy metals, photoblinking (physically on/off state of QDs) and a short blood circulation time [START_REF] Faucon | Bioconjugated fluorescent organic nanoparticles targeting EGFR-overexpressing cancer cells[END_REF]. Therefore, the functionalization of QDs with anti-EGFR ligands is a feasible solution to overcome these problems. As an example, Zhang et al conjugated anti-EGFR affibodies onto Ag2S quantum dots for photoacoustic imaging in EGFRoverexpressed tumors (A431 tumors). In vitro, the presence of EGFR-affibodies was shown to increase the cellular uptake of the obtained NPs by a factor of two in A431 cells. In A431 tumorbearing mice, after IV injection (10 mg/kg) of QDs, the targeted QDs not only presented a much higher fluorescence signal than the non-targeted NPs in tumor regions but also exhibited a longer half-life compared to bare QDs (3.21±0.12 h vs 11.8±1.4 min), and around 30% of the targeted NPs remained in blood after 8 h of injection. This prolonged blood circulation time of QDs due to the presence of anti-EGFR affibodies may be of great interest when it comes to monitoring the effectiveness of cancer treatment. Moreover, the targeted QDs were shown to be accumulated significantly in tumors compared to those in healthy tissues such as the kidney and lung, demonstrating their biocompatibility and suitability for in vivo applications [START_REF] Zhang | Affibodyfunctionalized Ag 2 S quantum dots for photoacoustic imaging of epidermal growth factor receptor overexpressed tumors[END_REF].

All previous studies have demonstrated the promising application of EGFR-targeted NPs in cancer diagnosis that involves various kinds of diagnostic methods (Table 3). These strategies represent a potential approach for the early detection of tumors, which resulted in an effective treatment plan. However, as the success of these strategies is strictly dependent on the choice of targeting ligands, the role of EGFR in the early stages of tumor growth needs to be clearly understood.

Theranostic tools

The term "theranostics" was first introduced in 2002 and refers to co-delivering therapeutic agent platforms (single use or combination of drugs/genes/drug + gene/ + photothermal probes/drugs + photosensitizers) and imaging probes (ultrasound/magnetic contrast/fluorescent agents). These multifunctional systems can deliver therapeutic agents, detect cancer cells, track the intracellular localization of drugs, and monitor the efficacy of treatment. Targeting NPs with anti-EGFR ligands are among the most widely used theranostic tools for cancer treatment.

EGFR-targeted nanoparticles for theranostic purposes have been developed and presented promising results. These targeting NPs can spontaneously deliver drugs + optical imaging agents, PTT agents + nuclear imaging, drugs + MRI agents, etc. As an example, Wang et al designed quantum dots (QDs)based polymeric micelles conjugated with anti-EGFR nanobodies and loaded with aminoflavone for EGFR-overexpressing breast cancer theranostics. On the one hand, the NIR fluorescence of QDs allowed in vivo tracking of these NPs' biodistribution. On the other hand, EGFR-nanobodies on QDs helped to improve the cellular uptake (67 times higher) and the cytotoxicity of aminoflavone in EGFRpositive breast cancer cells (MDA-MB-468 cells) in comparison with non-targeted NPs. Moreover, a better tumor accumulation resulting in better tumor inhibition without systemic in vivo toxicity was obtained with the targeted NPs [START_REF] Wang | Quantum-Dot-Based Theranostic Micelles Conjugated with an Anti-EGFR Nanobody for Triple-Negative Breast Cancer Therapy[END_REF]. In another study of Kao and colleagues, novel theranostic NPs for targeted radionuclide therapy and radioimmunotherapy in lung cancer was achieved with 131 I-labeled immuno-cetuximab-decorated AuNPs. With the help of around 124 cetuximab molecules per NP, the targeted NPs demonstrated an enhancement in endocytosis and cytotoxicity in high EGFR-expressed lung cancer cells (A549 cells). Furthermore, the microSPECT/CT images also confirmed a better tumor accumulation for the targeted NPs compared to the nontargeted ones [START_REF] Kao | Evaluation of EGFR-targeted radioimmuno-gold-nanoparticles as a theranostic agent in a tumor animal model[END_REF]. Kim et al formulated anti-EGFR aptamers-conjugated lipid NPs for siRNA and quantum dots co-delivery into triple negative breast cancer tumors. The finalized aptamers-conjugated NPs were able to effectively and specifically bind to MDA-MB-231 cells, whereas none of these results was observed in EGFR-negative MDA-MB-453 control cells. Moreover, in vivo fluorescence imaging of tumor-bearing mice treated with the targeted NPs showed a higher tumor accumulation and a better gene delivery for functionalized NPs than that of non-targeted NPs [START_REF] Kim | Cancer-targeted Nucleic Acid Delivery and Quantum Dot Imaging Using EGF Receptor Aptamer-conjugated Lipid Nanoparticles[END_REF].

Besides the dual-modality, the combination of more than two therapies and diagnosis methods can be achieved with EGFR-targeted nanomedicines. As an example, Liu et al developed multifunctional cetuximab-conjugated liposomes, encapsulating doxorubicine, AuNPs and SPIONs for an efficient combination of chemotherapy, photothermal therapy and MR imaging in EGFR-positive tumors. As a result, efficient cetuximab-mediated targeting with a higher drug uptake in A431 cells was observed for the antibody-decorated NPs compared to the non-targeted NPs. Moreover, after laser irradiation, the targeted NPs exerted higher cytotoxicity towards cancer cells than doxorubicin alone, revealing a synergistic effect between PTT and chemotherapy. In vivo, the targeted NPs promoted tumor destruction by laser activation without major morphological damages to normal tissues. The combination of MR and optical imaging also showed that the targeted NPs selectively targeted A431 tumor cells [START_REF] Liu | Development of a novel thermal-sensitive multifunctional liposome with antibody conjugation to target EGFRexpressing tumors[END_REF]. In another study, Li et al combined photodynamic therapy, immunotherapy, optical and MR imaging in their novel EGFR-targeted liposomal nanohybrid cerasomes. The theranostic NPs consisted of porphyrin-containing liposomes decorated with cetuximab, conjugated with the NIR dye for optical detection, DOTA-Gd as MRI contrast, and programmed death ligand-1 (PD-L1) as an adjuvant agent. The dual-modality imaging with the targeted liposomes provided better and selective images of EGFR-positive tumors than the non-targeted NPs. In addition, the antibody-decorated NPs were more effective against tumor growth compared to non-targeted NPs after laser irradiation and PD-L1 immunotherapy [START_REF] Li | EGFR-targeted liposomal nanohybrid cerasomes: theranostic function and immune checkpoint inhibition in a mouse model of colorectal cancer[END_REF].

These studies suggested a perspective approach to combine several therapies and imaging strategies in just one nanosystem. Nevertheless, the introduction of so many components onto NPs may lead to negative impacts on therapeutic or targeting functions of these components. Therefore, a careful consideration is required to have an appropriate design of theranostic NPs.

Conclusions

It is becoming more and more evident that EGFR is a potential target for the development of targeted nanomedicines in a wide range of cancers. Although such anticancer nanomedicines are still in early stages of development, their potential to overcome the current issues related to adjuvant treatment strategies and their innovative applications in diagnosis or even theranostics have been confirmed. Especially, their perspective results in certain drug-resistant cancers such as hypoxic ones provide a promising strategy of treatment and are worthy of further investigations. According to different clinical needs, various kinds of nanomaterials (liposomes, dendrimers, polymeric, micelles and inorganic NPs) and anti-EGFR ligands (EGF proteins, antibodies, antibodies fragments, peptides, aptamers) for EGFR-targeted nanomedicines have been successfully designed and demonstrated promising results both in vitro and in vivo. Nevertheless, an EGFR-targeted nanomedicine should be appropriately designed to tackle several issues as follows.

i) the type of cancer in which such targeted nanomedicines can offer additional advantages over nontargeted NPs. To be precise, EGFR-overexpression has been reported in a wide range of cancers but this level of EGFR-expression may not be sufficient to bring additional benefits from active targeting over the passive one. In addition, in case EGFRs are abundant in a kind of cancer, the therapeutic activities of conjugated anti-EGFR ligands on NPs seem to be interesting and may improve treatment efficacy. Therefore, this parameter requires deeper understanding of EGFR's involvement in each kind of cancer.

ii) the choice of nanomaterials, EGFR-targeting moieties, their binding affinity, density, and conformation on NPs are highly critical and directly affect the effectiveness of the obtained NPs.

With the help of advances in molecular engineering and nanotechnology, numerous types of nanomaterials and targeting ligands are available, which may lead to a dilemma in the design of nanomedicines. Therefore, the selection of these components should be based on the type of cancer and the needs of patients in terms of the loaded drug, and possibility for cancer detection or monitoring.

iii) even though EGFR-targeted nanomedicines have proven their improvements in terms of toxicity and side effects compared to conventional NPs, they might remain in the body for a long period of time. Therefore, further investigation on their long-term safety is needed before clinical trials are carried out.

iv) the large-scale production of these nanomedicines might be costly and time-consuming. As a result, these promising strategies remain in the laboratory scale. Studies on the transitional phase between laboratories and the industry are needed to ensure the quality and efficacy of these nanomedicines.

Taken altogether, targeted nanomedicines with anti-EGFR ligands are considered useful, potential and multifunctional tools to improve both the diagnostic and the treatment of cancer. It is highly expected that such promising nanomedicines will become commercialized and available for use in clinical settings in the near future.

Table 1 . Different kinds of NPs with their advantages and challenges used in EGFR targeting nanomedicines

 1

	NPs	Illustration	Structure	Anti-EGFR conjugation	Advantages	Limitations	Ref.
	Liposomes		Lipid bilayer with a central aqueous cavity	Via chemical conjugation or electrostatic interaction for cationic liposomes	Biocompatibility, high drug loading capacity, possibility to load large molecules, surface functionalization	Difficulty to sterilize, low stability	(Alavi et al., 2017; Dong et al., 2018; Riaz et al., 2018; Wang et al., 2020)
						Problems in
						heterogeneity of
	Extracellular vesicles (EVs)		Cell-derived nanosized vesicles made of cellular membrane surrounding an aqueous core	Via electrostatic interaction or chemical conjugation	Enhanced drug cellular uptake, safety, low risk of immunogenicity, increased half-life time of drugs	isolated vesicle populations, negative impacts of isolation methods on vesicle composition
						and properties,
						difficulty in scale-up
				Attached to the monomer of micelles via chemical conjugation	Biocompatibility and biodegradability, long half-life time, high drug stability and drug payload	Sophisticated synthesis that leads to difficulty in purification and large-scale manufacture	(Cheng et al., 2021; Huang et al., 2015; Milane et al., 2011a; Parveen and Sahoo, 2008)
				Mainly via			(Cheng et al.,
	Dendrimers		Artificial macromolecular nanocarriers with well-defined topological structures	electrostatic interaction but also via chemical	High payload capacity, high monodispersity, easy-to-control characteristics	Toxicity due to high positive charges, high-cost production	2011; Chis et al., 2020; Kim et al., 2018; Zhang et al.,
				conjugation			2012)

[START_REF] De Jong | Drug Delivery with Extracellular Vesicles: From Imagination to Innovation[END_REF][START_REF] Pi | Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression[END_REF][START_REF] Surman | Extracellular Vesicles as Drug Delivery Systems -Methods of Production and Potential Therapeutic Applications[END_REF]

Polymeric NPs

NPs made of polymers such as polysaccharides, poly (lactic-coglycolic acid) PLGA, amino acids, proteins (albumin), poly(methy methacrylate), poly(ethylenimines), polyesters, poly(methylene malonates), etc.

Table 2 .

 2 Applications of different kinds of EGFR-targeted nanomedicines in delivering chemotherapy or radiotherapy agents for various kinds of cancers

	Cancer	NPs	EGFR-ligands	API	IV dose (mg/kg)	Advantage of EGFR-targeted NPs compared to non-targeted NPs	Ref.
		Polymeric NPs	Peptides	Docetaxel	-	-Reduction in IC50 in MDA-MB-231 cells (5.1 ng/mL vs 30.1 ng/mL) for non-targeted NPs	(Clemons et al., 2018)
		Polymeric NPs	EGF proteins	Paclitaxel	2	-At 48 h, cell viability at 10.6% vs 18.2% of non-targeted NPs -A 93-fold higher drug tumor accumulation	(Venugopal et al., 2018)
						-Higher cellular uptake into cancer cells
	Breast	AuNPs	Panitumu mab	Radionuclide s ( 177 Lu)	-	-Cellular uptake corresponding to EGFR-expression level -Survival rate of MDA-MB-468 cells was 0.001% vs 8.4% of non-targeted	(Yook et al., 2015)
	(TNBC)					NPs
		Polymeric NPs	Peptides	Aminoflavon e	7		(Brinkman et al., 2016)
		AuNPs	EGF proteins	Radionuclide s ( 111 In)	-	-10-fold higher cellular uptake in MDA-MB-468 than MCF-7 cells -Surviving fraction of 17.1% vs 89.8% for MDA-MB-468 and MCF-7 cells	(Song et al., 2016)
		Polymeric NPs	Cetuxima b	Gemcitabine	-	-Two-fold higher cellular internalization -Enhancement of cytotoxicity (40%)	(Wang and Zhou, 2015)
	Lung (NSCLC)	Mesopor ous silica NPs	Cetuxima b	PLK1-siRNA	-	-Eight-fold higher cellular uptake in EGFR+ cells (A549 and H460 cells) than EGFR-ones (NL20 cells)	(Reda et al., 2019)
		Lipid NPs	EGF proteins	Cisplatin and doxorubicin	5 and 2.5	-Higher tumor accumulation of drugs after 24 h than that of non-targeted or free drugs -Tumor inhibition ratio up to 74.5% with a smaller tumor volume	(Nan, 2019)

-Better cellular uptake in EGFR+ cells (MDA-MB-468 cells) -No difference in cellular uptake in EGFR-cells (BT474 cells) -Stronger cytotoxicity in MDA-MB-468 + no cytotoxicity on BT474 cells -Greater tumor suppression than bare NPs or free drugs -No tissue damage.

Table 3 .

 3 Different diagnostic applications of EGFR-targeted nanomedicines in cancer

					Advantage of EGFR-targeted NPs compared to non-targeted
	Imaging	Type of NPs	Ligands	Type of cancer		Ref.
					NPs
					-Higher photo-stability than Cy5-cetuximab	(Lee et al.,
		QDs	Cetuximab	Skin	-Two-fold higher fluorescence intensity in EGFR-positive cancer cells than EGFR-negative cells	2010)
		QDs	Affibodies	Skin	-Two-fold higher cellular uptake	(Zhang et al.,
	NIR				-Higher in vivo fluorescence signal and longer half-life	2018)
		Fluorescent	EGF proteins	Breast	-Strong asymmetric clustering of targeted NPs at the	(Faucon et al.,
		organic NPs			membrane of MDA-MB-468 cells	2017)
		Virus-based NPs GE11 peptides	Skin, colon and	-Better cellular uptake into highly EGFR-expressed cells	(Chariou et al.,
				breast	-Similar cellular uptake in low EGFR-expressed cells	2015)
	Nuclear	Polymeric NPs	EGF proteins	Oesophagus	-Increased 111 In cellular uptake in cancer cells	(Gill et al.,
					-Reduced radiotoxicity of targeted NPs for normal cells	2018)
					-Specific binding to A431 cells	(Chu et al.,
	MR		Cetuximab	Skin	-Enhancement in MRI contrast -Better apoptotic activity of mAbs in cancer cells	2015)
		SPIONs	Cetuximab	Glioblastoma	-Possibility to distinguish EGFR-positive cancer cells from	(Chen et al.,
					EGFR-negative ones	2017)
			Bispecific		-Higher cellular uptake in EGFR-positive cells than EGFR-	(Wu et al.,
	MR + optical		antibodies	Breast	negative cells -61.5-fold better MRI contrast	2016)

Acknowledgments

This work was supported by the 'Cancéropôle Grand Ouest' and especially by the Emergence CGO 2019 NANOTIF project. This work was supported by the French National Research Agency under the program "Investissements d'avenir" Grant Agreement LabEx MAbImprove (ANR-10-LABX-53-01).