Dual-targeted photothermal agents for enhanced cancer therapy

Photothermal therapy, in which light is converted into heat and triggers local hyperthermia to ablate tumors, presents an inherently specific and noninvasive treatment for tumor tissues. In this area, the development of efficient photothermal agents (PTAs) has always been a central topic. Although many efforts have been made on the investigation of novel molecular architectures and photothermal materials over the past decades, PTAs can cause severe damage to normal tissues because of the poor tumor aggregate ability and high irradiation density. Recently, dual-targeted photothermal agents (DTPTAs) provide an attractive strategy to overcome these problems and enhance cancer therapy. DTPTAs are functionalized with two classes of targeting units, including tumor environment targeting sites, tumor targeting sites and organelle targeting sites. In this perspective, typical targeted ligands and representative examples of photothermal therapeutic agents with dual-targeted properties are systematically summarized and recent advances using DTPTAs in tumor therapy are highlighted.


Introduction
Cancer, as one of the most intractable diseases, has spoiled millions of lives due to its high morbidity and mortality. 1,2 Over the past decades, great efforts have been focused on the development of efficient therapeutics for cancer treatment, such as surgery, 3 radiotherapy, 4,5 chemotherapy, 6,7 photodynamic therapy (PDT), [8][9][10] photothermal therapy (PTT) [11][12][13] and immunotherapy. [14][15][16] In particular, PTT has attracted extensive attention due to its inherent advantages of minimal invasion and external light control. According to the mechanism of PTT for cancer therapy, it achieves the goal of killing cancer cells via converting light energy to heat in order to trigger local hyperthermia in tumor, so PTT does not suffer from the limitations in hypoxic environments compared to PDT. Up to now, PTT as an important and promising treatment option has been a focus of attention to develop potential photothermal agents (PTAs) over the past decades. A broad range of novel inorganic and organic materials have been designed as PTAs to ablate tumor tissues. Although great advances have been made in the development of PTAs, it has been found that hyperpyrexia can seriously damage surrounding normal tissues because of poor tumor aggregation and high intensity irradiation. Hence, the development of efficacious PTAs with excellent tumor inhibition and negligible side effect presents a challenging task for cancer therapy.
The rapid growth and proliferation of tumors result in abnormal demand and metabolism, which is benecial for distinguishing normal tissues from diseased tissues. Therefore, targeted therapeutic agents exhibit enormously potential applications in PTT and have received extensive attention. 17,18 Unfortunately, although single-targeted PTAs have made great achievements in cancer therapy, it is nonnegligible that offtargeted activities occur occasionally due to the dynamics and saturability of receptors on cancer cells. 19,20 For further access to the tumor tissues, dual-targeted photothermal agents (DTPTAs) have been developed to ablate solid tumors, with slightly adverse effects (Scheme 1). 21 Modied with two different targeted groups, DTPTAs show highly precise locating capability. Besides this, DTPTAs can trigger cell necrosis at low temperature and cause negligible damage to surrounding tissues when one of the targeted ligands tends to aggregate in the organelle, where heat-sensitive proteins and DNA are enriched.
In this perspective, typical targets are classied from three aspects based on the position of recognition sites, namely "extracellular targets", "intracellular targets" and "subcellular targets" (Table 1). Furthermore, the applications of dualtargeted photothermal materials are carefully introduced. DTPTAs are described on the basis of different materials, including noble metal materials, transition metal oxide/sulde materials, carbon materials, cyanine dyes, and organic polymers, as well as other materials. Photothermal conversion capability and tumor inhibition are also described in detail.

"Extracellular targets"
"Extracellular targets" refers to recognition sites present in the tumor microenvironment (TME). Because of the vigorous metabolism and rapid growth of the solid tumor, extreme environments, including hypoxic environments, acidic environments, angiogenesis and overexpression of enzymes, are generated in the surrounding tumor tissues. By taking advantages of these differences between normal tissues and cancers, extensive investigations have been made for the specic treatment of tumors. 71,72 Hypoxic environment. Although solid tumors contain intricate veins, oxygen and glucose are still in short supply due to their expanding tumor tissues and growth size. 73,74 As a result, a hypoxic environment is generated around the tumor tissues. To target this hypoxic environment, genetically modied anaerobions have become important carriers based on anaerobic orientation. [22][23][24] Additionally, typical ligands such as anti-CCL-28, 25 anti-LDLR 26 and hypoxia-specic drug tirapazamine 27 have also been applied to target hypoxia.
Acidic environment. The hypoxic environment of the tumor prevents adequate oxidation of glucose, which promotes tumor cells to derive energy not only from oxidative phosphorylation but also from glycolysis. During the process of glycolysis, a large amount of lactic acid is produced and excreted into the TME, resulting in an acidic environment. Various changes are prone to occur in an acidic environment, such as hydrolysis reactions; 28 protonation 29 and hydrophilic transformation. 30 In addition, the pH low insertion peptide (pHLIP) can transform into an regular a-helix in acid environment to insert into membrane, so it can be used for targeting tumor cells. 31,32 Angiogenesis. To relieve anoxia, vascular endothelial growth factor (VEGF) is released from cancer cells into the TME to promote angiogenesis. VEGF can specically combine with VEGF receptor (VEGFR) on the surface of surrounding endothelial cells, so both VEGF and VEGFR are potential targets for cancer therapy. Anti-VEGF monoclonal antibodies (anti-VEGFmAb), which behave amiably with VEGF were widely used for targeted PTT. 33 The antibody bevacizumab also shows the ability to accurately identify VEGF. 34 In addition, modied with the VEGFR-2 antibody, PTAs have also been developed to promote powerful TME aggregation and photothermal effect. 35 Matrix metalloproteinase (MMP). The dense extracellular matrix of the TME is mainly composed of collagen for slowing tumor invasion, migration and angiogenesis. To deal with this issue, carcinoma cells stimulate stromal broblasts and endothelial cells to secrete MMPs to degrade extracellular matrix. Multiple MMP subspecies including MMP-2, MMP-9, MMP-13 and MMP-14, can cut specic GPLGVRGC sequences, which have been widely applied for cancer stimulation therapy. 36 Besides this, the PG-6 peptide (PLGALG) 37 is another MMP discriminating peptide that can be identied and cut by the MMP-2 and MMP-9 subspecies.
Magnetic eld. Magnetic targeting is a method for articially controlling tumor aggregation based on an external magnetic eld. Magnetic materials, such as iron oxide nanoparticles (IONPs) 38 have been widely used for anti-cancer treatment due to their properties, such as non-toxicity, small size, and easy functionalization. In addition, magnetic materials demonstrate strong imaging capabilities in magnetic resonance imaging (MRI), which is benecial to guiding tumor treatment. The released iron ions can also catalyze the Fenton reaction to produce reactive oxygen species (ROS) for chemodynamic therapy. Moreover, the good biocompatibility, biodegradability, pharmacokinetics and bimodality of magnetic particles exhibit great potential applications in clinical trials.

"Intracellular targets"
"Intracellular targets" means that receptors exist on the surface of the tumor cytomembrane. Because cells are relatively separate spaces, substance ingestion requires the assistance of receptors that are embedded in the cell membrane, while specic receptors are produced to transfer different substances, including ions, small molecules and biomacromolecules. Tumor cells have abnormal metabolism, so many receptors are over-expressed. Many efforts have been focused on the development of specic drug-delivery systems based on these overexpressed receptors over the past decades.
Integrin a v b 3 . Integrins are crucial heterodimeric receptors for cell adhesion and intracellular signal transduction, then mediate tumor growth and invasion. Integrin a v b 3 as one of the integrin subspecies is closely associated with angiogenesis, wherein integrin a v b 3 is overexpressed in tumor tissues. A cell binding peptide RGD (arginine-glycine-aspartate) has been known to be able to identify integrin a v b 3 . Due to its advantages of being modiable by simple chemical synthesis, it has been recognized as the most commonly used targeting ligand over the past few decades. In addition, the monoclonal antibody against a v b 3 (integrin a v b 3 mAb) is also an important target to achieve the goal of targeting tumor cells. For this reason, various PTAs conjugated with RGD [39][40][41][42] or integrin a v b 3 mAb 43,44 have been applied for tumor targeted PTT.
Epidermal growth factor receptor (EGFR). EGFR as a kind of receptor is overexpressed on the surface of multiple tumor cells. Moreover, it can specically combine with epidermal growth factor, which is a cell growth factor with high physiological activity. Aer binding to the ligand, EGFR can inhibit apoptosis and trigger a series of enzymatic signalling to enhance cell growth, invasion, and also migration. Moreover, EGFR can specically bind to monoclonal antibodies EGFRmAb (Cetuximab, CET) 45,46 to achieve internalization. In addition, as a tyrosine kinase receptor, EGFR can be inhibited by corresponding small molecule inhibitors, such as getinib 47,48 and erlotinib. 49,50 Both monoclonal antibodies and inhibitors inhibit EGFR-mediated downstream signalling when providing targeting capabilities.
Folate receptor (FR) and biotin receptor (BR). Due to the rapid proliferation of malignant cells, vitamins are highly demanded to promote cell growth. For example, folate (FA) is mainly involved in cell growth and the formation of DNA, which enters carcinoma cells mediated via the FR. In order to bind and internalize FA, FRs are over-expressed on the surface of tumor membranes. Another typical vitamin in high demand is biotin (also called vitamin H), which must be ingested in the daily diet of humans. Both FA 51-53 and biotin 55,56 as natural small molecules have shown advantages of being low cost, biologically stable and easy to modify, so they have been widely used as targeted groups. In addition, the chemotherapeutic agent methotrexate (MTX) also shows an affinity to FR because of it having a similar structure to that of FA. 54 Transferrin receptor (TfR). A large amount of iron ions are needed to be transferred into cancer cells in order to synthesize heme for carrying oxygen. In this process, TfR as a kind of glycoprotein embedded in membrane has shown the signicant responsibility of binding transferrin (Tf) and endocytosis of Tf, which accelerates transport and internalization of iron ions in vivo. Meanwhile, tumor cells express TfR in high levels, which is benecial to cancer-targeted therapy. Through the camouage of Tf, plenty of nano-photothermal therapeutic agents have been used for targeting tumor cells. 57,58 Cluster of differentiation-44 (CD44). In the cluster of differentiation family, the CD44 receptor is a kind of transmembrane glycoprotein for extracellular matrix adhesion and signal transduction, as well as stem cell migration. Besides this, many studies have shown that CD44 can serve as a marker for cancer stem cells, which are closely associated with tumor recurrence, migration, and drug resistance due to their outstanding selfregulation. Hyaluronic acid (HA), as an endogenous polysaccharide, binds well to CD44 and then is mediated to internalize into cells. For these reasons, HA modied therapeutic agents 59,60 have been widely developed and applied in tumortargeted therapy and are considered to be an effective method to inhibit tumor metastasis.

"Subcellular targets"
"Subcellular targets" means that PTAs are conjugated with organelle-targeted ligands, so the photothermal materials can efficiently induce apoptosis of organelles aer endocytosis. Because of heat-sensitive proteins and genetic material in organelles, organelles are easily destroyed and cause cell death at low-dose irradiation, thereby reducing damage to surrounding normal tissues. Hence, organelle-targeted imaging and therapy have attracted wide attention in exploring technologies for tumor inhibition and life regulation. 64,75,76 Nucleus. The nucleus as the brain of cell contains most of the genetic material to control the replication and expression of genes, and takes part in multiple processes such as cell growth, division and apoptosis. Tumorigenesis is based on genetic mutations, which make the nucleus become an ideal target for tumor suppression. However, such an important organelle is protected by a two-layer-membrane, and the exchanges of materials mainly depend on the strict control of nuclear pores complex. Nuclear localization sequence (NLS) peptides 61 and TAT peptides 62 have been demonstrated to have excellent capability of targeting the nucleus, which have been exploited to overcome the difficulty of poor nuclear localization. Aptamers based on articially screened oligonucleotide or polypeptide sequences have the advantage of highly specic targeting properties. The AS1411 DNA aptamer 63 exhibits excellent nuclear recognition and has been used for nuclear localization therapy.
Mitochondria. Mitochondria, where high-energy ATP is generated via complex reactions, are recognized as the energysupplying organelles. Although glycolysis provides a large amount of energy to the cancer cells, mitochondria is involved in a variety of biological processes such as the maintenance of oxidative phosphorylation. The destruction of mitochondria will lead to the apoptosis of cells, so it has been regarded as an important target for cancer therapy. Extensive investigations illustrated that triphenylphosphonium (TPP) exhibited good mitochondrial aggregation by taking advantage of hydrophobicity and positive charge, thus TPP has been widely explored for guiding targeted therapy. 64,65 Meanwhile, cyanine cation 66 and mitochondria localization (MLS) peptides 67 can also target mitochondria, which have been applied to transport PTAs to mitochondria for cancer therapy.
Lysosomes. Due to low pH (ca. 5.0) and dozens of hydrolases, lysosomes are capable of degrading biological molecules, including foreign impurities and even necrotic organelles. Moreover, they also play important roles in autophagy, apoptosis and membrane repair. When lysosomes suffer from membrane rupture or dysfunctions, the internal hydrolases will be released, leading to cellular digestion, so lysosomes are regarded as a powerful target for cancer therapy. Compared with other organelles, nano-therapeutic agents can more easier enter lysosomes aer experiencing endocytosis. 77 Because positively charged agents are cannot penetrate the lysosomal membrane, pro-drugs bearing morpholine groups can aggregate in the lysosome. 68 Endoplasmic reticulum (ER). The ER is composed of a large biolm system, which is involved in protein assembly, calcium homeostasis regulation and intracellular environmental stabilization. When the stimulation of the ER occurs, it not only blocks the protein synthesis but also triggers the apoptotic signaling pathway. Therefore, the ER is related to the occurrence of a variety of diseases. Meanwhile, it is also considered as one of the key aims of tumor-targeted therapy. So far, sulfamides 69 and pardaxin (FAL) peptides 70 have been widely used as good ligands for ER-targeted therapy.

Dual-targeted photothermal agents
DTPTAs have got great advances over the past few years, and many materials including noble metal materials, transition metal oxide/sulde materials, carbon materials, cyanine dyes, and organic polymers as well as other materials that have been used for designing novel PTAs for tumor ablation ( Table 2).

DTPTAs based on noble metal materials
Noble metal nanoparticles (NPs) are well-studied inorganic PTAs because of their excellent photothermal and optical properties. Due to localized surface plasmon resonance (LSPR), they exhibit great talents for absorbing laser to reach electron excited state and release heat via nonradiative decay.
Gold-based NPs, as excellent PTAs with intense LSPR, have got diverse applications via the modication of resonance wavelength. Researchers have developed a variety of gold nanostructures, such as gold nanorod (GNR), gold nanoshells, gold nanoprisms and gold nanostars (GNS).
Although Au NPs can passively accumulate in tumors via enhanced permeability and retention effect, the efficiency of material utilization is low. Due to the convenience of surface modication, Au NPs can be functionalized with active targeting ligands (e.g., antibodies, aptamers and peptides) to realize selective accumulation in tumor tissues.
Recently, Sun's group successfully prepared and characterized dual-targeted gold nanoplatforms (Au-Apt-TPE@Zn, Apt: aptamer), which were used to identify early apoptotic cells and perform accurate PTT for tumors (Fig. 1a). 63 In their study, gold nanoprisms were coupled with phenanthroline derivatives (functionalized with tetraphenylethene (TPE)), and further stabilized with target peptide aptamers through Au-S bonds to obtain Au-Apt-TPE. The remaining nitrogen atoms of Au-Apt-TPE effectively chelated with Zn 2+ to synthesize Au-Apt-TPE@Zn. Moreover, Au-Apt-TPE@Zn selectively recognized  early apoptotic cells and bound cell membrane. Connected to AS1411 DNA aptamers, Au-Apt-TPE@Zn specically targeted the nucleus. The dual-targeted therapeutic agent for cell membrane and nuclei showed prominent photothermal conversion efficiency (PCE) and allowed the nanoplatform to achieve accurate treatment for tumor inhibition.
In addition, a dual-targeted photothermal material for targeting tumor cells and mitochondria was also studied by Zhang's group (Fig. 1b). 78 AuNS-pep/DOX@HA (pep: peptide) was composed of cationic peptide R8, TPP modied a-helical pro-apoptotic peptide (TPP-KLA), doxorubicin (DOX) and HA. The nanoplatform was internalized into tumor cells through CD44 receptor-mediated recognition. TPP-KLA was used to specically insert and destroy mitochondrial membranes, and then induce dysfunction of the mitochondria and cause mitochondrial-dependent apoptosis. In vitro and in vivo experiments showed that AuNS-pep/DOX@HA presented prominent non-resistant or resistant tumor inhibition. In 2018, the authors also designed a cellular/intranuclear dual-targeted nanoplatform for tumor therapy via PTT (Fig. 1c). 79 NLS peptides were used to modify GNS to give GNS-NLS for targeting the nucleus. Subsequently, HA was coated on the surface of GNS-NLS (GNS-NLS@HA) through electrostatic interactions, which then bound with CD44 to enhance internalization and then expose NLS aer degradation by hyaluronidase (HAase). GNS-NLS@HA showed high photothermal conversion (DT ¼ $15 C, 808 nm, 1.74 W cm À2 , 4 min) and prominent tumor suppression efficiency.
Moreover, Xu and co-workers explored the therapeutic effects of GNRs in different subcellular organelles with the modication of specic targeting peptides and RGD peptides (Fig. 1d). 80 Surface-enhanced Raman scattering spectroscopy promoted AuNRs to achieve super-resolution imaging of biomolecules and monitor the position of therapeutic agents. Under the same experimental conditions, AMT (AuNRs modi-ed with MLS and RGD) or NLS (AuNRs modied with NLS and RGD) have better tumor killing effect than ALT (AuNRs only modied with RGD) (Fig. 1e).
Another effective dual-targeted model is the combination of two different tumor cell-targeted ligands such as RGD, HA and FA. Recently, Xu and co-workers reported a mesoporous silica (mSiO 2 )coated nanoplatform (DOX-GNRs@mSiO 2 -HA-RGD) by combining HA, RGD and DOX (Fig. 2a). 81 Cell uptake studies proved that the internalization of the dual-targeted DOX-GNRs@mSiO 2 -HA-RGD was more effective than single-targeted and untargeted therapeutic agents. In addition, DOX-GNRs@mSiO 2 -HA-RGD showed excellent photothermal conversion properties, with a rapid increase to 43.5 C (808 nm, 2.00 W cm À2 , 4 min). Yao and coworkers designed a pH and near-infrared (NIR) dual-responsive drug release nanoplatform for the cooperative chemophotothermal therapy of breast cancer, which was prepared by combining GNR, FA, HA, dopamine, adipic acid dihydrazide and DOX (Fig. 2b). 82 Compared with the non-FA-modied NPs, the dual-responsive nanoplatform was more efficient to deliver GNR and DOX to MCF-7 cells. Moreover, tumor cells were killed more effectively through inducing apoptosis under NIR irradiation. Recently, Li and co-workers also studied the therapeutic effect of DOX-loaded FA/RGD dual-targeted GNR (denoted as FA/RGD-   DOX-hz-GNRs, hz: a pH-sensitive hydrazone) for B16-F10 xenogra tumors. 83 In vivo experiments showed that this dual-targeted nanoplatform exhibited excellent tumor therapeutic effects via the synergistic cooperation of DOX-induced apoptosis, heat induced necrosis and angiogenesis inhibition. Other than that, human epidermal growth factor receptor 2 (HER2) is also an important targeting site for tumor therapy, which is overexpressed in multiple tumors. Conjugated with HER2, a pH, GSH and HAase triple-responsive nanoplatform GNR-HA -ALA/Cy7.5 -HER2 (ALA: 5-aminolevulinic acid) was reported by Yao and coworkers. 84 In vivo experiments illustrated that tumor tissues could be completely eliminated without obvious side effects. This nanoplatform provided a potential strategy for HER2/CD44 targeted breast cancer PDT/PTT. Furthermore, dual-targeted therapy can also be achieved via simultaneously targeting the tumor environment and tumor cells. Liu's group modied ultrasmall superparamagnetic IONPs onto NaYF 4 -based upconversion NPs, and then coated a thin gold shell through seed-induced reduction growth, on which poly(ethylene glycol) (PEG) were anchored as linkers to conjugate FA to prepare the multifunctional platform (denoted as FA-PEG-MFNP) (Fig. 2c). 85 FA-PEG-MFNP could achieve dual-modal targeting capability mediated by magnetic eld and FR with the monitoring of upconversion luminescence and MR imaging. Aer that, Zhang's group constructed a MTX-Fe 3 O 4 -gold shell (MFG) nanosystem hybridized with LPM lipoic acid (LA)-PEG-MTX (LPM) to obtain MFG-LPM NPs for dual-targeted chemo-photothermal therapy. 86 MTX has a similar structure to FA, which can be used for cancer therapy as both targeted ligand and chemotherapeutic drug. This dual-targeted MFG-LPM had excellent healing talent that cured all tumor-bearing mice in a short time, while mice in none-magnetic group needed a second injection therapy.
The acidic microenvironment of tumor tissues has also been used for dual-targeted cancer therapy. Lee's group combined low molecular weight hyaluronic acid (LMWHA), the pHsensitive group sulfamethazine (SM) and GNR to synthesize dual-targeted therapeutic platforms from ligand-mediated cell uptake and acid-induced aggregation (Fig. 2d). 87 When the pH was lower than 7.4, the de-ionization of SM in AuNRs@SM/LA-LMWHA improved the surface hydrophobicity, resulting in particle aggregation and gradually increasing size (Fig. 2e). And then, the NPs were internalized into tumor cells through CD44 receptor-mediated recognition for tumor therapy.
Other noble metals (e.g., platinum) are also used as DTPTAs for enhanced tumor therapy by taking the advantages of photothermal stability and certain catalytic ability. In 2017, Lin's group developed Au@Pt NPs modied with a cell-targeting ligand FA and a mitochondria-targeting group TPP for PDT/ PTT synergistic therapy. 88 In this nanoplatform, the excellent catalytic activity of Pt NPs could enhance the peroxidase-like catalysis of H 2 O 2 to improve the efficacy of PDT.

DTPTAs based on transition metal oxide/sulde materials
Although noble metal NPs such as gold and platinum have shown great potential applications in the eld of tumor Chemical Science photothermal treatment, the high cost, difficult biodegradation and toxicity during long-term metabolic processes in the body have restricted further research and clinical practice application. Transition metal oxide/sulde NPs as another type of inorganic PTAs exhibit strong NIR-absorption, low cost and high PCE, which has attracted much attention in PTT for tumor tissues. Because of these advantages, copper sulde (CuS) NPs are widely used in the treatment of multifarious diseases such as cancer, 108,109 atherosclerosis 110 and diabetes, 111 and show excellent biological applications based on low cytotoxicity and controllable morphology. Tang and co-workers developed a nucleus-targeted PTT strategy based on CuS NPs to achieve intra-nuclear PTT and effectively prevent local cancer recurrence (Fig. 3a). 89 Through the surface modication of mesoporous silica coated CuS (CuS@MSN) by RGD and TAT peptides, CuS@MSN-TAT-RGD NPs directly targeted tumor cells and further entered the nucleus. Aer irradiation with a 980 nm NIR laser, the endonuclear genetic materials were destroyed by hyperthermia, resulting in cell necrosis. Under 980 nm laser irradiation for 5 minutes, the cell killing rate of the NPs with targeting ability was 84%, while that of non-targeting NPs was 42% (Fig. 3b). This PTT platform, with the capability of targeting nucleus, provides powerful possibilities for tumor ablation and anti-recurrence. WSSe nanosheets were also used in dual-targeted PTT for tumors by Zhang's group via the preparation of a WSSe/MnO 2 -INH nanocomposite (Fig. 3c). 90 In this study, Mn 2+ generated by the degradation of MnO 2 was used to catalyze the isoniazid (INH) to obtain hydroxyl radicals (cOH) for achieving PTT and PDT anticancer treatment. WSSe/MnO 2 -INH-TPP@CM (CM: cell membrane) was coated with cancer cell membrane extracted from the breast cell line MCF-7, and then it was modied with the mitochondrial-targeted group TPP to realize dual-targeted tumor therapy. WSSe/MnO 2 -INH-TPP@CM showed good anticancer effects both in vivo and in vitro.
Moreover, Yang and co-workers developed nucleus-targeted multifunctional magnetic NPs (IONP-20-TAT-Tf) bearing Tf and TAT peptides (Fig. 3d). 91 The conjugated monodisperse magnetic NPs exerted considerable photothermal stability and high PCE ($37%). Quantitative analysis showed that the accumulation of multifunctional IONP in the nucleus was 45-fold higher than that without TAT modication (Fig. 3e). During in vivo experiments, the composite NPs effectively inhibited xen-ograed tumors irradiated by NIR lasers, and are expected to be used in cancer treatment.

DTPTAs based on carbon materials
Carbon-based nanomaterials have great potential applications in the eld of tumor PTT. Graphene oxide, carbon dots and other carbon-based materials have extensive optical absorption and reasonable photothermal properties, which has aroused widespread interest. Carbon nanodots have outstanding characteristics, such as high photostability, low toxicity, high solubility, low cost and good biocompatibility. However, the low efficiency of the absorption in the visible to NIR window limits their application in light-sensitive cancer treatment strategies and in vivo imaging. Xu's group prepared a new type of super carbon dots (SCDs) (about 20 nm) with broad absorption from the visible light to NIR region. 67 The carbon dots (CDs) possess a high PCE for PTT, and can be functionalized with dualtargeted ligands (RGD and MLS) for cancer cells and mitochondria. The SCDs were constructed via the self-assembly of small sized CDs (around 5 nm) in an acidic environment, which could be located precisely in the mitochondria of cancer cells for cancer therapy (Fig. 4a and b). The experimental results showed that the difference in the survival rate between the tumor and normal cells was as high as 70%, reecting the high specicity and selectivity of these SCDs (Fig. 4c and d).
Due to its large specic surface area, excellent electrical properties and optical properties, graphene has attracted extensive attention in many elds in recent years. Graphene oxide can be obtained via the chemical oxidation of graphene, which has a good PCE for PTT. Kong's group successfully prepared multifunctional magnetic graphene as a nanomedical platform (denoted as MGMSPID) for MRI guided chemophotothermal glioma treatment (Fig. 4e). 92 This photothermal agent could be easily prepared via a PEGylated method and then modied with an interleukin-13-based peptide (IP). In addition, the MGMSPID system showed thermal stimulation, pHresponse and sustained release properties. All these features provide a powerful multi-functional therapeutic platform for visual glioma treatment. Chen's group also developed a pHsensitive dual-targeted magnetic nanocarrier for chemophototherapy in cancer treatment. 93 The authors prepared magnetic graphene oxide (MGO) via chemical coprecipitation, and then modied MGO with PEG and CET to obtain MGO-PEG-CET. MGO-PEG-CET was used for magnetic and receptormediated dual-targeted synergistic treatment to delivery DOX to EGFR over-expressed tumor cells. In vitro cytotoxicity tests showed that the half-maximum inhibitory concentration (IC 50 ) value of the dual-targeted nanoplatform MGO-PEG-CET/DOX on CT-26 cells was 1.48 mg mL À1 , which was lower than the group of MGO-PEG/DOX (2.64 mg mL À1 ).

DTPTAs based on cyanine dyes
Compared with inorganic PTAs, organic PTAs have the inherent advantages of biocompatibility and degradability, among which cyanine dyes are the most representative agents, which have widespread applications. Indocyanine green (ICG), one of the cyanine dyes, has drawn a lot of attention due to its great biosafety, and has been approved by the Food and Drug Administration for clinical uorescence imaging. 112 ICG also shows great properties for tumor PTT, PDT and uorescence imaging, with satisfying water solubility and potential mitochondrial targeting, as well as splendid PCE properties. However, since ICG is easily cleared in vivo and has a short cycle time, it is usually administered via a delivery system. Obviously, a dual-targeted drug delivery system is an ideal strategy to carry ICG to tumor tissues for imaging and therapy. In 2018, Wang et al. developed thermo-sensitive NPs to drive ICG, photosensitizer chlorin e6 (Ce6) and chemotherapy drug cisplatin into cancer cells with the help of tumor recognition groups. 94 Targeted FA molecules and cRGD peptides were covalently anchored to different amphiphilic polymers and the modied polymers self-assembled to form NPs to enhance the active targeting properties.
This intelligent drug release realized synergistic treatment and ablated tumors under NIR irradiation. To improve the cancer-targeted efficiency, Anilkumar et al. constructed dual targeted magnetic photosensitive liposomes (MPLs) to deliver ICG to tumor tissues driven by a magnetic eld and to internalize drugs into cells via the combination of HA and CD44. 95 In this study, citric acid-coated Fe 3 O 4 magnetic NPs (CMNs) and ICG molecules were loaded in MPLs via a solvent evaporation/ hydration technique, and then HA-PEG was connected by electrostatic interaction triggered self-assembly to fabricate HA-PEG-MPLs. These HA-PEG-MPLs particles not only achieved tumor aggregation enhanced therapy, but also increased the PCE (DT ¼ $30 C, 808 nm, 2.0 W cm À2 , 5 min) via the combined inuence of ICG and CMNs. Therefore, HA-PEG-MPLs showed superior inhibiting ability in a xenogra tumor model. Fang et al. constructed biocompatible selenium NPs (SeNPs) to deliver DOX and ICG to tumor cells (Fig. 5a). 37 In Chemical Science order to assure agents arrived in tumor cells successfully, chitosan was chosen as a linker to conjugate with RGD-derived RC-12 peptide and MMP-recognized PG-6 peptide to give SeNPs-DOX-ICG-RP. The internalization of tumor cells triggered by endocytosis offers great possibilities of accurate treatment. Besides this, the Se nanosystems showed amazing PCEs and temperatures that could increase to 78.2 C (808 nm, 3.0 W cm À2 , 100 s), and the excellent stability was also certied via a laser turn on/off experiment ve times (Fig. 5b and c).
Wu et al. constructed a tumor and mitochondria dualtargeted nanocarrier for mitochondrial locating PTT and chemotherapy. 96 In this case, FA and TPP were respectively connected to amphiphilic polymer DSPE-PEG, and the resulting polymers were mixed and self-assembled to form nanovesicles for encapsulating ICG and DOX. The vesicles displayed great colloid stability and released DOX via the rapid temperature rise (DT ¼ $58 C, 808 nm, 1.0 W cm À2 , 5 min) induced by NIR irradiation. Importantly, combined with FA and TPP, these nanoagents showed powerful mitochondria locating capability (p up to 0.84) and enhanced cytotoxicity, with a sharply reduced MCF-7 cell survival rate of as low as 3.8%.
IR 825 is a kind of heptamethine type NIR dye with perfect photothermal conversion performance. Despite possessing a similar structure to ICG, IR 825 has limited applications in the theranostic eld due to low water solubility, so it is usually delivered to tumor tissue via a micelle encapsulation technique. In 2017, Kang et al. described a FA and TPP modied polymeric core-shell therapeutic agent for specically transmitting IR 825 and 3-bromopyruvate (BP) to tumor mitochondria. 97 PEG grafted poly(dimethyl aminoethyl methacrylate) (PEG-g-PDMA) was connected with catechol and carbonized with sulfuric acid to obtain the core, and the shell was composed of PEG-g-PDMA, tumor-targeted FA, mitochondria-targeted TPP, IR 825 and phenylboric acid, which reacted with catechol to achieve coreshell binding. This therapeutic agent damaged tumor mitochondria and induced cell death triggered by hyperpyrexia (up to 65 C) and the glycolysis inhibition of BP under NIR irradiation (808 nm, 2.0 W cm À2 , 5 min).
Since the IR 825 molecules have lipophilicity cationic structure for mitochondrial aggregation, they are also used for mitochondrial-targeted therapy. Based on this feature, Wang et al. engineered a targeted theranostic agent (IR825@B-PPNs) for anaplastic thyroid carcinoma oncotherapy (Fig. 5d and e). 34 IR 825 and peruoropentane (PFP, an ultrasound contrast agent) were loaded in poly(lactic-co-glycolic acid) (PLGA) to form IR825@PPNs, and then, polyvinyl alcohol (PVA) and bevacizumab antibodies were modied (IR825@B-PPNs) in order to promote tumor aggregation. The conjugated bevacizumab specically recognized VEGF and blocked the combination of VEGF and receptors. With the monitoring and diagnosis of the photoacoustic, uorescence, and ultrasonic multi-mode imaging, tumor and mitochondrial aggregation enhanced the thermal damage of IR 825 to tumor cells. The well-merged uorescence signals of IR825@B-PPNs and Mito-Tracker indicated that the theranostic agent had a tendency for mitochondrial localization and in vivo experiments realized the complete curing of a C643 tumor ( Fig. 5f and g).
Other cyanine dyes also make a great contribution to dualtargeted PTT. In 2019, Tang and co-workers designed a novel small molecule (Bio-PPh 3 -PT) for highly effective and selective tumor mitochondrial PTT (Fig. 6a le). 55 In this study, quinoline and indole blocks were connected via continuous double bonds to obtain a solid compound that possesses the ability of photothermal conversion, and then conjugated it with biotin and TPP for targeting tumors and mitochondria. The DTPTAs showed a superb PCE (37.8%) with a temperature change of $25 C (635 nm, 0.5 W cm À2 , 5 min). Biotin was used to distinguish diseased tissue from normal tissue and TPP functioned as an important targeting group for locating in mitochondria. The dual-targeted photothermal material can cause irreversible damage at a light power as low as 0.5 W cm À2 . Besides this, during the treatment of tumor-bearing mice, apparent inhibition occurred in the dual-targeted group, while the therapeutic effect in the non-targeted group was negligible (Fig. 6b).
The photothermal conversion capability of small molecule PTAs was regulated by the electrical changes initiated from the regulation of specic substances in tumor cells. 113 An aggregation-enhanced PTT probe (termed as ETP) was developed by Yao et al. for mitochondria-targeted imaging and therapy, 98 which was activated by alkaline phosphatase (ALP) (Fig. 6a right). A phosphate ester group was introduced into the body of the probe for inhibiting molecular activity. Aer hydrolysis by ALP, which is overexpressed in prostate cancer, molecule absorption occurred with a large redshi, to allow active FL and photoacoustic imaging. Simultaneously, the photothermal performance and therapeutic effect were enhanced via molecular aggregation and mitochondriatargeting properties. The probes showed a 32.8 C temperature change in in vitro experiment aer incubation with ALP for 24 h, while the group without incubation only changed by 9.5 C (650 nm, 0.5 W cm À2 , 5 min) (Fig. 6c). Thermal imaging showed that the tumor temperature increased to over 50 C, which indirectly illustrated the effective tumor healing ability of the activated DTPTAs (Fig. 6d-f).

DTPTAs based on organic polymers
Organic polymers have better light stability than small molecules, which have attracted scientists' interest for PTT. Among these polymers, polydopamine (PDA) plays a crucial role in PTT due to it being in low cost and easy to synthesize. It is worth mentioning that PDA as a natural biodegradable polymer has been widely used in targeted PTT. Mitochondria-targeted PTAs were demonstrated by Wang et al. using magnetic materials and TPP as targeting ligands. 99 Magnetic Fe 3 O 4 NPs were coated with PDA to form core-shell structures, on which TPP and GSH responsive PEG were graed. At last, DOX was further adsorbed on the surface via p-p stacking to give the desired therapeutic agent Fe@PDA-TPP/SS/DOX. This multistage targeted therapeutic agent was guided by an external magnetic eld to reach tumor tissues, and experienced lysosome escape and GSH mediated degradation to expose TPP for mitochondrial aggregation. Under low power density irradiation, Fe@PDA-TPP/SS/ DOX exerted an excellent temperature increase of 19 C within 6 minutes (808 nm, 0.8 W cm À2 ) and released DOX to destroy mitochondrial DNA. Analogously, Guo et al. also constructed a Fe 3 O 4 @PDA@mSiO 2 core-shell material for targeted PTT (Fig. 7a). 100 In this study, tumor-identied Tf and mitochondriatargeted TPP were anchored on the surface of Fe 3 O 4 @-PDA@mSiO 2 materials to enhance their transport to tumor subcellular organelles. In addition, ICG molecules were loaded in the outermost mSiO 2 layer to improve photothermal conversion and produce ROS. This composite material showed a considerable capacity of heat production (DT ¼ $10 C, 785 nm, 0.5 W cm À2 , 500 s) under a low dose of light radiation and outstanding tumor ablation with complete healing efficacy ( Fig. 7b and c).
Aer carbonizing them with concentrated sulfuric acid, PDA ramied uorescent carbon NPs (FNPs-PDA) were synthesized by Park and co-workers. 101 FNPs-PDA, acting as a PTA and imaging agent, underwent a boracic acid mediated esterication reaction to give it a HA shell, which was then modied with TPP and b-cyclodextrin (b-CD) to obtain the nanocomposite drug FNPs-PDA@HA-TPP-CD-PTX. When the therapeutic agent arrived at the tumor tissues, the combination of HA and CD44 promoted endocytosis and TPP triggered mitochondrial aggregation aer lysosome escape. The low pH in the lysosome not only promoted the heat production of FNPs-PDA@HA-TPP-CD-PTX, but also facilitated the release of paclitaxel (PTX) in the b-CD hydrophobic cavity for multimodal therapy.
Zhu and co-workers reported magnetic polydopamine (MPDA), which was loaded with DOX and then coated with HA-MTX. 102 In this study, the magnetic Fe 3 O 4 NPs guided tumor aggregation in vitro in a magnetic eld, while HA as an active targeting ligand enhanced CD44 mediated endocytosis. Besides this, MTX played the dual roles of chemotherapeutic drug and ligand to FR. In vitro experiments showed that tumor cells could be efficiently killed by the synergistic treatment of PTT and chemotherapy based on a high PCE (DT ¼ $38 C, 808 nm, 1.0 W cm À2 , 5 min).
Semiconducting polymers (SP) are chain-like macromolecular materials with semiconductor properties. Due to their advantages of long wavelength absorption and efficient PCE, SP have been widely studied for PTT and photoacoustic imaging. [114][115][116][117][118][119][120] Cai et al. used SP as a photothermal agent for glioblastoma multiforme (GBM) ablation, and investigated the inuences of two targeted ligands in different ratios on tumor internalization ( Fig. 7d and e). 103 SP and aggregation-induced emission molecules were loaded in DSPE-PEG 2000 , meanwhile FA and RGD were conjugated on the surface to achieve specic tumor accumulation. In vitro and in vivo experiments showed Chemical Science that the SP exhibited a perfect light absorption and PCE in the NIR region, which resulted in a rapid temperature rise of $65 C aer 10 min of exposure (808 nm, 0.8 W). Moreover, NPs modied with FA : RGD (25 : 75) were proven to have superior endocytosis ability (Fig. 7f).

DTPTAs based on other materials
Prussian blue (PB) as an ancient pigment has also been used as a PTA to treat tumor in recent years. Taking advantage of the absorption of PB in the NIR region, a multifunctional PTA was designed and synthesized by Du et al. for dual-targeted PTT. 104 In their study, PB NPs were designed as a kind of PTAs, while magnetic Fe 3 O 4 NPs were adsorbed on the surface of upconversion NPs as magnetic targeting ligands. In addition, PEG-HA was then modied on the outermost layer to enhance the water solubility and active targeting of the material. Upon being exposed to NIR light, the composite materials rapidly increased in temperature from 20 to 50 C in 5 min (808 nm, 2.0 W cm À2 ). In vivo experiments showed that they exhibited around four-fold higher aggregation in tumor than control groups without targeting ligands.
The limited penetration depth of light and the thermal resistance caused by heat shock proteins signicantly restricts the therapeutic efficiency of PTT. To solve these problems, Zhang's group developed a nucleus-targeting strategy for lowtemperature PTT in the NIR-II region. 105 The authors rst modied V 2 C quantum dots with a nucleus-targeting TAT peptide (V 2 C-TAT), and then wrapped the V 2 C-TAT quantum dots into an endogenous exosome (Ex). Then, RGD was connected to quantum dots to obtain a cancer cell membrane and nucleus dual-targeted system (V 2 C-PEG-TAT@Ex-RGD). This V 2 C-PEG-TAT@Ex-RGD system exhibited good biocompatibility, long circulation time, endosome escape and amazing PCE in the NIR-II region. Furthermore, this type of PTA can target cells and enter the nucleus to achieve low-temperature PTT with good tumor destruction efficiency.
Boron dipyrromethene (BODIPY) has also been extensively studied due to its prominent extinction coefficient, photostability, photothermal and photodynamic capabilities. In 2013, Chen et al. described an aza-BODIPY derivative (termed as MeOABBr) for multimodal imaging and phototherapy. 106 The designed MeOABBr molecule exhibited a high singlet oxygen quantum yield (up to 84%) and excellent PCE (up to 40%), which made it possible to achieve tumor suppression by phototherapy. Aer mixing with PEG-NH 2 , PEG-TPP and PEG-FA, the molecules formed NPs via self-assembly for tumor elimination. In vivo experiments showed that the synergistic effect of PTT and PDT inhibited and eliminated HeLa tumors, and presented little damage to normal tissues and organs.
Melanin, a natural polymer that exists in many organisms, has been applied as a biocompatible therapeutic agent due to its excellent NIR light absorption and PCE. Wang and coworkers encapsulated melanin NPs (MNPs) and DOX into RGD-modied platelet vesicles to create a dual-targeted drug delivery system, which was used for treating multidrug resistant cancer and inhibiting metastasis. 107 In vivo experiments illustrated that the growth and metastasis of resistant breast cancer were efficiently inhibited by DOX and the MNPs (DT ¼ $14 C, 808 nm, 1.5 W, 10 min).

Conclusions
In recent years, PTT based on dual-targeted ligands has attracted extensive attention, and great advances have been made in this eld. In this perspective, typical targeting strategies can be divided into three parts, "extracellular targets", "intracellular targets", and "subcellular targets" according to the recognizing position. Compared with traditional PTAs, DTPTAs have the great advantage of being able to precisely target cancer cells or organelles. First, dual-targeted PTT shows better selectivity and decreased side effects to the surrounding tissues. Second, dualtargeted therapy makes tumor cells uptake DTPTAs more easily to improve therapy efficiency.
Although great efforts have been made to develop efficient DTPTAs, there are still some challenging problems to be resolved in the future. (a) Photosensitizers: the PCE and potential biological toxicity of photosensitizers should be carefully considered in advance. For examples, inorganic PTAs are known to have higher PCEs in general, but the degradation in vivo is still unclear. Although organic PTAs have inherent excellent biodegradability, they also suffer from the limitations of photostability and thermal stability. Besides this, PTAs that strongly absorb in the NIR-II region show superior potential due to their deep tissue penetration and high maximum permissible energy for the skin. For example, semiconducting polymers improve the possibility of clinical application. 121 (b) Conguration choice: it has been reported that NPs of around 100 nm in size have a longer circulating time in vivo, meaning that it is difficult for them to clear biological barriers. Moreover, rodshaped materials have better permeability in the tumor tissue, which is benecial for the treatment of solid tumors. Many investigations have illustrated that organic PTAs should be synthesized into nanoscale materials. (c) Targeting ligands: given the fact that targeting ligands have different compositions and aiming positions, it is necessary to reasonably design PTAs with targeting ligands. A synergistic targeting strategy is an ideal method by which to prepare specic PTAs, however, targeting ligands should not interfere with each other to identify tumor sequentially. It is worth noting that large ligands can shield small molecule ligands resulting in decreased targeting efficiency, so this should be avoided before the design of the multifunctional PTAs. (d) Methods for the modication of targeting ligands: the methods for the modication of targeting ligands also seriously inuences the efficiency of dual-targeted PTAs for cancer therapy. In general, multifunctional PTAs can be simply coated onto a targeted delivery system, which can easily cause side effects because of the risk of leakage. So, it is highly desirable to connect targeting ligands to PTAs using covalent bonds. Although synthetic methods may be more difficult than physical coating, they provides a safe strategy for in vivo therapy. In order to achieve optimal tumor delivery, in the preparation of an ideal dual-targeted agent the above problems should be considered in advance.
In conclusion, recent advances in dual-targeted PTT for tumors are summarized in this review. DTPTAs have provided an effective and non-invasive alternative with more accurate positioning capabilities compared to traditional PTAs, which possess the extensive advantages of low side effects and high efficiency in tumor therapy. Although scientists have made great efforts in this eld, there are still many challenges to overcome before realizing clinical applications. Prospectively, good biocompatible DTPTAs have low toxicity to organisms, and will be superior choices for clinical application. We believe that this report will provide valuable ideas and references for researchers to enable them to design novel DTPTAs to enhance cancer therapy.

Conflicts of interest
The authors declare no competing nancial interests.