Cancer is a serious threat to human health, and its morbidity and mortality are increasing year by year in the world, which is one of the main causes of death in the world^[1,2]. At present, cell therapy, immunotherapy and other emerging methods are not yet fully mature, surgery, radiotherapy and chemotherapy are still the three main means of cancer treatment^[3,4]. Among them, chemotherapy is mainly through oral, subcutaneous or intravenous injection of anti-tumor drugs, which are distributed throughout the body through blood circulation and produce therapeutic effects after reaching a certain blood concentration in the body. However, chemotherapy drugs have obvious toxic and side effects on normal tissue cells in the human body while eliminating tumor cells, and long-term chemotherapy can easily lead to multidrug resistance (MDR) of tumor cells, which may even lead to recurrence and metastasis of tumors and greatly reduce the therapeutic effect^[3,4].

The mechanism of MDR is complex, which can be roughly divided into cellular mechanism and non-cellular mechanism^[5,6]. Cellular mechanisms mainly include increased drug efflux, decreased drug uptake, defects in apoptotic pathways, repair of damaged DNA, and autophagy, while non-cellular mechanisms are mainly related to the overexpression of ATP-binding cassette (ABC) transporters. P-glycoprotein (P-gp) is one of the important transporters in the process of drug efflux, which has been widely studied since its discovery. The overexpression of P-gp on the tumor cell membrane leads to the efflux of anti-tumor drugs against the concentration gradient and the decrease of intracellular drug concentration. At the same time, the efflux of a drug will also affect the therapeutic effect of other chemotherapeutic drugs, resulting in MDR of tumor cells. Studies have found that Combination therapy with two or more drugs has gradually become a new trend in cancer treatment, and appropriate Combination of drugs can regulate signaling pathways by different mechanisms.Restore the sensitivity of tumor cells to chemotherapeutic drugs, reverse the MDR of tumor cells, effectively reduce the dosage of drugs while reducing the toxic and side effects of drugs, and play a better anti-tumor effect than single drug treatment^[7].

Doxorubicin (DOX), also known as Doxorubicin, is an anthracycline antibiotic with broad-spectrum anti-tumor activity. It was first isolated in 1969 when mutagenizing Streptomyces peucetius, which produces daunomycin. It has become one of the commonly used chemotherapy drugs for leukemia, lymphoma, breast cancer, uterine cancer, ovarian cancer and lung cancer^[8,9]. According to the ClinicalTrials website, as of January 2023, there are 2457 clinical trials on DOX in the world, and many products (such as Adriamycin^®, Doxil^®, Myocet^®, etc.) Have been approved for marketing in different countries. DOX is an orange-red crystalline powder with a density of 1.61 g/cm³ and a melting point of 205 ° C. It is soluble in dimethyl sulfoxide and tetrahydrofuran, insoluble in acetone, benzene, chloroform and ether, and can exist stably at normal temperature and pressure, but it needs to be stored in a dark and cool place. The water solubility of DOX is very low (~ 1.18 mg/mL), and its water solubility can be greatly improved (50.0 mg/mL) by converting it to the hydrochloride form (DOX · HCl)^[9]. As shown in Fig. 1, DOX has an amphiphilic chemical structure containing a water-insoluble adriamycin unit and a water-soluble amino sugar unit.

It has been reported that DOX acts on nucleic acids in dividing cells for therapeutic purposes mainly through two mechanisms: (1) inserting between base pairs of DNA strands to inhibit DNA and RNA synthesis in rapidly proliferating cells by blocking replication and transcription processes^[8,9]; (2) Production of iron-mediated free radicals to cause oxidative damage to cell membranes, proteins, and DNA. However, anthracyclines such as DOX can not only cause bone marrow suppression, but also damage the normal myocardial cell tissue of the human body, causing dose-cumulative cardiotoxicity and irreversible myocardial damage^[10,11]. At the same time, it has also been reported that DOX can cause mitochondrial dysfunction in normal cells, produce a large number of reactive oxygen species (ROS), lead to oxidative stress, neuroinflammation and apoptosis, and ultimately induce cognitive dysfunction^[12]. These toxic side effects largely limit the clinical application of DOX. As no other chemotherapy drugs have been found to completely replace anthracyclines, researchers have been committed to finding ways to reduce the toxic and side effects of anthracyclines for a long time, and the rapid development of nanotechnology in recent years has provided a powerful tool to solve these problems. More and more studies have shown that the combination of DOX with other chemotherapeutic drugs, gene drugs, gas molecules or natural drugs can reduce the toxic and side effects of drugs on normal cells and tissues, and can reverse the MDR of tumor cells to a certain extent, which is expected to achieve the synergistic therapeutic effect of "1 + 1 > 2"^[13⇓~15]. By introducing targeting molecules and stimuli-responsive groups into the co-delivery carrier, the enrichment of the drug delivery system at the tumor site and the internalization of specific cells can be improved, and the drug can be rapidly released in the lesion area in response to endogenous (pH, hypoxia, redox, enzyme) or exogenous (light, heat, ultrasound) stimuli at the tumor site^{[16⇓⇓⇓~20]}. In this paper, we will review the research progress of DOX combined with other chemotherapeutic drugs, gene drugs, gas molecules and natural drugs in the treatment of cancer in recent years, and look forward to its development trend.

Single chemotherapeutic drug therapy usually can only temporarily delay tumor growth, and it is easy to cause MDR and reduce the efficacy, which promotes the research of combination chemotherapy to overcome MDR. The cocktail therapy for AIDS is a successful example of drug combination therapy, which shows that the combination of drugs can effectively control the disease. As one of the most commonly used chemotherapeutic drugs, DOX is often used in combination with other chemotherapeutic drugs, which can regulate different signaling pathways in tumor cells at the same time, effectively overcome MDR of tumors, and reduce systemic toxicity by reducing the dose of a single drug, thus playing a synergistic therapeutic effect^[21].

Camptothecin (CPT) and paclitaxel (PTX) are natural antitumor drugs originally extracted from plants, and are also commonly used as first-line chemotherapeutic drugs in clinic, and have different antitumor mechanisms from DOX^{[22⇓⇓~25]}. Therefore, the combination of DOX and these two drugs can simultaneously use different mechanisms for synergistic therapy. Our research group designed and synthesized the side chain reduction-sensitive CPT-dextran prodrug (Dex-ss-CPT) and acid-sensitive DOX-dextran prodrug (Dex-hyd-DOX) (Figure 2a), which effectively improved the water solubility of CPT and DOX, and the mixed nanomicelles formed by the co-assembly of the two prodrugs could achieve the synergistic therapeutic effect and inhibit the MDR of tumor cells to a certain extent^[26]. Subsequently, we further coupled CPT and DOX to the end group and side chain of biodegradable polyphosphate, respectively, to prepare a pH/reduction dual stimulus-responsive prodrug nanoparticle, which can also release DOX and CPT simultaneously in the tumor microenvironment (TME)^[27]. This polymeric prodrug strategy effectively improves the stability of the drug in the blood circulation, solves the problem of drug leakage, and can achieve site-specific controlled release of the drug in the TME^[28,29]. Using a similar strategy, Li et al. Used reduction-sensitive disulfide bonds to connect DOX and PTX to the end of polyethylene glycol to prepare two polymer prodrugs, and then co-assembled to form mixed micelles to achieve drug co-loading and responsive release^[30]. In addition, by adjusting the polymer chain structure to control the proportion of drug loading, the synergistic controlled release of different proportions of drugs can be achieved, and the therapeutic effect can be further improved^[31].

Cisplatin (CDDP), also known as cisplatin dichloride, is effective for breast cancer, nasopharyngeal carcinoma, osteosarcoma and other tumors, and the combination therapy of DOX and cisplatin has also been widely studied. Cisplatin can induce biological alkylation reaction and cause DNA damage during tumor therapy, while DOX can act as an anthracycline topoisomerase Ⅱ (TOP2) inhibitor to prevent effective DNA repair to a certain extent, thus reflecting the synergistic therapeutic effect^[32⇓~34]. However, the side effects and pharmacokinetic differences of the two drugs are still important problems to be overcome in the realization of combination therapy. In this regard, researchers suggest that nanoparticle systems have the potential to improve the current problems. Zhang et al. constructed a cross-linked nanogel based on natural hyaluronic acid (HA) in an aqueous system to co-load DOX and cisplatin. DOX was loaded on HA through electrostatic adsorption, while cisplatin could coordinate with the carboxyl group on HA to stabilize the nanoparticles (Figure 2b)^[35]. The self-crosslinked nanogel shows good blood circulation stability and intratumoral enrichment ability, and can simultaneously transport DOX and cisplatin into tumor cells and release the drug in a weak acidic environment in the cells. The mouse tumor inhibition experiment showed that compared with the blank group and the combination of the two free drugs, the self-crosslinked nanogel showed more excellent performance of inhibiting the growth of osteosarcoma. Yang et al. Also used electrostatic adsorption and coordination to connect DOX and cisplatin with thermosensitive triblock copolymer to form nanogels, and cisplatin formed a compact three-dimensional crosslinked network shell around the DOX core, so that DOX and cisplatin could be released in parallel to maximize their synergistic therapeutic effects^[36]. In addition, due to the good thermosensitivity of the designed triblock copolymer, the nanogel can form a macroscopic hydrogel after intratumoral injection, which further improves the intratumoral retention time of DOX and cisplatin.

MDR and toxic side effects caused by long-term use of DOX limit the further development of drug therapy. With the in-depth understanding of the physiological mechanisms and cellular activities related to tumorigenesis, anti-tumor therapies have become more diversified and targeted^[37]. Gene therapy refers to the use of appropriate vectors to introduce exogenous genes into tumor cells, through silencing the genes that regulate the function of tumor cells and down-regulating the expression of related proteins to achieve the purpose of eliminating tumor tissues. However, gene therapy is also limited by gene degradation, low cell transfection efficiency and short blood circulation time, so its application needs to rely on more efficient nano-carrier system^{[38⇓⇓⇓⇓⇓~44]}. Chemotherapy with a single drug can only block one signaling pathway in the process of tumor growth, and it is difficult to achieve sustained and complete anti-tumor effect. Chemotherapy and gene therapy are used for combined anti-tumor, and specially modified nanocarriers are used to transport chemotherapeutic drugs and genes to tumor lesions at the same time, which can effectively inhibit tumor MDR, reduce drug dosage, reduce carcinogenic protein expression, and improve anti-cancer effect, with good prospects for development^[45]. There are many types of nanocarriers for co-delivery of chemotherapeutic drugs and genes, including liposomes, nanoparticles, and polymeric micelles. These nanocarriers can increase the enrichment of chemotherapeutic drugs and genes in tumor tissues through enhanced permeability and retention (EPR) effect or active target direction, and then selectively release them at the lesion site^[38,39].

Compared with normal cells, tumor cells are unable to undergo normal apoptosis, because the expression of tumor suppressor genes in tumor cells is often inhibited or down-regulated, and the induction of apoptosis requires the expression of tumor suppressor genes. P53 gene is the first discovered important tumor suppressor gene, which can slow down or monitor cell division, and its damage or mutation is closely related to the occurrence of many tumors.Therefore, the use of plasmid DNA (pDNA) has become a classical gene therapy, which can correct the disorder of human gene structure or function, inhibit the replication of pathogenic genetic material, and achieve the purpose of disease treatment. Zhang et al. Used acid-sensitive hydrazone bond to connect DOX to polyethyleneimine (PEI) to prepare PEI-hyd-DOX prodrug, and added pGL-3 plasmid to obtain PEI-hyd-DOX/pGL-3 co-carrier nanoparticles. The study found that the co-carrier system could reduce the toxicity of DOX and improve the transfection efficiency of pGR-3^[46]. Nie et al. Used graphene oxide (GO) to immobilize PEI to form a GO-PEI skeleton, and then stacked DOX on the surface of GO through π-π bonds, while the negatively charged p53 gene could be co-loaded with the positively charged PEI chain segment through electrostatic interaction, and finally the DOX/GO-PEI/p53 complex was obtained^[47]. The results showed that the co-carrier system released DOX faster under acidic conditions than under neutral conditions, and had lower toxicity than PEI/DNA nanoparticles, and the complex could release DOX and express p53 in sequence. Our research group used various reactions such as ring-opening polymerization, reversible addition-fragmentation chain transfer (RAFT) polymerization and "click" chemistry to construct a multifunctional carrier based on polyphosphate for co-loading DOX and p53 (Figure 3A), and the results showed that the carrier had good biocompatibility and could play a synergistic role in inhibiting tumor cell proliferation^[48]. Targeted molecular bonding to the carrier surface will further increase antitumor efficacy, as tumor cells often overexpress certain specific receptors on their surface. Chen et al. Designed and synthesized an amidated cell-penetrating peptide (aTAT) -modified chitosan/polylysine graft copolymer for targeted delivery of p53 and DOX^[49]. The results show that the targeted modification vector can effectively and actively target the tumor site, increase the tumor enrichment of the vector, achieve high gene transfection efficiency and drug delivery, and also has high anti-tumor effect in vivo.

Small interfering RNA (siRNA) is usually a double-stranded RNA of 20 to 25 nucleotides in length, which can inhibit the expression of specific genes by blocking their translation or transcription. Tumors are characterized by uncontrolled cell growth due to abnormalities caused by gene mutations, and overexpression of some genes is also one of the causes of tumors^[50]. In addition, P-gp, an important transporter in drug efflux, is mainly encoded by MDR1 gene, and the overexpression of P-gp is the main cause of MDR in tumor cells. Therefore, the combination of specific siRNA and chemotherapeutic drugs can promote the synergistic effect of chemotherapy and gene therapy through targeted delivery, down-regulate the expression of specific genes or transporters, increase the accumulation of chemotherapeutic drugs in cells, and bring about stronger cell killing effect^[42,51]. Shuai et al. Designed and synthesized a class of complex micelles composed of two block copolymers polyethyleneimine-poly (ε-caprolactone) and folate-polyethylene glycol-polyglutamic acid for targeted co-delivery of hydrophobic DOX and hydrophilic BCL-2 siRNA, which played a good synergistic effect in both human hepatocellular carcinoma cell Bel-7402 and mouse glioma C6^[52,53]. Yin and Tang et al. Also combined hydrophobic interaction and electrostatic interaction to construct a multifunctional composite carrier based on modified chitosan for co-loading DOX and siRNA, which achieved good synergistic anti-tumor effect in cell and animal experiments^[54,55]. Cheng et al. Prepared mPEG-PCL-g-PDMAEMA micelles for co-delivery of DOX and Cy5-siRNA by nanoprecipitation technique (Figure 3B), and the confocal images showed that both of them appeared not only in the cytoplasm of tumor cells but also in the nucleus, proving that the micelles had great potential as a carrier for efficient delivery of DOX and siRNA^[56]. Li et al. Used PEI and PEG to modify molybdenum disulfide nanosheets with high near-infrared photothermal conversion efficiency, and co-loaded DOX and siRNA to obtain a multifunctional nano-co-delivery system with chemical, genetic and photothermal therapy^[57]. The results showed that the system had good biocompatibility, photoresponsiveness and stability, achieved faster drug release under low pH and near-infrared light triggering conditions, and had stronger killing effect on drug-resistant tumor cells. The above literature reports need to use hydrophobic interaction and electrostatic interaction to co-load DOX and siRNA, and the synthesis route of the vector involved is relatively complex. Recently, Wang and Yang et al. Developed a carrier-free nanoassembly PEG @ D: siRNA composed of a prodrug of PEG and DOX linked by an acid-sensitive hydrazone bond (PEG-hyd-DOX) and siRNA^[58]. PEG-hyd-DOX prodrug can load siRNA through π-π stacking and electrostatic interaction to form carrier-free nanoassemblies with high drug content (siRNA content of 4.13%, DOX content of 21.67%). The findings suggest that this siRNA-loaded nanoassembly can significantly increase tumor-infiltrating T lymphocytes and enhance interferon-γ expression, thereby enhancing the immunogenic cell death effect of DOX prodrug, and finally significantly enhance the anti-tumor immune response and effectively inhibit tumor growth. Moreover, the facile synthetic route of this nanoassembly is expected to be a versatile strategy for the efficient preparation of co-loaded chemotherapeutic drugs and genes.

MicroRNA (miRNA) is a non-coding microRNA of 19-24 nucleotides in length, which can participate in the regulation of tumor cell dynamics by binding to complementary sequences of target messenger RNA (mRNA) and promoting its translational inhibition or degradation. The combination of miRNA with specific sequences and small molecule chemotherapeutic drugs can promote tumor cell apoptosis and autophagy, reverse tumor epithelial-mesenchymal transition, inhibit tumor angiogenesis, and down-regulate the expression of ABC transporters, thus showing synergistic anti-tumor effects^[59]. Gao et al. Prepared a disulfide-cross-linked amphiphilic polyamino acid copolymer for co-loading DOX and miR-34a to synergistically treat prostate cancer^[60]. The polyarginine on the outer side of the block copolymer structure has excellent endocytosis and gene compression ability, the hydrophobic octadecyl chain is used for loading DOX, and the pH-sensitive polyhistidine in the middle of the structure can be protonated in the cytoplasm to facilitate the release of drugs. The results showed that the composite micelle system not only reduced the cardiotoxicity of DOX, but also down-regulated the expression of silent information regulator 1 (SIRT1) and inhibited the proliferation of human prostate cancer cells DU145 and PC3, showing better anti-tumor effect than DOX or miR-34a administration alone. Torchilin et al. Designed a dual matrix metalloproteinase-2 (MMP-2) and reduction-sensitive prodrug micelle to deliver DOX and miR-34a^[61]. The composite prodrug micelle mainly consists of three parts: DOX and PEG2k bonded by MMP-2-sensitive peptide, miR-34a and phospholipid bonded by disulfide bond, and PEG-PE modified by cell-penetrating peptide TAT. The results showed that the micelles showed strong cytotoxicity in HT1080 cells overexpressing MMP-2, and could release miR-34a molecules under the condition of high concentration of glutathione (GSH) reduction in tumor cells, which promoted the down-regulation of oncogenes such as Bcl2, notch1 and survivin. Shi et al. Recently reported an amphiphilic phosphorus-containing dendrimer micelle co-loaded with DOX and miR-21i for the combined treatment of triple-negative breast cancer (Figure 3C), verifying the synergistic efficacy of the co-loaded system in cell and animal trials^[62]. The surface of the nanoparticle is modified by natural or biomimetic cell membrane materials, and the obtained cell membrane-coated nanoparticle not only retains the original physical and chemical properties of the nanoparticle,At the same time, the modified nanoparticles can be protected from the attack of the immune system by the function of proteins and polysaccharides on the surface of the cell membrane, so that the modified nanoparticles have longer circulation time in vivo and tumor targeting ability^{[63⇓⇓~66]}. Paulmurugan et al. Constructed a cancer-platelet fusion membrane vesicle (CPMV) using cell membranes extracted from breast cancer cells and platelets, and loaded miRNAs (antimiR-10b and antimiR-21) with microfluidic technology to treat triple-negative breast cancer^[67]. It has been found that CPMVs can efficiently recognize their source cells and avoid phagocytosis by macrophages. After systemic administration in mice, CPMVs showed longer circulation time and site-specific accumulation effect, and the delivered miRNA could improve the sensitivity of triple-negative breast cancer to DOX, thereby enhancing the synergistic therapeutic effect.

Suicide gene is an exogenous gene fragment that can express toxic substances from different sources in target cells, and targeted delivery of appropriate suicide genes to tumor cells or surrounding tissues can effectively cause their death^[68]. Hajitou et al. Developed a non-pathogenic phage vector containing human adeno-associated virus DNA in the shell to combine with DOX for anti-tumor therapy. The results showed that DOX could promote the accumulation of suicide genes in the nucleus by destroying the stability of the nuclear membrane, thus changing the intracellular trafficking of the vector^[69]. Genes can not only be used in cancer therapy, but also be designed as drug delivery carriers, which provides a new idea for the development of new drug delivery systems. Yang et al. First used the rolling circle amplification method to obtain long single-stranded DNA (lsDNA) from tumor cell-associated TK1 mRNA, and then self-assembled the lsDNA, fluorescent indicator probe HP, and DOX into DNA nanospheres (Fig. 3D), which can react with intracellular mRNA in two steps to release DOX, and can effectively overcome the efflux of drug-resistant cells, thereby improving the therapeutic effect of DOX^[70]. Li et al. Used DNA tetrahedron as a carrier to co-load DOX and CpG oligonucleotides for immunotherapy, and the formed nanoparticles showed synergistic effects in enhancing immunostimulatory activity and improving anti-tumor efficiency^[71].

Gas therapy, as a new therapeutic strategy, has been paid more and more attention in cancer treatment. Many gaseous molecules, such as O₂, CO, NO, H₂S, and SO₂, play important regulatory roles in various physiological processes of cells, tissues, or organisms, and thus exhibit great potential in the treatment of a variety of diseases, including tumors^{[72⇓⇓⇓⇓~77]}. Studies have shown that appropriate concentrations of physiologically relevant gas molecules can effectively reduce the Warburg effect of tumor cells, inhibit the proliferation of tumor cells and accelerate their apoptosis without damaging normal cells, so as to achieve the therapeutic effect of cancer. In addition, it has been found that some physiologically active gas molecules can cooperate with other conventional cancer treatment methods through different mechanisms to improve the efficacy. A variety of nanosystems have also been designed to co-carry DOX and gas molecules (or donor molecules capable of producing gas) to achieve combined treatment of tumors.

As mentioned above, the antitumor effect of DOX itself has been recognized, but the dose-cumulative cardiotoxicity limits its wide clinical application. Therefore, finding suitable cardioprotective drugs has also become a way to expand the clinical application of DOX. Dexrazoxane, as a clinically approved cardiotoxic protective drug, can prevent the cardiotoxicity of anthracycline chemotherapy while maintaining the anti-tumor activity of the drug^[111]. However, because the cardiotoxicity of DOX has the characteristics of dose accumulation, the therapeutic intervention time of dexrazoxane is still controversial, and there are some side effects, so the cardioprotective drugs of anthracycline chemotherapy drugs such as DOX are still being studied^[112].

With the development of research, the cardioprotective effect of natural compounds has been paid more and more attention. Natural compounds in some plants can enhance the efficacy and reduce the cardiotoxicity of DOX, and their combination with DOX can enhance their clinical application value. Flaxseed, a common health food, has been shown to have the same level of preventive effect as Perindopril on DOX-induced cardiotoxicity^[113]. Costantini et al. Found that the combination of phenolics (PEFSO) extracted from linseed oil and DOX could inhibit the proliferation of MCF-7 and MDA-B231 breast cancer cells at the same time, and reduce the effective dose of DOX to reduce its toxic and side effects^[114]. Berberine, the main component of Rhizoma Coptidis, has its own anti-tumor and cardioprotective effects, and its combination with DOX can prevent the heart injury caused by Berberine, but its mechanism is not yet clear^[115]. The existing experiments show that the cardioprotective effect of berberine may involve multiple mechanisms. Su et al believed that berberine may show cardioprotective effect by increasing the level of intracellular Ca²⁺ and reducing mitochondrial dysfunction^[115]. Xiong et al. Found that berberine could protect cardiomyocytes through the SIRT1/p66Shc-mediated pathway^[116]. Zhang et al. Have recently shown that low doses of berberine have cardioprotective effects by stabilizing Bcl-xL protein levels affected by DOX and dissociating Bcl-xL and Beclin1 to restore mitophagy in cardiomyocytes^[117].

Oxidative stress is one of the main mechanisms of DOX-induced cardiotoxicity^[118]. DOX activates NADPH oxidase (NOX) in the heart to produce excessive ROS, which causes DNA damage, p53 protein phosphorylation and cardiomyocyte apoptosis, and ultimately leads to different degrees of myocardial disease^[119]. Karimi et al. Found that natural flavonoid small molecules such as Luteolin, Quercetin and Paeoniflorin can reduce the generation of ROS by generating stable free radicals and preventing the aggregation of NOX subunits (mainly NOX2 and NOX4), and ultimately effectively attenuate the cardiotoxicity caused by DOX, thus becoming a better "antioxidant" to participate in the adjuvant treatment of chemotherapy^[120]. However, it is not known whether the combination of flavonoids and DOX will produce drug-drug interactions. Researchers examined the pharmacokinetic characteristics of this type of therapy through in vitro experiments of drug-metabolizing enzymes, and found that the consequences of drug-drug interactions depend on whether the enzyme inhibitors are reversible, that is, the drug-drug interactions produced by the use of class I and class II chemotherapy-related CYP-metabolizing enzymes are not the same^[121]. At the same time, current studies have shown that the bioavailability of flavonoids in human body is low, and there is still a long way to go before they are put into clinical use^[120].

Anti-tumor combination therapy is a widely concerned research direction in recent years, which is expected to overcome the common problems of tumor heterogeneity, complexity and drug resistance in traditional chemotherapy, and minimize the toxic and side effects on normal tissues and organs on the basis of achieving synergistic therapeutic effect. In this paper, the broad-spectrum anti-tumor drug DOX is used as the core, and its combined anti-tumor treatment system with other chemotherapeutic drugs, gene drugs, gas molecules and natural drugs is summarized. The different components of these combination therapy strategies can eliminate tumor cells through their own mechanisms, and the stimuli-responsive targeted therapy system can be designed with the help of the characteristics of TME to achieve site-specific drug release at the lesion site.

However, while the current combination therapy strategy has made some progress, it also faces many challenges. (1) In addition to the combination treatment strategies summarized above, there are many other options. For example, the combination of DOX and protein drugs, anti-vascular therapy drugs, immunotherapy drugs, photodynamic therapy reagents, etc., the drug combination that can target primary tumors and metastatic tumors respectively, the drug combination that can target tumor tissues and circulating tumor cells, etc.^[122⇓~124]; (2) How the co-loaded drug carrier maintains high stability and less drug leakage in the blood circulation, and how it can overcome the obstacles of dense extracellular matrix and high tissue osmotic pressure to efficiently penetrate into the tumor tissue and release drugs after reaching the tumor site^[125⇓~127]; (3) Anti-tumor drug combination is not a simple superposition of drugs, but requires full consideration of the mechanism of action of different drugs, cell kinetics, drug toxicity, as well as the timing of drug administration, dose optimization, drug interactions and other issues, rigorous analysis and experiments to obtain the optimal solution of the combination strategy; (4) With the rapid development of combination therapy, many nanosystems with different functions have emerged. However, considering the complexity of biological systems and the metabolism of nanomaterials, it is necessary to develop nanosystems with simple structures and fewer components to reduce their uncertainty in vivo, which is also conducive to the next step of clinical translation^[128]. Anti-tumor combination therapy is an emerging field with broad application prospects. Although it still faces many difficulties, it is believed that with the vigorous development of interdisciplinary integration of tumor biology, nanotechnology and targeted drug delivery, more breakthroughs will be made in the near future.