Cancer is still a worldwide malignant disease. According to the latest statistics of CA 2021 (CA: A Cancer Journal for Clinicians), by 2020, the number of cancer deaths in the world had reached 9.96 million (Sung et al., 2021). With the developments in cancer pathology, the research on chemotherapeutic drugs developed for cancer has obtained tremendous advances. However, due to its inherent characteristics, chemotherapy still faces some challenges, such as lack of sufficient targeting ability and limited bioavailability (Subramanian et al., 2016; Rodriguez-Nogales et al., 2018; Mu et al., 2020). More importantly, repeated treatments of chemotherapeutics lead to multiple drug resistance of tumor cells (Kibria et al., 2014) so that patients with malignant tumors (such as non–small cell lung cancer and triple-negative breast cancer) may initially respond to the first-line chemotherapy strategy; their tumor growth is obviously inhibited, but they often have cancer metastasis or recurrence; then, second-and third-line chemotherapy or other treatments are needed (Wu et al., 2010; Shapira et al., 2011; Brufsky et al., 2012). Consequently, the response of tumor cells to the subsequent chemotherapy using various cytotoxic drugs becomes ineffective. Therefore, finding a broad and effective treatment strategy to reverse multidrug resistance is a very important topic in tumor treatment research.

It has been proven that the mechanisms of tumor multidrug resistance mainly include ATP-dependent drug efflux, DNA repair, inhibition of the apoptosis pathway, and tumor tissue heterogeneity in recent years (Figure 1) (Shapira et al., 2011; Housman et al., 2014; Bar-Zeev et al., 2017; Wei et al., 2021). In addition to that, the hypoxic tumor microenvironment (Zeng et al., 2015; Jing et al., 2019), cancer stem cell regulation (Donnenberg and Donnenberg, 2005; Mihanfar et al., 2019), endoplasmic reticulum stress (Bahar et al., 2019), and the immune suppression microenvironment of tumors (Vasan et al., 2019) were also proven to be closely related to the progress of MDR. At present, although the severity of multidrug resistance can be alleviated by various therapeutics, which can mainly increase the sensitivity of tumor cells to chemotherapeutic drugs by reducing the drug efflux of tumor cells, these small molecular drugs still have the same disadvantages as mentioned above, such as lack of targeting and bioavailability (Milane et al., 2011; Gao et al., 2012). More importantly, it is difficult for these drugs to approach the tumor site along with chemotherapy drugs simultaneously, for achieving an ideal synergistic effect and eventually a better clinical treatment effect (Duan et al., 2013; Meng et al., 2013; Zhang M. et al., 2017; Wang and Huang, 2020).

With the continuous development of nanotechnology, rationally designed nanoscale drug delivery systems (NDDSs) with their distinct advantages can be employed to deliver various bioactive payloads to tumor sites for resolving the aforementioned problems (Acharya and Sahoo, 2011; Bertrand et al., 2014; Guo and Huang, 2020; Xu et al., 2021). Through evading or inhibiting tumor MDR mechanisms, NDDSs can effectively improve the enrichment process of chemotherapeutics, or sensitivity of tumor cells, to obtain obvious therapeutic effects (Lim et al., 2019; Wang L. et al., 2019; Ji C. et al., 2021). Therefore, the development of more ideal NDDSs, which can synergistically reverse multidrug resistance, has become one of the important topics in the field of tumor therapy. This review will focus on the different mechanisms of multidrug resistance and systematically summarize the relevant progress of NDDSs which are utilized for synergistically treating tumors effectively and reversing multidrug resistance in recent 3 years, to provide references for exploring the next generation NDDSs targeting MDR.

As illustrated by Figure 1, among these mechanisms involved in MDR, the drug efflux mechanism is the most important one which obtains the most common and intensive research. After continuous stimulation by chemotherapeutic drugs, tumor cells will upregulate drug efflux–related transporters, especially including multidrug resistance protein 1 (MDR1), multidrug resistance–associated protein 1 (MRP1), and breast cancer resistance protein (BCRP) (Shapira et al., 2011; Housman et al., 2014). The physiological processes of these transporters are ATP energy-dependent, so they belonged to the ATP-binding cassette (ABC) transporter family. After recognizing chemotherapeutic drugs with a molecular weight less than 2,000 Da, ABC proteins will pump these drugs out immediately (Ambudkar et al., 2003). As a result, the intracellular drug concentration will be reduced significantly, with decreased or even completely lost chemotherapeutic efficiency. Therefore, targeting the ABC proteins and engineering a novel kind of NDDS for regulating or evading the MDR pathway has become an important research topic for reversing the MDR of tumors.

In addition to the drug efflux mechanism discussed above, tumor cells can also effectively repair the damaged DNA by excision repair or homologous recombination under the stimulation of chemotherapeutic drugs and eventually weaken or even eliminate the efficacy of chemotherapy (Galluzzi et al., 2012; Housman et al., 2014; Hu et al., 2016). Studies have shown that the process of DNA repair by tumor cells often depends on O6 methylguanine DNA methyltransferase (MGMT), suggesting that MDR caused by DNA repair can be reversed through function or expression inhibition to restore the efficacy of chemotherapeutic drugs, but there were few ideal results at present (Galluzzi et al., 2012; Housman et al., 2014; Hu et al., 2016). Recently, Wang, L. et al. proposed a novel systematic combination strategy for overcoming cisplatin resistance using near-infrared (NIR)-light–triggered hyperthermia. The NDDSs, named F-Pt-NP, were composed of two kinds of polymer. Induced by an 808 nm NIR laser, F-Pt-NPs generated mild hyperthermia (43°C) for enhancing the cellular membrane permeability to promote the uptake of drugs and activating cisplatin by accelerating the glutathione consumption. Moreover, it could increase the Pt-DNA adduct formation and possibly the formation of a portion of irreparable Pt-DNA interstrand crosslinks, which significantly inhibited the repair of DNA. In vivo, on a patient-derived xenograft model of multidrug-resistant lung cancer (A549DDP), the efficacy of the F-Pt-NPs treatment group showed a tumor inhibition rate of 94% (Wang L. et al., 2021) (Figure 5A). Similarly, Xin, J. et al. reported a carrier-free aquo-cisplatin arsenite multidrug nanomedicine loaded with cisplatin and arsenic trioxide prodrugs simultaneously. This nanomedicine achieves a high loading capacity and pH-dependent controlled release of the drugs. Cisplatin and arsenic trioxide in this nanocomposite can induce massive DNA damage and inhibit the activity of PARP-1, which is closely related to the DNA damage repair in cisplatin-resistant tumor cells. Gene expression profiles demonstrated the expressions of proto-oncogenes, and DNA damage repair–related genes MYC, MET, and MSH2 were reduced, respectively. On the other hand, the expressions of tumor suppressor genes PTEN, VHL, and FAS were increased. This nanocomposite showed strong cytotoxicity against cisplatin-resistant ovarian tumor cells and could overcome cisplatin resistance effectively (Xin et al., 2019).

The reactivation of the anti-apoptotic process of tumor cells plays another important role in resisting the attack in chemotherapeutics, which involves promoting survival, anti-apoptotic regulator Bcl-2, and its downstream nuclear factors-κB (NF-κB), and so on. More than 50% of cancers have defects in the mechanism of apoptosis. The most characteristic of these abnormalities is the increased expression of Bcl-2 family proteins (Housman et al., 2014; Hu et al., 2016). Targeting these anti-apoptotic factors to improve the killing efficiency of chemotherapeutics against tumor cells is a promising tumor therapy strategy (Pan et al., 2020; Wang H. et al., 2020; Xie et al., 2020; Sivak et al., 2021). For instance, Sivak, L. et al. described a polymer biomaterial composed of the antiretroviral drug ritonavir derivative (5-methyl-4-oxohexanoic acid ritonavir ester; RD), covalently bound to the HPMA copolymer carrier via a pH-sensitive hydrazone bond (P-RD). Importantly, RD inhibited STAT3 phosphorylation in CT26 cells (murine colon adenocarcinoma) in vitro and the expression of the NF-κB p65 subunit, Bcl-2, and Mcl-1 in vitro. P-RD nanomedicine showed significant antitumor activity in CT26 and B16F10 tumor-bearing mice (Sivak et al., 2021). Additionally, Pan. Q. et al. constructed a multifunctional DNA origami-based nanocarrier for co-delivery of a chemotherapeutic drug (doxorubicin, DOX) and two different antisense oligonucleotides [ASOs; B-cell lymphoma 2 (Bcl2) and P-gp] into drug-resistant cancer cells for enhanced therapy. Experiments revealed that the origami could protect ASOs against nuclease degradation in 10% FBS. Moreover, with the synergetic effect of co-delivery of multi-ASOs and DOX, the anticancer assay showed that Apt-DOA (Apt-DOX-origami-ASO) could circumvent multidrug resistance and significantly enhance cancer therapy in Hela/ADR and MCF-7/ADR cells (Pan et al., 2020) (Figure 5B).

Recent studies have shown that a small number of tumor cells have the characteristics of stem cells and usually have drug resistance ability, due to the heterogeneity of tumor tissues (Housman et al., 2014). In addition, another small number of adult tumor cells also have the same ability (Housman et al., 2014). In conventional tumor treatment, these drug-resistant tumor cells can survive and continue to proliferate as seeds over time, leading to recurrence (Housman et al., 2014). Some of these drug-resistant cancer cells may enter the blood circulation and may form metastases in distant organs (Mansoori et al., 2017; Bukowski et al., 2020). Therefore, through the rational design and construction of new NDDS, accurately targeting cancer stem cells or adult cancer cells that have developed drug resistance can effectively improve the efficiency of reversing multidrug resistance and inhibiting tumor growth. As discussed before, the cancer stem cell-specific–targeted mesoporous silica (mSiO₂)-dendritic polyglycerol (dPG) nanocarriers co-delivered the chemotherapy drug DOX and the P-gp inhibitor tariquidar (Tar) for reversing MDR and enhancing chemotherapy to breast cancer stem cells (Pan et al., 2021). In addition to the active targeting strategy, relying on the intelligent change of nanomedicine size for deeply penetrating the tumor tissue and improving the clearance of cancer stem cells is also an important strategy for reversing MDR efficiently. Based on that, a particular kind of morphology-tunable nanomedicine was developed. Chemotherapy and immune checkpoint blocking therapy for large tumor cells and cancer stem cells were integrated into a drug delivery system. The particle size shrank when the nanoparticle transferred from circulation to tumor tissues, favoring both pharmacokinetics and cellular uptake, meanwhile achieving the sequential drug release where needed. This nanomedicine reduced the proportion of cancer stem cells and enhanced the therapeutic efficacy against the tumor and thus prolonged the survival time of mice (Lang et al., 2019) (Figure 5C).

With the continuous development of nanotechnology, the application of NDDSs in cancer treatment has received extensive attention all over the world. In fact, according to the NIH, there are 110 clinical trials involving the application of nanotechnology for cancer treatment nowadays (Gonzalez-Valdivieso et al., 2021). Among them, Doxil^® is a representative example, which is the first FDA-approved NDDS for clinic application since 1995, consisting of PEG-liposome and doxorubicin. Compared to free doxorubicin, Doxil^® has some important advantages, such as a 100-times longer half-life inside the body circulation and a reduction in cardiotoxicity. Due to that, Doxil^® has been applied in several clinical therapies of malignant tumors, such as myeloma and ovarian cancer (Barenholz, 2012). Similarly, another FDA-approved NDDS is Abraxane^®, which is composed of albumin particles and paclitaxel. The results of clinical studies showed that, compared with Taxol^®, Abraxane^® can significantly reduce the toxic side effects caused by Cremophor^® in Taxol^® and increase the maximally tolerated dose of patients by 80% (Green et al., 2006; Desai, 2016).

In addition to these, other novel NDDSs, which are constructed based on different strategies, have entered the clinical research from preclinical research in the laboratory, including passive-targeting tumor lesions via the EPR effect or active-targeting tumor sites relying on the antibody technology, such as PK2, an HPMA polymer doxorubicin conjugate, was the first targeting nanotherapeutic agent reaching the clinic (Julyan et al., 1999; Hopewell et al., 2001; Kopeček and Kopečková, 2010; Gonzalez-Valdivieso et al., 2021). Notably, in addition to the primary tumor-targeting NDDSs, some NDDSs aiming at reducing tumor metastasis have also been gradually translated into clinical studies, such as DaunoXome^® which was employed for the treatment against metastatic ovarian cancer and metastatic breast cancer (Eckardt et al., 1994; Forssen, 1997). Unfortunately, for patients with MDR, although in preclinical laboratory studies, researchers have developed many potential NDDSs described above, which can obtain exciting in vivo/vitro therapeutic effects in animal models, especially mouse models; there is still no NDDS for reserving MDR that has been transferred into the clinic due to the great differences between human and animal models, as well as the significant challenges brought by the complex mechanism of MDR to the construction strategy of NDDSs.

Thus, novel strategies for overcoming these following obstacles that limit the clinical translation process of NDDSs are needed. First, although nanoscale carriers can enhance drug penetration and accumulation within tumor lesions, the actual number of drugs that can eventually enter tumor tissues is still limited due to the generally low drug content in NDDSs, and the vehicles themselves have clinical risks (Stiegler et al., 2010; Yang et al., 2019; Chen S. et al., 2020). At present, some nanoscale carriers are related to oxidative stress, adverse inflammatory reaction, and genotoxicity (Liu et al., 2013; Zuo et al., 2013; Yuan et al., 2020). Based on this, researchers began to focus on the development of carrier-free NDDSs. For example, siRNA itself was used as the carrier for drug delivery by using gene origami technology and a kind of carrier-free NDDS composed of the drug-chemogene conjugate. This drug-chemogene was formed by two paclitaxel (PTX) molecules with a floxuridine (FdU)-integrated antisense oligonucleotide (terminated chemogene), which take fluorescent dithiomaleimide (DTM) as a linker. This carrier-free drug delivery system obtained the ability to knock down the expression of P-gp and the synergistic inhibitor effect (Zhu et al., 2020). Similarly, we recently combined the COX-2 inhibitor indomethacin (IND) with the chemotherapeutic drug paclitaxel through a simple self-assembly to obtain a series of nanomedicine with different morphologies and achieved ideal results in preliminary antitumor therapy (Zhang C. et al., 2019). Because COX-2 inhibitors can effectively inhibit the production of ATP, this carrier-free NDDS has the potential to reverse tumor multidrug resistance and was of great value for further research. The above research can not only effectively reduce the use of inactive carrier materials and effectively improve the drug content but also greatly reduce the preparation difficulty of NDDSs and then has a broader prospect of clinical transformation. However, how to rationally integrate the existing concept of developing NDDSs for multidrug-resistant tumors with carrier-free NDDSs is still an urgent problem to be solved by researchers, which needs to make great efforts.

In addition, because the mechanism of MDR is very complex and changeable, simply targeting one mechanism is far from meeting the clinical needs. Therefore, exploring a reasonable drug combination to achieve multi-channel reversal of multidrug resistance and tumor treatment is a further research direction in this field. At present, researchers have made some corresponding exploration. For instance, Liu, X. et al. developed a novel kind of gold nanoparticle (AuNP) modified by multifunctional molecular beacons (MBs), which was utilized as a vehicle for loading three different drug resistance–related mRNAs (MDR1 mRNA, MRP1 mRNA, and BCRP mRNA). After uptake by cells, MBS AuNP would inhibit the translation of drug resistance–related mRNAs and reduce the flux protein expression. This NDDS was provided to have the ability of synergistic inhibiting MDR and in situ imaging of drug resistance–related mRNAs in living cells (Liu X. et al., 2019). Delivering drugs by passing the drug efflux mechanisms of MDR is also a solution to those problems discussed above. For example, telomerase, as a biomarker of tumor cells, is expressed in more than 90% of tumor cells. The inhibition of telomerase can effectively increase the chemosensitivity of tumor cells. Therefore, the construction of new NDDS-targeting telomerase has also achieved an obvious therapeutic effect on multidrug-resistant tumors (Wu Y. et al., 2020).

In conclusion, even though key steps are still needed to improve their possibility of clinical translation, NDDSs are becoming more and more promising to eventually solve MDR of tumors in clinical application. Based on this fact, we have reason to believe that in the foreseeable future, there will be an NDDS that can effectively solve the MDR of tumors and be translated into clinical application.