Traditional Chinese medicine-combination therapies utilizing nanotechnology-based targeted delivery systems: a new strategy for antitumor treatment

Abstract

Cancer is a major public health problem, and is now the world’s leading cause of death. Traditional Chinese medicine (TCM)-combination therapy is a new treatment approach and a vital therapeutic strategy for cancer, as it exhibits promising antitumor potential. Nano-targeted drug-delivery systems have remarkable advantages and allow the development of TCM-combination therapies by systematically controlling drug release and delivering drugs to solid tumors. In this review, the anticancer activity of TCM compounds is introduced. The combined use of TCM for antitumor treatment is analyzed and summarized. These combination therapies, using a single nanocarrier system, namely codelivery, are analyzed, issues that require attention are determined, and future perspectives are identified. We carried out a systematic review of >280 studies published in PubMed since 1985 (no patents involved), in order to provide a few basic considerations in terms of the design principles and management of targeted nanotechnology-based TCM-combination therapies.

Keywords:

• cancer
• codelivery
• combination therapy
• nanotargeted drug-delivery system
• tumor targeting
• TCM

Introduction

Cancer is the leading cause of disease-associated death in China,¹ and is now the world’s leading cause of death.² According to the Global Cancer Report 2018 on the trend of 36 cancers in 185 countries worldwide by the WHO, the global burden of cancer is increasing at an alarming rate (one in eight deaths on average are due to cancer). The report also pointed out that the incidence and mortality rate of cancer continues to rise each year, with developing countries accounting for approximately 60% of the world’s new cases and 70% of annual deaths. In 2018, nearly half the world’s new cases of cancer occurred in Asia, most of which occurred in China.³ Bray et al² provided a status report on the global burden of cancers using GLOBOCAN 2018. It is estimated that there will be 18 million new cases of cancer and 9.6 million cancer deaths in 2018. Lung cancer and breast cancer are the most frequent cancers in men and women, respectively, and the two leading causes of cancer death. Due to the high incidence and mortality rate of cancer, the global health care burden is also increasing rapidly.

Surgical treatment, chemotherapy, and radiotherapy are the primary treatment methods for cancer.⁴ If cancer patients are diagnosed early and receive timely surgical treatment, the probability of surviving for 5 years after surgery is greatly improved. However, when cancers are diagnosed late, the vast majority of patients are already in the terminal stages, and thus may have lost the opportunity of surgical treatment. In addition, due to adverse reactions caused by radiotherapy, such as fatigue, gastrointestinal reactions, skin damage, bone-marrow suppression, and cardiotoxicity,⁵ chemotherapy is still the main method of cancer treatment.

Nevertheless, due to lack of specificity and poor targeting, chemotherapy drugs not only kill tumor cells but also act on normal tissue, causing a reduction in immunity, significant side effects, and low drug efficacy. In addition, cancer patients can develop resistance to a single chemotherapy drug in clinical practice, resulting in a decrease in the subsequent curative effect. Multidrug resistance (MDR) was once considered the leading cause of chemotherapy failure, and may also promote tumor metastasis and recurrence.⁶ Based on recent statistics from the American Cancer Society, >90% of cancer patients die from different levels of MDR.

Therefore, the treatment of cancer should be changed from an initial single medication to combination therapy. The combination of two or more active antitumor ingredients plays a crucial role in complementarity and synergy, and has become the preferred scheme in cancer treatment. Notably, the combination of traditional Chinese medicines (TCMs) with chemotherapeutic drugs and the combination of various TCMs, which involves multiple targets and multiple signaling pathways, have improved efficacy compared with drugs with a single molecular target and become a new strategy for tumor therapy in recent years.⁷ Due to a great deal of investment and rapid development, nanotechnology is already used in various fields of biomedical science.⁸ Novel nanoformulation-based drug-delivery systems, such as liposomes, nanoparticles (NPs), vesicles, mesoporous silica NPs (MSNs), and micelles, provide promise in overcoming current limitations, including poor targeting, insufficient absorption, poor pharmacokinetics and bioavailability, and limited biodistribution.^9–11

In this review, the anticancer activity of TCM compounds is introduced. The combined use of TCMs for antitumor therapy is analyzed and summarized. These combination therapies using a single nanocarrier system, namely code-livery, are analyzed to determine their potential in prolonging drug duration in vivo, targeting drug delivery, and reducing toxicity (Figure 1). Matters requiring attention and future perspectives in this field are also reviewed, in order to accelerate the clinical application of combination antitumor therapy using targeted nanotechnology.

Figure 1 Advantages of targeted nanotechnology-based TCM-combination therapy.

Notes: (A) Antitumor effects of TCM; (B) antitumor effects of TCM-combination therapy; (C) antitumor effects of targeted nanotechnology-based TCM-combination therapy.

Abbreviations: TCM, traditional Chinese medicine; MDR, multidrug resistance.

Antitumor effects of TCMs

Herbs, animals, and minerals are used widely as health foods and medicines to remedy various diseases in Asia, and have been collected and recorded as effective and traditional therapies in the TCM literature. For example, artemisinin was isolated by Youyou Tu at the China Academy of Traditional Chinese Medicine in Beijing, and is now an effective medicine in the treatment of malaria. As a result, Tu won the Nobel Prize in Physiology or Medicine in 2015. In most developing countries, 80% of the population continue to use traditional medicines for primary health care.¹² From 2016 to 2017, the total amount of TCM herbal medicines and other related products exported to the Belt and Road Initiative countries reached US$295 million. In addition, the WHO also recognized traditional medicine in its influential global medical compendium.¹³

Worldwide, including in Western countries, TCM has been increasingly used in the past few decades, and is well known for its vital role in cancer prevention and treatment. A number of studies have confirmed that the active ingredients in TCM (curcumin [Cur], gambogic acid, and baicalein [BA], among others) are able to effectively induce apoptosis, interfere with tumor progression, inhibit tumor development, inhibit angiogenesis, cause cell-cycle arrest, and block metastasis. A summary of the antitumor effects of drugs isolated from TCMs is shown in Table 1. Structures of TCM compounds are shown in Figure 2.

Table 1 Antitumor effects of traditional Chinese medicine

Monomer composition	Source (Chinese name)	Anticancer category	Anticancer property
Curcumin (C21H20O6)	Curcuma longa (jiang huang)	Breast cancer, neurological cancers, lymphoma, lung cancer, melanoma, sarcoma, leukemia, gastrointestinal cancers, ovarian cancer, genitourinary cancers^Citation14	It can inhibit the initiation step of cancer and malignant- cell proliferation during the promotion and progression of carcinogenesis.^Citation15
Arsenic trioxide (As2O3)	Arsenic (pi shuang)	Acute promyelocytic leukemia,^Citation16 renal cancer, prostate cancer, hepatocellular carcinoma,^Citation17^–^Citation19 lung cancer^Citation20,Citation21	As2O3 can induce the accumulation of cellular ROS, causing DNA damage and leading to cell-cycle arrest and apoptosis in various solid tumors.^Citation17^–^Citation19
Resveratrol (C14H12O3)	Polygonum cuspidatum (hu zhang)	Osteosarcoma cells,^Citation22 23 breast cancer, colon cancer,^Citation24 cervical cancer, blood cancer, kidney cancer, liver cancer, bladder cancer, thyroid cancer, esophageal cancer, prostate cancer, brain cancer, gastric cancer, bone cancer, ovarian cancer^Citation25	It can induce apoptosis and inhibit proliferation by modulating the PI3K–Akt–mTOR and MAPK pathways.^Citation22,Citation26,Citation27
Ginsenoside Rg3 (C42H72O13)	Panax ginseng (ren shen)	Breast cancer cells,^Citation28 colorectal tumor,^Citation29 ovarian cancer^Citation30	Ginsenoside Rg3 can inhibit tumor development by inhibiting tumor angiogenesis, regulating apoptosis of tumor cells, controlling the proliferation, invasion, and metastasis of tumor cells, and inhibiting multidrug resistance.^Citation28^–^Citation30
Baicalein (C15H10O5)	Scutellaria baicalensis (huang qin)	HCT116 human colon cancer,^Citation31 pancreatic cancer stem cells,^Citation32 bladder cancer,^Citation33 breast cancer,^Citation34 gastric cancer,^Citation35 hepatocellular carcinoma^Citation36	It can inhibit a variety of cyclins or cyclin-dependent kinases, regulate the cell cycle,^Citation37 scavenge oxidative radicals, weaken MAPK, Akt, or mTOR activities,^Citation38 inducing apoptosis^Citation39 and inhibiting the invasion and metastasis of tumors.^Citation40
Gambogic acid (C38H44O8)	Garcinia hanburyi (teng huang)	Gastric cancer,^Citation41 prostate cancer,^Citation42 lung cancer,^Citation43 pancreatic cancer,^Citation44 hepatocarcinoma^Citation45	Gambogic acid can induce apoptosis,^Citation46 enhance the accumulation of ROS,^Citation47 and inhibit telomerase activity.^Citation41,Citation43
Gambogenic acid (C38H46O9)	Garcinia hanburyi (teng huang)	Human nasopharyngeal carcinoma CNE1 cells,^Citation48 U251 glioblastoma cells,^Citation49 breast cancer,^Citation50 lung cancer^Citation51	Gambogenic acid is associated with inhibition of proliferation of A549 cells by apoptosis induction and cell-cycle arrest,^Citation48,Citation52,Citation53 and can mediate apoptosis in human nasopharyngeal carcinoma CNE1 cells.^Citation49
Quercetin (C15H10O7)	Hypericum ascyron (hong han lian)	Melanoma,^Citation54 HL60 cells^Citation57 prostate cancer,^Citation55 breast cancer,^Citation56 human leukemia	Quercetin can induce cancer-cell apoptosis by modulating signaling pathways and blocking cell-cycle progression,^Citation58 as well as actively suppress cancer proliferation and cancer metastasis.^Citation59
Triptolide (C20H24O6)	Tripterygium wilfordii (lei gong teng)	Medulloblastoma,^Citation60 lung cancer cells,^Citation61 blood cancer cells, colon cancer cells, breast cancer cells, brain cancer cells, ovary cancer cells, kidney cancer cells, prostate cancer cells^Citation62,Citation63	Triptolide can inhibit cell proliferation and arrest the cell cycle,^Citation64 increase LC3α expression levels,^Citation65 and inhibit the PI3K–Akt–mTOR pathway. While ERK1/2 is activated, autophagy of cell death is induced.^Citation66,Citation67
Berberine (C20H18NO4)	Coptis chinensis (huang lian)	Human malignant pleural mesothelioma NCIH2452 cells,^Citation68 human colon cancer cells,^Citation69 hepatocellular carcinoma,^Citation70 breast cancer cells,^Citation71,Citation72 human HSC3 oral cancer cells,^Citation73 human epidermoid carcinoma A431 cells^Citation74	It can inhibit cancer-cell proliferation, induce cell-cycle arrest at the G1/G0 phase, induce apoptosis in cancer cells,^Citation69 cause cell-growth inhibition, and suppress cell migration and invasion.^Citation71^–^Citation74
Cryptotanshinone (C19H20O3)	Salvia miltiorrhiza (dan shen)	Prostate cancer,^Citation75 breast carcinoma,^Citation76 lung cancer^Citation77	It inactivates STAT3 activity,^Citation75 arrests the cell cycle in the G1/G0 phase, and inhibits expression of cyclin D1.^Citation76
Tetramethylpyrazine (C8H12N2)	Ligusticum striatum (chuan xiong)	Lung cancer,^Citation78 osteosarcoma^Citation79	It can reduce activities of COX2 and MMP2/TIMP2,^Citation78 upregulate cytosolic NFκB p65 expression, and downregulate protein expression of nuclear NFκB p65, BCL2, and cyclin D1.^Citation79
Dihydroartemisinin (C15H24O5)	Artemisia annua (huang hua hao)	Pancreatic cancer,^Citation80 osteosarcoma,^Citation81 liver cancer,^Citation82 leukemia^Citation83,Citation84	It can activate caspase 3, caspase 8, and caspase 9, upregulate the expression of Fas, cyclin D1, and Bax, downregulate the expression of CDC25B, Bcl2, and cyclin B1, and inhibit NFκB activity.^Citation85
Ginsenoside Rh2 (C36H62O8)	Panax ginseng (ren shen)	Breast cancer,^Citation86 ovarian cancer,^Citation87 prostate cancer,^Citation88 leukemia,^Citation89 colorectal cancer,^Citation90 hepatocellular carcinoma^Citation91	Some signaling pathways, such as TNFα,^Citation92 EGFR,^Citation93 JAK–STAT3,^Citation94 and PI3K–Akt^Citation95 have been implicated in the regulatory process of Rh2 in cancer cells.
Plumbagin (C11H8O3)	Plumbago zeylanica (bai hua dan)	Prostate cancer,^Citation96 breast cancer cells,^Citation97 melanoma cells^Citation98	It can induce apoptosis and autophagy, destroy the cell cycle, inhibit invasion and metastasis, and prevent angiogenesis, with anticancer potential in different cancer cells in vitro and in vivo.^Citation99

Figure 2 Chemical structures of the active components in traditional Chinese medicine.

Antitumor effects of TCM-combination therapy

Initially, cancer therapy consisted of a single drug, which could involve a single target. However, malignant disease is caused by many complicated factors, and treatment with a single drug is not adequate. Patients are usually susceptible to drug resistance after sequential cycles of therapy with these chemotherapy drugs,¹⁰⁰ and a single medication frequently causes serious side effects. For instance, although cisplatin is clinically effective, it lacks selectivity for tumor tissue, resulting in serious side effects, such as kidney-function damage,¹⁰¹ neurotoxicity,¹⁰² ototoxicity,¹⁰³ and the emergence of MDR, resulting in the failure of chemotherapy.¹⁰⁴ In addition, long-term or high-dose cisplatin treatment can also cause severe anemia.¹⁰⁵ Therefore, the clinical application of single drugs, such as cisplatin, has been greatly restricted.

Cancer therapy urgently requires a new therapeutic approach to overcome these shortcomings. Combination-drug therapy is a new mode of treatment, and has gradually gained the attention of researchers.¹⁰⁶ Combination therapy involves the simultaneous or sequential use of two or more medicines for therapeutic purposes, and gradually plays a meaningful role in a complementary way, has synergistic action, and alleviates adverse reactions. It can not only produce a better therapeutic effect by regulating multiple signaling pathways in abnormal cells and act on multiple targets simultaneously but also reduce the occurrence of MDR and reduce both the dosage and side effects. The combination of two or more active antitumor ingredients is now a vital treatment method for tumors, and has received US Food and Drug Administration (FDA) approval.¹⁰⁷

Based on classic TCM theory, the combination of antitumor TCMs exhibits promising potential in cancer treatment such as: 1) enhancing the therapeutic efficacy of chemotherapeutic drugs – due to the combined effects of Cur and cisplatin determined in vitro and in vivo, experimental results demonstrate that Cur can enhance the antitumor effect of cisplatin in A549 cells in vitro, the combination markedly inhibiting tumor growth and promoting apoptosis in the A549-xenograft mouse model;¹⁰⁸ 2) achieving synergistic therapeutic effects – resveratrol and Cur synergistically cause apoptosis in breast cancer cells by p2 (Waf/Cip1)-mediated inhibition of the Hedgehog–Gli cascade;¹⁰⁹ 3) reversing drug resistance – the combination of cryptotanshinone and cisplatin leads to cell death and apoptosis, and cryptotanshinone reverses cisplatin resistance in human lung carcinoma A549 cells by downregulating the Nrf2 pathway;¹¹⁰ 4) reducing the dose of drugs – combination therapy with triptolide and cisplatin completely suppresses tumor growth, suggesting that lower concentrations of cisplatin and triptolide may produce a synergistic anticancer effect;¹¹¹ and 5) prolonging survival – As₂O₃ combined with ginsenoside Rg₃ can significantly inhibit the proliferation of NCIH1299 cells and prolong survival of tumor-bearing nude mice, with a significant effect on lung cancer treatment.¹¹² In addition, TCM-combination therapy results in good prognosis, has fewer adverse reactions, has long-lasting curative effects, regulates the expression of intracellular marker proteins, and reduces the side effects of drugs.^112–115 Further superior effects are shown in Table 2. The anti–lung cancer and anti–breast cancer mechanisms of TCM-combination therapy are shown in Figures 3 and 4. TCM-combination therapy achieves the effects that single chemotherapeutic drugs fail to achieve, and has become the main direction in clinical and experimental research on antitumor therapy.¹¹⁶

Table 2 Antitumor effects of TCM-combination therapy

Monomer composition	Combination therapy	Cellular, animal levels	Advantages of combination therapies	Reference
Curcumin (Cur)	Cur + cisplatin	Human non-small-cell lung cancer (NSCLC) A549, A549-xenograft mouse model	Cur can strengthen the antitumor effect of cisplatin in A549 cells in vitro. Cur combined with cisplatin can inhibit tumor growth and promote apoptosis in xenograft mouse model.	^Citation108
	Cur + paclitaxel	Breast cancer cell line MCF7, MCF7-xenograft mouse model	Cur combined with paclitaxel can inhibit the growth of MCF7 cells synergistically, induce significant apoptosis in MCF7 cell lines, and exert increased antitumor efficacy in mouse models.	^Citation117
	Cur + Rsv	Breast epithelial MCF10A-Tr, tumor xenograft in mice	Cur and Rsv causes apoptosis synergistically in breast cancer cells by p2 Waf/Cip1-mediated inhibition of Hedgehog–Gli cascade.	^Citation109
		Colon cancer HCT116 (wild type) cells, xenografts in SCID mice	The combination of Cur and Rsv has stronger inhibitory effects on the growth of p53-positive (wild type) and p53-negative (HCT116) colon cancer cells in vitro and in vivo than either agent alone.	^Citation119
	Cur + Rsv–diallyl disulfide	Malignant rhabdoid (SJRH4, RD/18) and osteosarcoma (SAOS2) cell lines	Cur induces apoptosis in rhabdomyosarcoma and osteosarcoma cells, which is potentiated when Cur is combined with Rsv or diallyl disulfide.	^Citation120
	Cur + As2O3/lonidamine	U937 and HL60 human acute myeloid leukemia cells, K562 chronic myelogenous leukemia cells	Cur plus As2O3 or lonidamine stimulates events typical of the mitochondrial executioner pathway. Cotreatment with Cur may be an effective way to improve the efficacy of As2O3 and lonidamine as antineoplastic agents for myeloid leukemia cells.	^Citation121
	Cur + vesicular stomatitis virus	PC3 prostate cancer cells, mouse model of prostate cancer	Cur makes prostate cancer cells sensitive to the oncolytic effects of vesicular stomatitis virus through modulating antiviral responses and components of the intrinsic apoptotic pathway.	^Citation122
	Cur + Qct	Human gastric cancer MGC803 cells	The combination of Cur and Qct has a significant inhibitory effect on cell proliferation and a stronger effect on gastric cancer MGC803 cells than individual therapy.	^Citation123
Arsenic trioxide (ATO)	ATO + artesunate	Human K562 cell lines	Artesunate combined with ATO can significantly promote apoptosis of K562 cells induced by artesunate.	^Citation124
	ATO + dihydroartemisinin	A549 cells	Dihydroartemisinin effectively sensitizes A549 cells to ATO, increasing the effectiveness of ATO-induced cell death, and can cooperate with ATO to exert synergistic effects in promoting apoptosis in A549 lung cancer cells.	^Citation85
	ATO + cisplatin– doxorubicin (Dox)	HepG2 cell lines, hepatic cancer stem cells	This study was critical in realizing the potential for synergy between ATO and chemotherapeutic protocols, such as cisplatin and Dox.	^Citation125
		Human hepatoma BEL7402 cells, mouse hepatoma HepA	Low-dose cisplatin or Dox and ATO in combination can increase the antihepatocarcinoma effect.	^Citation126
	ATO + Dox	SGC7901/S human GC cell line, Dox-resistant SGC7901/ADM cell line	ATO can reverse MDR to Dox in human GC cells. This may be relevant to decreased Pgp expression.	^Citation127
	ATO + matrine	Myeloma cell lines RPMI 8226 and U266	Matrine shows an antimyeloma effect by inducing apoptosis, and ATO combined with matrine enhances apoptosis induced by matrine, having synergistic effects.	^Citation128
Resveratrol (Rsv)	Rsv + cisplatin	A549 cells	Rsv combined with cisplatin synergistically induced apoptosis by modulating autophagic cell death in A549 cells.	^Citation104
	Rsv + Dox	MCF7 and MDA-MB231 cell lines, Ehrlich ascitic carcinoma–bearing mice	Rsv combined with Dox can inhibit tumor volume and prolong the life of Ehrlich ascitic carcinoma cell–bearing mice.	^Citation129
	Rsv + paclitaxel	HepG2 human liver cancer cells	Rsv can enhance the anticancer effect of paclitaxel on HepG2 cells and be used as a sensitizer of paclitaxel.	^Citation130
		NSCLC cell line A549	Rsv enhances the anticancer effects of paclitaxel in A549 cells, and thus Rsv might be used as an excellent sensitizer for paclitaxel.	^Citation131
	Rsv + temozolomide	Human glioblastoma U87 MG and GBM8401 cell lines, GBM-SKH (GBM) cell line, orthotopic xenograft model	Rsv can enhance temozolomide-mediated antitumor effects in glioblastoma multiforme in vitro and in vivo by ROS-dependent AMPK–TSC–mTOR signaling pathway.	^Citation132
	Rsv + Qct	Human hepatoblastoma HepG2 cells	Rsv plays different roles in autophagy according to cellular energy state, and can represent a promising strategy to sensitize cancer cells to Qct therapy.	^Citation133
	Rsv + As2O3	HeLa, MCF7, and NB4 cells, HeLa-cell mouse model	Rsv can significantly raise the antitumor effect induced by As2O3 in vitro, and As2O3 combined with Rsv can significantly inhibit tumor growth and angiogenesis in nude mice.	^Citation134
Ginsenoside Rg3	Rg3 + As2O3	NCIH1299 lung cancer cells, nude mice bearing hepatoma	As2O3 combined with Rg3 can dramatically inhibit proliferation of NCIH1299 cells and prolong the survival of nude mice bearing tumors, having a significant effect on lung cancer therapy.	^Citation112
	Rg3 + paclitaxel	Triple-negative breast cancer lines MDA-MB231, sMDA-MB453, BT549, mouse MDA-MB231-xenograft model	Rg3 promotes cytotoxicity and apoptotic effects of paclitaxel on triple-negative breast cancer through inhibition of NFκB activity and regulating Bax/Bcl2 expression.	^Citation113
	Rg3 + paclitaxel/cisplatin	Mouse tumor-xenograft models	The combined use of Rg3 can remarkably raise the inhibitory effects of chemotherapy on tumor growth, and expression levels of Ki67 in the chemotherapy + Rg3 group were significantly lower than in the other groups (Rg3 alone, paclitaxel alone, and cisplatin alone).	^Citation114
	Rg3 + docetaxel	Colon cancer (HCT116 and SW620) cells	Compared to treatment with Rg3 or docetaxel alone, combination therapy can inhibit the growth of cancer cells and induce apoptosis of cancer cells more effectively, accompanied by significant inhibition of NFκB activity.	^Citation135
Baicalein (BA)	BA + taxol	Human ovarian cancer A2780 cells, SKOV3 cells, and OVCAR cells	BA combined with taxol at low concentrations can play a synergistic antitumor role via the Akt–β-catenin signaling pathway and mitochondria-mediated cell apoptosis in ovarian cancer cells.	^Citation136
	BA + 10-hydroxycamptothecin (Hcpt)	BGC823, MCF7, and SMMC7721 cells, mice xenografted with BGC823 tumors	In BGC823, MCF7, and SMMC7721 cells, BA significantly enhances the anticancer activity of Hcpt at an atoxic dose. Hcpt with BA is a novel and effective combination therapy, which synergistically target Topo I and upregulate p53 to induce cell-cycle arrest and cell apoptosis.	^Citation115
Gambogic acid (GA)	GA + cisplatin	Human NSCLC A549, NCIH460, and NCIH1299 cell lines	GA synergizes the growth-inhibitory activity of CDDP and enhances the apoptosis-induced effect of CDDP on NSCLC cells, meanwhile sensitizing lung cancer cells to CDDP in vitro and in vivo.	^Citation137
	GA + gemcitabine	PANC1 and BxPC3 cells, mouse xenograft model of human pancreatic cancer	GA inhibits the activation of ERK–E2F1–RRM2 signaling pathway to sensitize pancreatic cancer cells to gemcitabine in vitro and in vivo.	^Citation138
	GA + sunitinib	Renal carcinoma cell lines 786O and CAKI1, mouse xenograft models	Therapy of 786O and CAKI1 cells with GA or sunitinib can reduce the proliferation of tumor cells, especially when combined with the two medicines. The combination of GA and sunitinib has greater antitumor efficacy than either drug alone.	^Citation139
Gambogenic acid (GNA)	GNA + adriamycin (Adr)	MCF7 cell lines, MCF7/ADR cell lines	GNA can increase the chemosensitivity of breast cancer cells to Adr. This modulatory role is mediated by suppression of the PTEN–PI3K–Akt pathway, leading to apoptosis in MCF7/ADR cells.	^Citation140
	GNA + bortezomib (Btz)	Myeloma MM1S cells, tumor-xenograft models	Btz and GNA have strong synergistic effect in combination therapy, inducing apoptosis of MM.1S cells. The combination of Btz and GNA is superior to single drug on MM.1S-xenograft models.	^Citation141
Quercetin (Qct)	Qct + tamoxifen	Prostate-tumor xenografts in mice	Tamoxifen combined with Qct can effectively delay the occurrence of tumors, inhibit the final volume of tumors, and reduce the weight of the end-point tumor (P<0.05).	^Citation142
	Qct + metformin	PC3 and LNCaP cells, prostate cancer xenografts in nude mice	Combined application of metformin and Qct has a significantly stronger effect on apoptosis of prostate cancer cells than monotherapy, and combination therapy can inhibit the VEGF–Akt–PI3K signaling pathway to play a synergistic antitumor effect.	^Citation143
	Qct + trichostatin A	Mouse xenograft model of A549 cells	Qct enhances the antitumor effect of trichostatin A and prevents trichostatin A-induced muscle wasting.	^Citation144
	Qct + Hcpt	MCF7, BGC823, and HepG2 cells, MCF7 xenograft in nude mice	The combination of Qct and Hcpt can inhibit Topo I in MCF7 cells, which synergistically induces cell-cycle arrest and apoptosis by triggering DNA damage.	^Citation145
	Qct + irinotecan–SN38	The AGS human gastric adenocarcinoma cell line, AGS xenograft mouse model	Qct combined with irinotecan has a superior regulatory effect on angiogenesis and EMT-related factors. Qct may enhance the efficacy of irinotecan–SN38 in the human AGS cell line.	^Citation146
Triptolide (TL)	TL + sorafenib	Huh7 and PLC/PRF/5 cells, Huh7 tumor-xenograft mouse model	Sorafenib combined with TL is superior to single-drug therapy in increasing cell death and apoptosis in vitro.	^Citation147
	TL + cisplatin	Human gastric adenocarcinoma SCM1 cells, xenografts in SCID mice	TL can raise the cytotoxicity of cisplatin to SCM1 cells, and the combined therapy can significantly inhibit the growth of tumors.	^Citation111
		Urothelial cancer cells, PC3 prostate cancer cell line, mouse xenograft model	Cancer-specific enhancement of cisplatin-induced cytotoxicity with TL by the interaction of inactivated GSK3β with p53.	^Citation148
	TL + oxaliplatin	Colon cancer cell line SW480, nude mouse model	Combination therapy with TL and oxaliplatin exerts synergistic antitumor effects at low concentrations in colon cancer cells, with less cytotoxicity, which exhibits high potency for clinical applications.	^Citation149
	TL + fluorouracil	Colon carcinoma HT29 cells, tumor xenografts in nude mice	The combined effects of TL and fluorouracil on the growth of colon carcinoma are better than that of single drug, whether in vitro or in vivo. TL combined with fluorouracil has synergistic effects at lower concentrations and promotes apoptosis, but the side effects of chemotherapy are not increased.	^Citation150
	TL + gemcitabine	Pancreatic cancer BxPC3 and PANC1 cell lines	TL can increase gemcitabine-induced cell-growth inhibition and apoptosis synergistically. When TL is combined with gemcitabine, the potential for therapy of pancreatic cancer increases markedly.	^Citation151
Berberine (Bbr)	Bbr + Dox	Human breast cancer MCF7/MDR cells, tumor xenografts in the BALB/c nu/nu mice	Bbr can increase the sensitivity of drug-resistant breast cancer to Dox chemotherapy and can directly induce apoptosis in vitro and in vivo by dose-orchestrated AMPK signaling.	^Citation152
		NSCLC NCIH460, NCIH1975 cells	Bbr inhibits Dox-mediated STAT3 activation and enhances the cytotoxic effect in lung cancer cells of Dox treatment.	^Citation153
	Bbr + evodiamine	Human breast cancer MCF7 cells, mice with MCF7 human breast cancer xenografts	Bbr combined with evodiamine plays a synergistic role in inhibiting the proliferation of MCF7 cells through inducing cell-cycle arrest and apoptosis.	^Citation154
	Bbr + galangin	ECA9706 cells, nude mice with xenograft tumors	Galangin combined with Bbr synergistically exerts cell-growth inhibition, apoptosis, and cell-cycle arrest in esophageal carcinoma cells and also exhibits outstanding synergistic anticancer role in vivo.	^Citation155
	Bbr + tamoxifen	MCF7 and MCF7/TAM cells	Bbr combined with tamoxifen is more effective than tamoxifen alone in inducing cell-growth inhibition, inducing G1-phase arrest and activating cell apoptosis.	^Citation156
	Bbr + sorafenib	SMMC7721 and HepG2 cells	The combination of Bbr and sorafenib exhibits a synergistic inhibitory effect on the proliferation of SMMC7721 and HepG2 cells.	^Citation157
Allicin (AN)	AN + IL2	BXPC3 cell line, pancreatic cancer xenograft in mice	The combination of AN and IL2 can inhibit the growth of tumors and prolong survival, possibly by activation of CD4^+ T, CD8^+ T, and NK cells.	^Citation158
	AN + fluorouracil	SK-Hep1 and BEL7402 cell lines, hepatocellular carcinoma-xenograft models in nude mice	By ROS-mediated mitochondrial pathway, AN can increase the sensitivity of hepatocellular carcinoma cells to fluorouracil-induced-apoptosis. Combination of AN and fluorouracil can be used as a novel chemotherapeutic regimen for hepatocellular carcinoma.	^Citation159
Dihydroberberine (Dhb)	Dhb + sunitinib	Human NSCLC NCIH460, NCIH460 cell-xenografted nude mice	Sunitinib shows synergistic effects on proliferation, colony formation, and growth of transplantable tumors of NCIH460 cells, suggesting that Dhb can increase the sensitivity of lung cancer to sunitinib.	^Citation160
Oxymatrine (Omt)	Omt + oxaliplatin	Colon cancer lines (HT29 and SW480), SW480 xenograft mouse model	A combined index value <1 for Omt and oxaliplatin has a synergistic effect. Compared with oxaliplatin or Omt alone, combination therapy has more effective inhibition on tumor weight and volume of SW480-xenotransplant mice.	^Citation161
	Omt + NM3	Human gastric cancer cell lines SGC7901, MKN45, MKN74, human gastric cancer xenografted in mice	Omt combined with NM3 can synergistically inhibit the growth of human gastric cancer cell line SGC7901 in vitro and the growth of xenograft human gastric cancer cell line SGC7901 in vivo.	^Citation162
Tetraarsenic oxide (TAO)	TAO + cisplatin	Human cervical carcinoma cell line HPV16, CaSki cells, mice bearing CaSki-cell tumors	TAO can induce apoptosis. Combined with cisplatin, it can significantly increase the number of apoptotic cells, exerting a synergistic role.	^Citation163
Cryptotanshinone (Cts)	Cts + cisplatin	A549 cells, A549/DDP cells	Compared with cisplatin monotherapy, the combination of Cts and cisplatin induces cell death and apoptosis by increasing the sensitivity of A549/DDP cells to cisplatin. Cts can reverse the resistance of human lung cancer A549 cells to cisplatin by downregulating the Nrf2 pathway.	^Citation110
Tetramethylpyrazine (Tmp)	Tmp + cisplatin (CDDP)	Lewis lung cancer mice	The inhibitory effect of Tmp combined with DDP on tumor growth is enhanced compared to Tmp- or CDDP-alone treatment. Tmp with CDDP had additional or synergistic effects to inhibit tumor growth effectively.	^Citation164
Catechin (RQC)	RQC + gefitinib	Human metastatic breast cancer cell line MDA-MB231, hairless SCID female mice	Combination of RQC and gefitinib can significantly reduce the viability of gefitinib- resistant breast cancer cells. Compared with RQC or gefitinib alone, it can significantly inhibit the growth and metastasis of breast tumors in nude mice.	^Citation165
Dihydroartemisinin (Dha)	Dha + gemcitabine	BxPC3 and PANC1 cell lines, BALB/c nude mice	Dha significantly enhances the inhibition and apoptosis of gemcitabine on BxPC3 and PANC1 cell lines in vitro. Combination therapy can significantly enhance the antitumor effect of gemcitabine.	^Citation80
Epigallocatechin- 3-gallate (EGCG)	EGCG + Dox	Human prostate-cell lines, mouse tumor modeling with PC3ML cells	EGCG can be effectively used to improve the efficacy of Adr through improving its ability to eradicate highly aggressive, metastatic, or primary tumors.	^Citation166
		Human HCC cell line BEL7404/Dox, xenograft mouse model	EGCG can enhance Dox-induced cytotoxicity and increase the sensitivity of drug-resistant hepatocellular carcinoma cells to Dox at an atoxic dose.	^Citation167
Ginsenoside Rh2	Rh2 + paclitaxel–mitoxantrone	LNCaP prostate-tumor cells, human LNCaP-tumor xenograft	Rh2 combined with paclitaxel shows stronger inhibition and synergistic effect on growth in a cultured LNCaP cell line.	^Citation88

Abbreviations: SCID, severe combined immunodeficiency; MDR, multidrug resistance; CDDP, cis-diamminedichloroplatinum; EMT, epithelial–mesenchymal transition.

Figure 3 Anti–lung cancer mechanism of TCM-combination therapy.

Abbreviations: Cur, curcumin; Rsv, resveratrol; Tmp, tetramethylpyrazine; Cts, cryptotanshinone; Qct, quercetin; Bbr, berberine; Dhb, dihydroberberine; GA, gambogic acid.

Figure 4 Anti–breast cancer mechanism of TCM-combination therapy.

Abbreviations: Cur, curcumin; Rsv, resveratrol; Bbr, berberine; Hcpt, hydroxycamptothecin; Qct, quercetin; GNA, gambogenic acid; BA, baicalein.

Nevertheless, there are three possible interactions in drug combinations: antagonistic, additive, and synergistic effects. Therefore, if we do not understand interactions among drugs, blindly combining drugs will not only fail to achieve the desired response but also lead to reduced efficacy and increased toxicity, and even produce drug-borne diseases. For instance, the combination of paclitaxel (Ptx) and BA shows antagonism in breast cancer MCF7 cells,¹¹⁷ and Liu et al¹¹⁸ suggested that the combination of gambogic acid and bortezomib should be avoided in patients. In addition, attention should be paid to the proportion and sequence of the two drugs in combination.

Applications of targeted nanotechnology in TCM-combination therapy

During the early 20th century, Paul Ehrlich proposed the concept of targeted drugs, which consisted of three components: the drug, targeting moiety, and drug carrier. The main aim was to deliver the drug to the specific target organ under the specific guiding mechanism.¹⁶⁸ Targeted preparations are characterized by increasing the intensity of pharmacological action in target tissue, controlling drug release, and decreasing systemic adverse reactions. Targeted drug-delivery systems have become one of the important high-profile topics in modern pharmacy. Nanotargeted drug-delivery systems have remarkable advantages in improving the bioavailability of drugs, enhancing the targeting ability of drugs, improving the distribution and pharmacokinetic properties of antitumor drugs in vivo and in vitro, increasing the stability of drugs, solubilizing poorly soluble drugs, protecting drugs from degradation in vivo, intelligently regulating the release of components, enhancing efficacy, and reducing toxicity.^169–172 Moreover, metastasis of neoplastic cells is the major cause of death in cancer patients,^173,174 and nanosize drug-delivery systems also provide an encouraging strategy for lymphatic metastases.¹⁷⁵ In 2004, the National Cancer Institute (NCI) launched the NCI Cancer Nanotechnology Alliance, which aims to use nanotechnology to combat cancer.¹⁷⁶

In recent years, many types of nanopreparations of TCMs, which involve the combination of nanotargeted drug-delivery systems and the advantages of TCM components in the treatment of tumors, have been reported.^177–179 Simultaneously, nanotargeted drug-delivery systems are also promising multidrug carriers and allow the development of drug combinations by systematically controlling drug release and delivering drug to solid tumors.¹⁸⁰ Codelivery of multiple antitumor agents in a single well-designed nanocarrier has significant advantages over monotherapy.^181,182 Generally, drug targeting can be classified into three categories: passive targeted preparations, active targeted preparations, and other physicochemical targeted drug-delivery systems.

Passive targeted drug-delivery systems

In a passive targeted drug-delivery system, lipids, adipoids, proteins, and biodegradable high-molecular-weight polymers are mainly used as carriers, and the drug is encapsulated or embedded into various colloidal systems, forming stable structures, such as polymeric NPs (PNPs), micelles, nanovesicles, and liposomes, to increase drug concentration in tumor cells, decrease drug distribution in blood and other organs, and prevent toxicity and adverse reactions. This spontaneous accumulation, or “passive” targeting, is particularly effective against tumors, due to leaky angiogenic vessels and poor lymphatic drainage of the tumor, which is currently referred to as the enhanced permeability and retention (EPR) effect. That is to say, the high permeability of tumor blood vessels allows nanosystems to enter the interstitial spaces of the tumor, while impaired lymphatic filtration allows these nanosystems to remain there. This phenomenon does not exist in normal tissue. Currently, EPR-mediated drug delivery is considered effective in delivering drugs into tumors, especially nanocarriers (Figure 5). The size of the particles is also closely related to their distribution.¹⁸³ The different sizes of these nanosystems decide the in vivo distribution behavior. Nanopreparations of <100 nm can be slowly accumulated in bone marrow; nanocarriers of 100–200 nm are apt to become enriched in the solid-tumor site; nanosystems of 0.2–3 μm are taken by macrophagocytes in the liver and spleen and particles of >7 μm are often intercepted by pulmonary capillary beds and enter the pulmonary tissues or alveoli.¹⁸⁴

Figure 5 Passive (A) and active (B) targeting of tumors.

Abbreviation: EPR, enhanced permeability and retention.

Liposomes

Liposomes are microvesicles with one or more aqueous cavities formed by the encapsulation of one or two amphiphilic molecular double-layer membranes, and the drug is encapsulated or embedded into the liposomes to form the liposome drug. Due to the similarity between the structure and the biological membrane, the encapsulation of water-soluble and fat-soluble drugs can reduce the drug dose, lower drug toxicity, delay release, lower in vivo elimination speed, change in vivo distribution of the drug, and achieve targeted release. Due to these advantages, liposome drugs have attracted considerable attention, and many studies have been carried out on them.¹⁸⁵

Plumbagin is a quinonoid isolated from the roots of Plumbago zeylanica (bai hua dan in Chinese).¹⁸⁶ It has high antip-roliferative activity against a variety of tumor cell lines,^187,188 and its anticancer properties have also been demonstrated in vivo.¹⁸⁶ Celecoxib and plumbagin are two antitumor drugs that synergistically kill melanoma cells instead of normal cells. The combined use of these two agents in traditional approaches was not possible, due to their poor bioavailability and toxicity concerns. In order to circumvent these challenges, Raghavendra et al¹⁸² developed a nanoliposome containing celecoxib and plumbagin, named CelePlum 777, which has good stability and can release these two drugs at optimal proportion to achieve the maximum synergistic killing effect. Compared to nanoliposomes containing individual drugs, CelePlum 777 can enhance the inhibition of melanoma-cell proliferation in vitro and reduce the growth of melanoma tumors, with negligible systemic toxicity in nude mice. The goal of loading different individual drugs into a nanoliposome that releases the drugs at synergizing ratios was realized.

Previous studies have shown that Cur can reduce side effects caused by cisplatin (cis-diamminedichloroplatinum [CDDP]), including ototoxicity,¹⁸⁹ nephrotoxicity,¹⁹⁰ and neurotoxicity.¹⁹¹ In addition, Cur can also overcome resistance to CDDP and improve the sensitivity of hepatocellular carcinoma cells to CDDP.¹⁹² However, due to poor water-solubility and the different pharmacokinetics of CDDP and Cur,¹⁹³ the cocktail of both drugs controlling the drug proportions and dose regimen at target cancer cells would be challenging. Based on a previous approach and advantages of the liposome, Cheng et al¹⁹⁴ developed a liposomal delivery system using a reverse-microemulsion and film-dispersion method, which coencapsulated CDDP and Cur and transplanted them into hepatocellular carcinoma cells. The anti-tumor activity of CDDP-Cur liposomes against HepG2 cells was higher than that of free drug or encapsulated-monodrug therapy, and retention was prolonged (t_½=2.38 hours). Therefore, coloaded liposomes can be used as an effective treatment for tumors, with great clinical application potential.

Polymeric nanoparticles

NPs are solid colloid particles 10–100 nm in size formed by drug dissolution, encapsulation, or adsorption on macromolecular materials. NPs within the particle-size range of 10–100 nm can hide the physicochemical characteristics of the drug, and the in vivo process of the drugs depends on the physicochemical characteristics of the carriers.¹⁹⁵ NPs are characterized by a relatively simple preparation process, a significant solubilization effect on active components, significantly improved drug targeting to tumors, and proneness to surface modification. They can improve drug stability, reduce digestive tract stimulation, achieve sustained release or controlled release,¹⁹⁶ effectively overcome multiple physiological barriers encountered in vivo, and achieve accurate, safe, efficient, and targeted therapeutic effects. Coating the surface of NPs with polyethylene glycol (PEG), or “PEGylation”, is a commonly used approach to improve the efficiency of drug delivery to target cells and tissue. PEGylation is capable of achieving prolonged blood circulation of nanocarriers, and can improve colloidal stability by providing steric surface hindrance. In addition, it has the ability to improve particle dispersion and decrease hemolysis.^197,198 Polylactic-co-glycolic acid (PLGA), a biodegradable polymer, is atoxic to final degradation products and has been approved by the FDA.^181,199 The antitumor effects of codelivered PNPs in TCM combinations are shown in Table 3.

Table 3 Polymeric nanoparticles (NPs) for codelivery of antitumor agents

Codelivery ingredients	Delivery-system materials	Method	Advantages	Reference
Epigallocatechin gallate (EGCG) + paclitaxel (Ptx)	Polylactic-co-glycolic acid (PLGA)–casein polymer– protein hybrid NPs	Emulsion precipitation	The core–shell nanoconstruct sequentially released EGCG followed by Ptx, which was sustained for 7 and 12 days, respectively. Compared with the bare drugs, dual-loaded NPs significantly increased plasma concentration, residence time, and circulatory half-life of nanoencapsulated Ptx and EGCG.	^Citation181
Camptothecin (CA) + ABT737	PEGylated polymeric NPs	Nanoencapsulation	Dual-loaded NPs exerted synergistic cytotoxic effects against HCT116 cells, RKO cells, and HT29 cells at a range of NP concentrations.	^Citation200
Gambogic acid (GA) + docetaxel (Dtx)	PLGA NPs	Nanoprecipitation	GA and Dtx were released synchronously in blood from the NPs in vivo. Compared with the saline control group, free Dtx solution and free Dtx-GA solution, the cocarrier NP-preparation group had the strongest inhibitory effect on the MCF7/ADR human breast-tumor xenograft.	^Citation201
Quercetin (Qct) + tamoxifen (Tmx)	PLGA NPs	Emulsion–diffusion solvent evaporation	NPs coencapsulated with Tmx and Qct had higher cellular uptake, cytotoxicity, and tumor suppression in female rats compared to free Tmx citrate, free Qct, and their combination.	^Citation202
Piperlongumine (PL) + Ptx	PLGA and -α-tocopheryl D PEG succinate NPs	Organic solvent evaporation	Compared with free Ptx, the double-loaded NPs had a sustained drug-release rate, showing increased cytotoxicity and cellular uptake in vitro. Ptx/PL-PT NPs suppressed tumor growth more efficiently with less toxicity than Ptx solution.	^Citation203
Qct + etoposide (Etp)	PLGA NPs	Single-emulsification (oil in water) solvent evaporation	With sustained release shown by NP pharmacokinetic parameters, bioavailability increased gradually. Cytotoxicity assays showed that the IC50 values of Etp-loaded NPs and Etp + Qct double-loaded NPs were nine- and elevenfold lower than free Etp.	^Citation199
Piperine + rapamycin	PLGA NPs	Nanoprecipitation	Dual-loaded NPs exhibited sustained release, with potential for long-term therapeutic action with less dosing frequency, and would result in a reduction of dosing and improved bioavailability compared to single-drug administration.	^Citation204
Vincristine (VI) + verapamil (VE)	PLGA NPs	Oil-in-water emulsion- solvent evaporation	The toxicity of coencapsulated NPs was lower than that of the free VI–VE combination. Compared with normal saline, free VI, free VI–VE combination, and single-drug NP combination, the coencapsulated NP group had the best tumor growth–inhibition effect in the MCF7/ADR human breast-tumor xenograft.	^Citation205

Abbreviation: PEG, polyethylene glycol.

Lipid–polymer hybrid nanoparticles

As indicated, it has been demonstrated by the increasing numbers of research reports that biodegradable PNPs and liposomes have become the two main types of active TCM nanocarriers. Lipid–polymer hybrid NPs (LPNs) are nuclear-shell NPs formed by polymer core–lipid/lipid–PEG shells^206,207 that have the advantages of biodegradable PNPs and bionic liposomes.

Li et al²⁰⁸ prepared LPNs and PNPs loaded with cisplatin and Cur. Results indicated that LPNs had higher anticancer efficacy than PNPs and free drugs. Cytotoxicity was highest in vitro and antitumor effect best in vivo. Therefore, LPNs can be used as a targeted and synergistic nanodrug codelivery system for tumor chemotherapy. In a similar study, Zhu et al²⁰⁹ developed vincristine–quercetin (Qct) dual-loaded LPNs. The experimental results proved that the LPNs loaded with both drugs exhibited better antitumor efficacy in vitro and in vivo.

Ruttala et al²¹⁰ developed nanocarriers loaded with Ptx and Cur using a method different from the previously mentioned studies. Ptx-loaded albumin NPs were prepared and encapsulated in PEGylated hybrid liposomes containing Cur by a thin-film hydration method. This combination guaranteed the release of Ptx and Cur in a sustained and sequential manner. Compared with a cocktail combination, the dual-drug-loaded nanocarrier had a better cytotoxic effect at a lower dose. Therefore, such coloading drug-delivery systems can be used as a promising treatment method to improve clinical efficacy in various malignant tumors. NPs containing genistein and Ptx have also showed similar experimental results.²¹¹

Nanostructured lipid carriers

Nanostructured lipid carriers (NLCs) are novel lipid NPs and mixtures of solid and liquid lipids, which have the advantages of excellent drug-loading capacity and sustained-release properties. Jiang et al²¹² prepared etoposide (Etp)- and Cur-loaded NLCs by the solvent-injection technique. Etp-Cur NLCs had the highest cytotoxicity in vitro and higher accumulation in tumor tissues in vivo compared with other preparations, including Etp NLCs, Etp + Cur solution, Etp solution, and NLCs. In addition, Etp-Cur NLCs displayed low cytotoxicity in normal tissue in vivo, suggesting that NLCs could serve as a promising therapeutic strategy in the treatment of tumors.

Mesoporous silica nanoparticles

MSNs have attracted much attention due to their potential biomedical applications. MSNs possess many attractive features for application in the delivery of TCMs, such as the size of tuning particles/pores, large surface, large pore volume, high loading capacity, mass producibility, biocompatibility, and chemical inertia.^213,214 TCMs can be dissolved in surfactant micelles, simultaneously hydrolyzed, and concentrated with silica to form NPs.

Jia et al²¹⁵ prepared NPs using the self-assembly in situ drug-loading approach, in order to deliver the anticancer drug Ptx and the MDR-reversal agent tetrandrine (Tet) to increase the intracellular concentration of Ptx, enhance its antitumor effect, and minimize the exposure of normal cells to Ptx and Tet. The study demonstrated that Tet significantly increased the accumulation of NPs in cells. Furthermore, Ptx-Tet–cetyltrimethylammonium bromide MSNs suppressed tumor-cell growth more efficiently than the delivery of Ptx (Ptx–cetyltrimethylammonium bromide MSNs) or free Ptx alone. The prepared NPs released the drugs easily in the acidic environment of tumors, and thus, side effects and toxicity in normal tissue and organs were reduced. This nano-carrier may have important potential in clinical applications to avoid MDR by codelivering multiple TCMs. Solid self-nanoemulsifying drug-delivery systems containing tamoxifen and Qct have also shown similar experimental results.²¹⁶

PEGylated lipid bilayer–supported mesoporous silica nanoparticles

The anticancer drug axitinib (Axt) is a small-molecule tyrosine kinase–receptor inhibitor of VEGFR1, -2, and -3.^217,218 Another anticancer drug, celastrol (Cst), can induce the suppression of angiogenesis²¹⁹ and enhance the antitumor activity of standard chemotherapeutic drugs.²²⁰ Choi et al²²¹ loaded Cst into an MSN carrier and subsequently coated it with a lipid bilayer containing Axt, denoted by “ACML”, to increase the synergistic efficacy of the two agents. The difference in drug loading resulted in a sequential-release pattern where Axt was released first to exert its anticancer effect, and then Cst was released to further induce a synergistic effect. The experimental results showed that the synergistic apoptotic effect of ACML against cancer cells was stronger than the Axt-Cst cocktail. Moreover, ACML had a greater tumor-inhibitory effect than either drug administered alone in a tumor-xenograft mouse model. It has been proved that PEGylated lipid bilayer–supported MSNs have the potential to be used as an effective therapeutic strategy for malignant tumors.

Micellar systems

Self-assembled polymeric micelles have been studied widely, due to their excellent role in cancer treatment. Polymeric micelles have a core–shell structure, where hydrophobic drugs are soluble and remain stable in the hydrophobic core of the micelles, and the hydrophilic shell can prolong internal circulation and improve spatial stability by reducing opsonization during blood circulation. Furthermore, polymeric micelles can selectively and effectively accumulate in tumor tissues due to the EPR effect, thus enhancing the therapeutic effects of the loaded chemotherapeutic drugs. As such, codelivery micellar systems have attracted considerable attention.^222,223

Doxorubicin (Dox) has extensive antitumor activity against various solid tumors, including lung cancers, melanoma, neurological cancers, sarcoma, leukemia, lymphoma, gastrointestinal cancers, genitourinary cancers, breast cancers, and ovarian cancers.¹⁴ Due to the rapid elimination of drugs in vivo, the cocktail combination of free Dox and Cur often fails to provide enough antitumor efficacy or low systemic toxicity. Furthermore, the combination of Dox and Cur has not been realized clinically. In recent years, a few studies have proposed that codelivering Dox and Cur may result in less toxicity, good drug-release profiles, and improved drug distribution in tumor tissue.^224–226 Zhang et al²²³ prepared dual-loaded micelles with coencapsulated Dox and Cur. The experimental results showed that Dox delivered by this method prolonged systemic circulation and increased its accumulation in the tumor, resulting in a lower level of the toxic metabolite doxorubicinol in heart tissue than free Dox alone or the cocktail combination. In addition, Gu et al²²⁵ assembled micelles loaded with Dox and Cur. The micelles prolonged the circulation of Dox or Cur when compared with the individual administration of either, and exhibited strong inhibition of tumor growth and reduced Dox side effects.

Ptx has a broad spectrum of activity against various tumors. It has been used clinically for more than two decades. However, it is poorly soluble and has considerable limitations in clinical applications. In addition, Ptx extravasation of cancer cells caused by Pgp activity is also a main factor limiting its clinical efficacy.^227,228 Abouzeid et al²²⁹ prepared micelles loaded with Ptx and Cur using the thin-film hydration method. Cur–Ptx-loaded micelles released the entrapped drugs with a slow pattern, and resulted in a threefold inhibition of tumors in vitro. The combination of Cur and Ptx was shown to reverse MDR in a resistant human ovarian adenocarcinoma model. Therefore, these combinations of micelles have significant advantages in vitro and in vivo over individual drug treatment, especially in drug-resistant tumors.

Microemulsions

A microemulsion is a transparent or semitransparent oil– water system with low viscosity, isotropism, and thermodynamic stability, and is spontaneously formed by an oil phase, water phase, emulsifier, and coemulsifier. As an ideal drug carrier, it has the advantages of solubilizing components with different solubility properties, good dispersibility, excellent absorbability, and increased bioavailability.²³⁰

BA is one of the most commonly used traditional chemotherapeutic drugs for the treatment of various cancers, including HCT116 human colon cancer,³¹ pancreatic cancer stem cells,³² and bladder cancer.³³ It is known that BA has the ability to inhibit the function of Pgp.²³¹ Meng et al²³⁰ developed nanoemulsions (NEs) coencapsulating Ptx and BA using rotary evaporation. The research showed that compared with other Ptx preparations, Ptx-BA NEs had a better antitumor effect on MCF7/Tax cells. Studies on cellular uptake have shown that Ptx-BA NEs accumulate effectively in cancer cells. More importantly, Ptx-BA NEs have a higher antitumor effect than other Ptx formulations used in antitumor in vivo studies. The combined delivery of Ptx and BA by NEs may provide a potential combination-treatment strategy to overcome MDR.

Nanovesicles

Nanovesicles are microvesicles with a quasiliposome duallayer structure formed by the self-assembly of synthesized or naturally modified amphiphilic polymers and cholesterol in hydrophilic media. In contrast to other micromolecular vesicles, the polymer vesicle is characterized by good molecular designability, high intensity of the vesicle, excellent stability, and strong permeability.^232,233 As a TCM carrier, it can improve histocompatibility and cell permeability and encapsulate hydrophilic drugs or lipophilic drugs.

Alemi et al²³⁴ loaded both Cur and Ptx into cationic PEGylated niosomal formulations using thin-film hydration method to enhance efficacy in MCF7 human breast adenocarcinoma cells. The combination of Ptx and Cur, particularly in the nanoniosome formulations, improved the effectiveness of cancer therapy. The novel cationic PEGylated niosome delivery of combined Ptx and Cur is an effective strategy in the treatment of breast cancer.

Schematic illustrations of these nanosystems for drugs are shown in Figure 6. In addition, graphene oxide,²³⁵ carbon nanotubes,^236,237 nanorods,²³⁸ nanosponges,²³⁹ solid lipid NPs,²⁴⁰ nanometal–organic frameworks,²⁴¹ metallosupra-molecular nanogels,²⁴² and microspheres²⁴³ all provide new opportunities for the antitumor effects of TCM-combination therapy.

Figure 6 Schematic illustrations of several nanosystems for drugs.

Despite the EPR effect, most nanosystems fail to find their way toward tumor sites.²⁴⁴ Under most circumstances, 90% or more of the administered nanosystems end up in the liver or spleen, increasing adverse systemic reactions and causing low therapeutic efficacy.^245,246

Active targeted drug-delivery systems

An active targeting preparation is a drug-delivery system which can utilize the modified drug carrier as a “missile” and deliver the drug selectively to the target area to allow the drug to accumulate and exert its efficacy. The mechanism of active targeting is that after surface modification with the specific targeted antibody or ligand via covalent or noncovalent binding, the nanodelivery system can avoid recognition and phagocytosis by macrophages and change natural in vivo distribution, so as to deliver the drug to the targeted tumor site and exert its active tumor-targeting effects.²⁴⁷ For instance, due to the difference between tumor cells and normal cells in terms of receptor expression or other biological characteristics, the tumor-targeted drug-delivery system has been developed to ensure that the drug acts only on tumor cells²⁴⁸ and induces the off-target effect in normal tissue, which has become a high-profile topic in studies on drug-delivery systems.

Transferrin (Tf)-modified nanocarriers

The Tf receptor is commonly present in normal cells and tumor cells. However, expression of the Tf receptor is approximately four to five times that on the surface of tumor cells than on normal cells.²⁴⁹ Transferrin can bind with the Tf receptor and be internalized into the cells under mediation by the receptors to reach the targeted site.

Cui et al²⁵⁰ designed Tf-decorated NPs (Tf-PEG Cur–Dox NPs) to codeliver Cur and Dox for breast cancer therapy. Results showed that the combination of Tf-PEG-Cur and Dox NPs exerted higher cytotoxicity in MCF7 cells compared with Tf-PEG-Cur NPs alone. Higher accumulation of Tf-PEG Cur–Dox NPs was observed in tumors compared to the Cur-Dox injection. Therefore, Tf-PEG Cur–Dox NPs displayed higher efficiency in vitro and in vivo, and resulted in efficient tumor-targeted drug delivery, reduced cytotoxicity, and a stronger antitumor effect.

Folic acid–modified nanocarriers

Similarly to distribution of the Tf receptor on surfaces of tumor-cell membrane, folic acid receptors on tumor cells are overexpressed compared with normal cells, and their activity is also significantly higher than that on normal cells. In addition, folic acid is characterized by low immunogenicity, high modifiability, and high storability. Utilization of the difference in folic acid–receptor expression between tumor sites and normal tissue can achieve targeted delivery of a folic acid–modified drug to cancer cells.^251,252

Prodrugs of Ptx and baicalein containing dual-targeted ligands of folate and hyaluronic acid have been synthesized. NPs loaded with these prodrugs (Ptx–baicalein) have also been prepared and the synergistic antitumor effect evaluated in vitro and in vivo. The results showed that the Ptx–baicalein NP drug-delivery system delivered Ptx–baicalein prodrugs to drug-resistant human lung cancer cells, and the delivery was proven to be effective. In addition, Ptx–baicalein NPs exerted an enhanced synergistic anticancer effect, which also overcame MDR to Ptx.²⁵³

Low-density lipoprotein–modified nanocarriers

The low-density lipoprotein (LDL) receptor is widely present on the surface of various cells and tissue types, but is overexpressed in tumor cells. LDL is an endogenous NP with good biocompatibility, good biodegradability, and low immunogenicity, and can avoid being recognized and cleared by the in vivo endogenous reticuloendothelial system.^254,255 Therefore, LDL is an ideal potential ligand for tumor targeting.

A novel nanocarrier containing Ptx-loaded micelles and siRNA-loaded LDL has been developed. Results showed that the delivery system delivered siRNA and Ptx directly to cancer cells, enhancing the intracellular release of drugs and genes, increasing intracellular drug concentration, decreasing drug efflux, prolonging circulation, and reversing MDR.^256,257

Cell-penetrating/tumor-targeting peptide–modified nanocarriers

Nanocarriers using cell-penetrating and/or tumor-targeting peptides for functionalization are a promising strategy, and have attracted the attention of researchers. In our previous report, we reviewed the classification of polypeptide- and polypeptide-modified nanocarriers in detail.²⁵⁸ In this report, recent research progress is summarized in the following paragraphs.

Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, has been widely studied as a potential anticancer drug. Narayanan et al²⁵⁹ prepared targeted drug-loaded core–shell NPs using anti-EGFR and anti-HER2 antibodies, and entrapped a combination of Ptx and EGCG at different doses in the core and shell, respectively, using emulsion precipitation. Cellular uptake in MDA-MB231 cells was higher for targeted NPs than untargeted NPs at 24 hours. The sequential release of EGCG followed by Ptx from this core–shell nanocarrier sensitized Ptx-resistant MDA-MB231 cells to Ptx, induced their apoptosis, and inhibited NFκB activation. In addition, EGFR-peptide (GE11)-targeted, pH-sensitive docetaxel Dtx–Cur NPs²⁶⁰ and arginylglycylaspartic acid–modified lipid-coated PLGA NPs targeting delivery of both sorafenib and Qct²⁶¹ achieved significant inhibition of tumor growth in vitro and in vivo.

In addition, galactosamine can recognize and bind to the asialoglycoprotein receptor on the surface of hepatocellular carcinoma cells, and a galactosamine-mediated drug-delivery carrier was significant for targeted liver cancer therapy.^262,263 Glycyrrhizin, glycyrrhetinic acid, and mannose can serve as the guiding group in liver-targeted drug-delivery systems, with good potential.^264–270 As natural endogenous ligands, bile acids have good biocompatibility and are ideal routes for targeting hepatocellular cancer.²⁷¹ In addition, sialic acid,²⁷² human Nanog,²⁷³ and hyaluronic acid²⁷⁴ are excellent targets in cancer therapy. Vapreotide is a somatostatin analogue and can be also used as a ligand for targeted drug delivery based on its high affinity to somatostatin receptors, which are overexpressed in many tumor cells.²⁷⁵ Several studies have shown that double-modified nanocarriers have also attracted considerable attention in anticancer drug research.^276,277 Dual or multiple targeting also provides a new approach for antitumor therapy.

Physicochemical targeted drug-delivery systems

Physicochemical targeting refers to the binding of magnetic, pH-sensitive, temperature-sensitive, or electromagnetic wave–responsive materials onto the surface of drug-delivery systems (such as NPs and liposomes) to make them respond to various stimuli in vitro and in vivo (such as pH, temperature, applied magnetic fields, ultrasonic waves, infrared rays, and electromagnetic radiation) to ensure that the drug acts directly on the target area, increases drug concentration at the lesion site, and reduces adverse reactions. Most studies have used magnetic NPs, temperature-sensitive NPs, and pH-sensitive NPs.

NPs that are pH-sensitive have been designed to promote uptake in tumor cells²⁷⁸ and accelerate drug release at tumor sites, as the extracellular pH (6.5–7.2) of the tumor is different to that of normal tissue.²¹⁶ Zhang et al²⁷⁸ developed a codelivery system for Dox and Cur using pH-sensitive NPs. Enhanced release in the acidic environment of cancer cells and enhanced cellular internalization of the cargoes delivered from Dox–Cur NPs were observed in SMMC7721 cells and human umbilical vein endothelial cells compared to the free drugs. Therefore, pH-sensitive NPs can provide a promising strategy for the effective inhibition of cancer in a synergistic manner. Danafar et al²⁷⁹ achieved codelivery of Cur and sulforaphane (SF) with PEGylated gold-coated Fe₃O₄ magnetic NPs as an effective and promising antitumor agent. Results showed that SF–Cur coloaded Fe₃O₄@Au NPs caused a decrease in cell viability and induced apoptosis by increasing the Bax:Bcl2 ratio. Moreover, photosensitizer NPs,²⁸⁰ thermosensitive NPs,²⁸¹ and redox-sensitive NPs²⁸² also provide new opportunities for nanosystems with anti-tumor TCM combinations.

Conclusion

The significant challenge posed by cancers, as well as adverse reactions and drug resistance induced by long-term treatment of a single drug, compels us to change our focus from a single target to the regulation of networks in vivo. Many complex factors cause cancer; therefore, it is rational that treatment should involve multiple components, genes, systems, and target pathways. The combination of drugs has resulted in a new approach to cancer treatment. Rational combinations of drugs not only result in synergy but also reduce the occurrence of drug resistance and adverse reactions, which has resulted in combination therapy, thus becoming a significant antitumor treatment in the clinic and in research. As such, this significant research direction may allow medical researchers to identify a chemotherapeutic combination regimen with high efficacy and low toxicity.

The combination of TCMs for clinical therapy has increased. TCM combinations can exert improved synergistic antitumor effects by adjusting the multiple signaling pathways of tumor cells. Compared with single-drug therapy, combinations of TCMs can reduce the toxicity and side effects of chemotherapy drugs and increase the antitumor effect of drugs. Simultaneously, TCM combinations and chemical drugs can improve immunofunction, relieve clinical symptoms, improve patient survival and quality of life, and improve the efficacy of chemotherapy drugs.

It should be noted that there are usually three approaches in targeted nanotechnology-based TCM-combination-therapy: nanodrugs combined with conventional preparations, coloading of two or more anticancer drugs in a single nanocarrier system (recorded as codelivery), and combined administration of different nanosystems. The loading of two drugs into a single well-designed nanocarrier synchronizes the pharmacokinetic and biological distribution of different drugs to achieve a synergistic effect, which has distinct advantages. This method has been summarized in detail. An increasing number of studies have shown that dual nanosystems have distinct advantages in antitumor treatment and can provide drugs for different targets or sites of action, as they are administered flexibly using different dose/time schedules. Consequently, the remaining two methods urgently require further investigation. In addition, with further comparative analyses of these three research methods, the most suitable form of drug use for cancers can be identified to provide basic considerations in terms of design principles and management progress.

Acknowledgments

This study was financially supported by the National Natural Science Foundation of China (81673605).

Disclosure

The authors report no conflicts of interest in this work.