Anticancer activities of phytoconstituents and their liposomal targeting strategies against tumor cells and the microenvironment

Abstract

Various bioactive ingredients have been extracted from Chinese herbal medicines (CHMs) that affect tumor progression and metastasis. To further understand the mechanisms of CHMs in cancer therapy, this article summarizes the effects of five categories of CHMs and their active ingredients on tumor cells and the tumor microenvironment. Despite their treatment potential, the undesirable physicochemical properties (poor permeability, instability, high hydrophilicity or hydrophobicity, toxicity) and unwanted pharmacokinetic profiles (short half-life in blood and low bioavailability) restrict clinical studies of CHMs. Therefore, development of liposomes through relevant surface modifying techniques to achieve targeted CHM delivery for cancer cells, i.e., extracellular and intracellular targets and targets in tumor microenvironment or vasculature, have been reviewed. Current challenges of liposomal targeting of these phytoconstituents and future perspective of CHM applications are discussed to provide an informative reference for interested readers.

1. Introduction

Cancer is a leading cause of death worldwide. It is an uncontrolled and excessive growth of cells that can metastasize to a number of organs [1]. Cancer therapy has remained challenging for centuries, although therapeutic strategies such as surgery, chemotherapy and radiotherapy, have been developed. Until now, the main clinical treatment has been chemotherapy in addition to surgery. However, most chemotherapy drugs have high toxicity, low specificity, and are accompanied by painful side effects [2]. In addition, multidrug resistance could be induced by chemotherapeutics, causing treatment failure upon disease recurrence [3].

Phytoconstituents have emerged as a prospective approach for cancer treatment and are also widely used, especially in Asia [4,5]. These bioactive molecules are of great interest due to their high range of biological activity, minimal side effects, and low cost [6].

The current reviews on the developments of phytochemical delivery systems for cancer treatment mainly focus on specific types of delivery systems or compounds, e.g. polyphenols [5,7,8]. Bahrami’s group have provided a summary of the anticancer effect of natural agents, either alone or in combination with natural drugs, by modulation of regulatory T cells (Tregs) [9]. However, to the best of our knowledge, there is no comprehensive review using the syndrome differentiation, and treatment of traditional Chinese medicine (TCM) as the guiding principle which classifies plants according their functions and indications in Pharmacopoeia of the People’s Republic of China (ChP) 2015 edition, and covers preclinical stage liposomal targeting compound delivery strategies. Moreover, this review is both limited to the anti-tumoral efficacy of plant-derived medicines, and also includes their effects on the tumor microenvironment (TME), which is subdivided into tumor immune and non-immune microenvironments [10].

The aims of this review were to summarize the history of treating cancer in Chinese medicine and to critically analyze the potential anti-tumoral effects of Chinese medical plants recorded in ChP 2015 edition and their active components, which function in counteracting toxin with toxin, heat-clearing and detoxifying, promoting blood circulation and removing blood stasis, resolving phlegm and eliminating dampness, strengthening healthy energy and consolidating body resistance, and suppressing tumor aerobic glycolysis, by comparing the treatment theory of TCM with modern medicine. Because modulating the stromal TME (tumor associated macrophages (TAMs), TAFs, and endothelial cells) or immune microenvironment by either small molecules or nanodrugs can facilitate the remodeling of blood vessels or extracellular matrix (ECM) at tumor sites [11], we also summarize recent developments and strategies for liposomal delivery systems of promising anticancer phytoconstituents and discuss the opportunities of further improvement.

It is worth mentioning that our review summarizes the anti-tumor effects of natural products and their impact on the TME, together with the targeted liposomal drug delivery system. In fact, the specific pharmacological effects of each monomer compound on tumor cells and TME, and liposomal targeting formulations in this review are all based on the Western medicine theory and summarized by searching for literatures with systematic data sets. The research on the action mechanism of monoconstituents and their delivery system is the same as that of Western medicine, except that we use the traditional Chinese medicine classification standard for the classification of Chinese medicines. This classification is secondary, not the focus of our review. In fact, traditional Chinese medicine and Western medicine have their own characteristics in diagnosis and treatment. Not every concept of traditional Chinese medicine treatment can be explained in terms of Western medicine theory. In this review, the classification we use is an attempt to combine the research of traditional Chinese medicine monomers with the theory of traditional Chinese medicine.

2. The connotation of TCM in tumor treatment

“Tumor” was recorded in the oracle bone inscriptions of the Yin and Zhou era more than 3,500 years ago. Miraculous Pivot in the Han Dynasty mentioned the pathogenesis and symptoms of “carbuncle”. Until the Ming Dynasty, the occurrence and development of breast tumors were discussed in detail [12]. Physician Zhang Xichun explained the treatment and prescription of phrenic disease syndromes (mainly including esophageal cancer or gastric cardia cancer) in detail in his book “Records of Tradition Chinese and Western Medicine in Combination” at the end of the Qing Dynasty. As it was stated in Yellow Emperor’s Inner Cannon (written from the Warring States period to the Qin and Han Dynasties), deficient vital qi and excess pathogenic qi is the most important basic pathogenesis of cancer. Qi refers to the nutritive and refined substances circulating in the body and the functional state of organs and tissues [13]. Qi correlates with longevity and qi depletion is linked to death [14]. Qi is divided into vital qi and pathogenic qi. The so-called “vital qi” is the body’s resistance to pathogenic microorganisms and its ability to adjust and adapt. Deficiency of vital energy is deficiency of vital qi [15]. Pathogenic qi refers to various pathogenic factors, including wind, cold, summer-heat, dampness, dryness, and heat. Disease results from the struggle between the vital qi and the pathogenic qi in the body [16]. Therefore, treatment is divided into two aspects: removing pathogenic factors and strengthening the vital qi. Using the syndrome differentiation and treatment of TCM as the guiding principle, treatment to eliminate pathogens refers to counteracting toxin with toxin, heat-clearing and detoxifying, promoting blood circulation and removing blood stasis, resolving phlegm and eliminating dampness; treatment to reinforce healthy qi refers to strengthening healthy energy and consolidating body resistance [17–20]. Here, the TCM classification is not independent or irrelevant. On the contrary, the use and function of TCM are often intersected and need to be used as a whole.

Among the natural products eliminating pathogenic factors, counteracting toxin with toxin, and heat-clearing and detoxifying herbal medicines are most extensively used. According to the pathogenesis theory of cancerous toxins [21], it is suitable to use heat-clearing and detoxification therapy to eliminate cancer toxins at the early stages of cancer. If the cancer toxin has the potential to spread, then the method of attacking poison with poison should be used to attack and stop the development of cancer toxins; in the middle stages of cancer, it is better to use the synergistic therapy of heat-clearing and detoxification, and attacking toxin with toxin; for patients with advanced cancer, heat-clearing and detoxification is the only appropriate method to remove cancer toxins continuously.

3. Anti-tumor mechanism of different classification of Chinese herbal medicines (CHMs) and their active components

In the treatment of tumors, CHMs can play multi-channel and multi-target roles, and have the advantages of less adverse reactions and good tolerance. We summarized the anti-tumor efficacies of the following five categories CHMs and their active components in the aspects of inducing apoptosis, inhibiting cell proliferation, regulating autophagy, reversing multidrug resistance, inhibiting angiogenesis, targeting cancer stem cells (CSCs), blocking tumor invasion and metastasis, and regulating immune state (Figure 1). Some natural products target glycolysis pathway in cancer cells, which could control cancer growth, multidrug resistance, and metastasis. The regulatory effect of CHMs on TME including inhibiting tumor-associated fibroblasts (TAFs) and their released cytokines, decreasing angiogenic cells and their pro-angiogenic factors, degrading ECM and regulating on immune cells and their cytokines. The involving immune cells include T lymphocytes (T cells), B lymphocytes, natural killer cells (NK cells), tumor-associated macrophages (TAMs), dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs). Some monoconstituents could induce immunogenic cell death (ICD) on cancer cells, which mediated by damage-associated molecular patterns (DAMPs) and thus increased the levels of costimulatory signals on DCs to promote the antitumor T-cell response.

Figure 1:

Overview of mechanisms for monoconstituents of CHMs on tumor cells and the TME (Abbreviation: MRP, multidrug resistance-associated protein; GS-conjugates, glutathione conjugates; P-gp, P-glycoprotein; BCRP, breast cancer resistance-relative protein; TGF-β, transforming growth factor-β; IL-10, interleukin-10; IFN-γ, interferon gamma; TNF-α, tumor necrosis factor-alpha).

3.1. CHMs that counteract toxin with toxin and their active components

There are 70 kinds of toxic herbs and decoction pieces are recorded in ChP (2015 edition, volume I). Of these, eight had great toxicity, 33 had general toxicity, and 29 had small toxicity. Among them, 52 are reported to exhibit anti-cancer effects, 30 are used as extraction, and 22 as both extraction and active components.

The “cancer toxin” is thought to be an important reason for the formation of cancer. TCM uses the theory of the cancer toxin to describe viruses, bacteria, chemical pollution, and invisible carcinogens. Cancer toxins can directly lead to dysfunction of viscera, induce phlegm, blood stasis, heat toxin and other pathological factors similar to inflammation. Heat toxin refers to external toxin characterized by redness, heat pain, hemorrhage, dizziness, abnormal movement of limbs, and sudden illness [22]. Due to the slow process of tumor formation, and the deep accumulation of toxins, conventional Chinese medicine is challenging. As such, some toxic products, by virtue of their fierce nature, are needed attack the cancer poison in the so-called “fight fire with fire” approach. TCM that uses poison to attack poison can activate blood stagnation, soften hardness, and dissolve lumps [23]. The active components of the crude drugs CHMs for counteracting toxin with toxin and their effects on tumor cells are summarized in Table 1.

Table 1.

Mechanism of the CHMs that counteract toxin with toxin and their active components on tumor cells.

CHMs	Active components	Tumor cells					TME	References
		Apoptosis induction	Proliferation inhibition	Autophagy regulation	Anti-multidrug resistance	Migration and invasion inhibition	Anti-angiogenesis
Radix aconiti kusnezoffii (cao wu)	Aconitine	√	√		√	√		[30–33]
	Hypaconitine				√	√		[33,34]
	Mesaconitine	√			√			[33,35]
Nux vomica (ma qian zi)	Strychnine	√					√	[36]
	Brucine	√	√		√	√	√	[37,38]
Stellera chamaejasme (lang du)	Chamaejasmenin B and Neochamaejasmin C	√	√			√		[39,40]
Fructus gleditsiae sinensis (zao jiao)	Echinocystic acid	√	√					[41]
Fructus toosendan (chuan lian zi)	Toosendanin and Other triterpenoids	√						[42]
Sinopodophylli fructus (xiao ye lian)	Podophyllotoxin, Deoxypodophyllotoxin, 8-Isopentenyl kaempferol, and Kaempferol	√	√					[43,44]
Zanthoxylum nitidum (liang mian zhen)	Nitidine chloride and Zanthobungeanine	√	√		√			[45]
Fructus evodiae (wu zhu yu)	Evodiamine and Rutaecarpine	√	√	√		√		[46]
Semen armeniacae amarae (ku xing ren)	Amygdalin	√	√					[47]
Fructus cnidii (she chuang zi)	Osthole	√	√		√	√	√	[48]
Cortex pseudolaricis (tu jing pi)	Pseudolaric acid B	√	√					[49]
Momordicae semen (mu bie zi)	p-Hydroxylcinnamaldehyde		√					[50]
Herba chelidonii (bai qu cai)	Chelidonine and Sanguinarine	√						[51]
	Chelerythrine		√					[52]
Fructus xanthii (cang er zi)	Xanthium		√			√		[53]
Cortex periplocae (xiang jia pi)	Periplocin	√	√				√	[54]
	Baohuoside I	√						[55]

From Table 1, most of the CHMs that counteract toxin with toxin function by directly damaging tumor cell DNA, inhibiting tumor cell division and gene expression to induce tumor cell apoptosis. Apoptosis mainly includes the cell death receptor-mediated extrinsic and mitochondrion-mediated intrinsic pathways [24,25], which are affected by the B-cell lymphama-2 (Bcl-2) protein family.

The cell cycle can be divided into G1 (preceding initiation of DNA synthesis), S (DNA synthesis), G2 (preceding mitosis), and M (mitosis) stages. G1/S and G2 /M are the most important steps of cell cycle regulation, while cyclin dependent kinases (CDKs) are key cell cycle regulators [26]. CHM active components could decrease expression of proliferative genes, such as cyclin, protein 53 (p53), and k167, proliferating cell nuclear antigen (PCNA), telomerase, and CDKs [27].

The signaling pathways for inducing apoptosis and inhibiting proliferation include the Wnt pathway (such as resveratrol, berberine, and curcumin), transforming growth factor-β (TGF-β) pathway (such as resveratrol and baicalein), signal transducer and activator of transcription 3 (STAT3) pathway (such as nitidine chloride, scutellarin, isoliquiritigenin and cryptotanshinone), p53 pathway (such as magnolol, ligustrazine, emodin, and evodiamine), nuclear transcription factor-κB (NF-κB) pathway (such as evodiamine), p38 mitogen activated protein kinase (MAPK) pathway (such as matrine and baicalein), extra cellular signal regulated kinase (ERK) pathway (such as scutellarin, luteolin, curcumin and pistil isoflavone), c-Jun N-terminal kinase (c-Jun) pathway (such as tanshinone ⅡA and celastrol), and phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway (such as resveratrol glycoside) [28,29].

3.2. CHMs that clear heat and detoxify and their active components

There are 115 kinds of heat-clearing and detoxifying CHMs and slices recorded in ChP (2015 edition, volume I). Of these, 88 are reported to exhibit antitumor effects in vitro or in vivo. Eight are used as crude drugs, 37 as extraction, and 43 as active components.

Zhongying Zhou, a master of Chinese medicine, reported that heat is an important pathogenic factor for tumor development, as cancer patients often have fever, pain, thirst, local burning, constipation, red tongue with yellowish fur, and so on. Clinical manifestations of heat syndrome in microenvironments include increased oxygen consumption, higher leukocyte and neutrophil count, and higher consumption of cortical alcohols and catecholamines [56,57]. As recorded in Yellow Emperor’s Inner Cannon, body’s organs, limbs, skin, muscles and bones are connected into an organic whole through meridians and keep it relatively coordinated and unified to complete various functions, where meridians refer to channels that provide energy for human metabolism, treat diseases, and transmit diseases. “Heat toxin” will block the meridians of the viscera and heat-clearing and detoxification must continue through the whole process of cancer treatment. It is clearly put forward in the Yellow Emperor’s Inner Cannon that “treating heat syndrome with drugs of cold nature and treating warm syndrome with drugs of cool nature” is the proper approach [23]. Experimental results show that the most effective way to clear away heat and detoxify is to use cold and cool natured drugs to eliminate or degrade the heat toxin in the body and control inflammation.

Chronic “non-resolving inflammation” contributes significantly to the pathogenesis of malignancies. This relationship between inflammation and cancer was first put forward by Rudolph Virchow. The process consists of two parts, initiation and promotion. First, precancerous inflammation can cause irreversible DNA alteration in proliferating cells. Second, in the persistent presence of aberrant activation of inflammatory oncogenes in chronically inflamed tissues, the abnormal cell replication and proliferation continues, and achieve the full malignant phenotype, such as ECM remodeling, angiogenesis, metastasis, and suppressed innate immune responses [58–62].

Inflammation is recognized to play a major role in the development and progression of malignancies and is closely related to the heat toxin of TCM in disease occurrence and development [63–65]. Modern studies have shown that heat-clearing and detoxifying drugs exhibit as anti-inflammatory properties to clear heat, detoxify, resist bacteria, and improve immunity. They can control and eliminate inflammation of the tumor and its surroundings, proving that heat-clearing and detoxifying drugs used in tumor treatment are key to inhibiting tumor development [66]. The active components of crude drugs of heat-clearing and detoxifying CHMs and their effects on tumor cells and the TME are summarized in Table 2.

Table 2.

Mechanism of the CHMs that clear heat and detoxify and their active components on tumor cells and the TME.

CHMs	Active components	Tumor cells					TME							References
		Apoptosis induction	Proliferation inhibition	Autophagy regulation	Anti-multidrug resistance	Migration and invasion inhibition	TAFs	Anti-angiogenesis	TAMs	NK cells	T cells	DCs	MDSCs
Saururus chinensis (san bai cao)	Sauchinone	√	√	√		√								[67–69]
Solidago decurrens (yizhi huang hua)	6β-Angeloyloxykolavenic acid and 6β-Tigloyloxykolavenic acid		√											[70]
Rhizoma coptidis (huang lian)	Berberine	√	√	√	√	√	√	√	√		√			[71–74]
Sargentglory vine stem (da xue teng)	Chlorogenic acid	√	√		√	√		√	√	√	√			[75–79]
Folium isatidis (da qing ye)	Indirubin	√	√		√	√		√						[80–83]
Rheum palmatum (da huang)	Emodin	√	√	√	√	√		√	√					[84–88]
	Rhein	√												[89]
Radix sophorae tonkinensis (shan dou gen)	Cytisine	√												[90]
	Matrine and Oxymatrine	√	√	√	√	√		√						[91–95]
	Oxysophocarpine		√											[96]
Flos lonicerae (shan yin hua)	Macranthoside B	√	√	√										[97,98]
Portulace oleracea (ma chi xian)	Betacyanins	√	√											[99,100]
Fructus arctii (niu bang zi)	Arctiin and Arctigenin	√	√	√		√								[101–104]
Belleric terminalia fruit (mao he zi)	Gallic acid	√	√			√								[105–107]
Rhizoma cimicifugae (sheng ma)	Actein and 23-epi-26-Deoxyactein	√	√	√		√								[108,109]
	Cimigenol	√												[110]
	Ferulic acid and Isoferulic acid	√	√	√	√	√								[111–114]
Silybum marianum (shui fei ji)	Taxifolin	√			√						√	√		[115–118]
	Isosilybin A/B	√	√											[119,120]
	Silibinin	√	√	√	√	√	√	√	√		√	√	√	[121–130]
Caulis mahoniae (gong lao mu)	Jatrorrhizin		√			√		√						[131–133]
Radix liquiritiae (gan cao)	Liquiritigenin and Liquirtin	√	√					√						[134]
	Isoliquiritigenin, Soliquiritin	√	√	√				√	√					[135,136]
	Licochalcone A/E	√		√										[137]
	Glabridin	√				√								[138]
	Glycyrrhetinic acid	√	√	√	√	√		√			√			[139–141]
	Glycyrrhizic acid	√				√								[142]
Pulsatilla chinensis (bai tou weng)	Isorhamnetin	√	√	√										[143,144]
	Quercetin and Isoquercitin	√	√		√	√	√	√			√	√	√	[145–150]
	Oleanolic acid	√	√			√		√						[151–153]
	Hederagenin	√	√			√								[154,155]
	Anemoside B4	√	√											[156]
	Pulsatilla saponin A	√												[157]
	Pulsatilla saponin D	√	√											[158]
	23-Hydroxyl betulinic acid	√												[159]
Rhizoma imperatae (bai mao gen)	Caffeic acid	√	√			√								[160]
	Arundoin		√											[161]
Radix ampelopsis (bai lian)	Catechin	√	√		√	√		√						[162–164]
	Resveratrol	√	√	√	√	√	√	√	√		√			[165–168]
Rabdosia rubescens (dong ling cao)	Oridonin	√	√	√		√								[169]
Radix scrophulariae (xuan shen)	Cinnamic acid		√											[170]
Radix sanguisorbae (diyu)	Ellagic acid		√			√					√			[171]
	Ziyuglycoside I and II	√	√					√						[172]
Polygonum cuspidatum (hu zhang)	Polydatin	√	√											[173]
Fagopyrum dibotrys (jin qiao mai)	Rutin				√									[174]
	Luteolin	√	√		√	√		√			√			[175,176]
	Procyanidin B1 and B2	√	√			√		√						[177]
Radix scutellariae (huang qin)	Baicalein	√	√	√		√		√	√					[178,179]
	Baicalin	√	√	√		√								[178]
	Wogonin	√	√	√	√	√		√			√			[180]
	Wogonoside	√	√			√		√						[181,182]
Calyx seu fructus physalis (jin deng long)	Physalins A and B	√	√	√										[183–185]
Radix arnebiae (zi cao)	Shikonin	√	√	√		√		√						[186–188]
Radix bupleuri (chai hu)	Saikosaponins A and D	√	√	√	√	√	√	√						[189]
Centella asiatica (ji xue cao)	Asiaticoside	√	√	√		√								[190]
	Asiatic acid	√	√			√		√						[191]
Herba leonuri (yi mu cao)	Leonurine hydrochloride	√												[192]
Andrographis paniculata (chuan xin lian)	Andrographolide	√	√	√		√		√						[193,194]
	Neoandrographolide	√	√											[195]
Cortex fraxini (qin pi)	Esculetin	√	√			√		√	√					[196]
	Aesculin	√	√											[197]
Fructus brucea (ya dan zi)	Brusatol, Bruceantinol, Bruceine A/D, Bruceantin	√	√	√	√	√								[198–201]
Fructus gardeniae (zhi zi)	Genipin	√	√											[202]
	Ursolic Acid	√	√	√		√		√						[203]
	Apigenin	√	√			√		√						[204]
	β-Sitosterol	√	√	√										[205]
Scutellaria barbata (ban zhi lian)	Aloe emodin	√	√	√										[206]
	Rhein	√	√		√	√					√			[207]

Heat-clearing and detoxifying plant-derived drugs can directly inhibit tumor cell proliferation, induce cell apoptosis, regulate and enhance immune levels, induce differentiation and reversion, regulate cell signaling pathways and transduction, resist mutation, inhibit angiogenesis, and reverse multidrug resistance. The possible mechanisms for their antitumor effects mainly derived from their anti-inflammatory properties. Anti-inflammatory drugs might suppress the release of inflammatory cytokines, inhibit protein kinases and growth factors, then retard tumor cell proliferation and transformation. Flavonoids have the abilities to inhibit proinflammatory enzymes, including inducible nitric oxide synthase and cyclooxygenase-2 (COX-2), and suppress COX-2-induced prostaglandin E2 (PGE2) expression. The combination of baicalin and baicalein can promote apoptosis of human breast cancer cells through the ERK/p38 MAPK pathway. Most phenylpropanoids are reported to suppress proinflammatory cytokines (tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), IL-6, IL-12 and IL-2). Saponins might induce cleavage of poly-ADP ribose polymerase and activate of caspases by downregulating inflammatory factors. They would also inhibit tumor growth, invasion and metastasis through matrix metalloproteinases (MMP-2, −9 and −14), STAT3, and NF-κB. Quinones suppress cell proliferation by inhibiting of protein kinase C and epidermal growth factor-receptor tyrosine kinase, associating cyclin D1 with cyclin-dependent kinases, downregulating protein activated kinase 1, and phosphorylating CDK-mediated retinoblastoma protein. Alkaloids inhibit angiogenesis by modulating the expression of vascular endothelial growth factor. The broad range of molecular targets provides a molecular basis for the therapeutic action of these anti-inflammatory plant medicines.

3.3. CHMs that promote blood circulation and remove blood stasis and their active components

There are 54 blood circulation and removing blood stasis herbs and decoction pieces recorded in ChP (2015 edition, volume I). Of these, 42 are reported to exhibit anticancer effects, 16 are used as extraction, and 26 as both extraction and active components.

According to the theory of TCM, tumor occurrence caused by the deficiency of vital energy, phlegm coagulation, blood stasis, toxins, etc. Among them, blood stasis is one of the main pathological mechanisms of tumor formation and development, and can be seen in all stages of tumor course. Modern research confirmed that the hemorheology of tumor patients shows high coagulation, high viscosity, and peripheral microcirculation disorders [208,209]. Most TCM for promoting blood circulation and removing stasis function by regulating vascular growth factor, improving microcirculation, and improving hemorheology and coagulation [210]. The active components of the crude drugs promoting blood circulation and removing blood stasis and their effects on tumors are summarized in Table 3.

Table 3.

Mechanism of the CHMs that promote blood circulation and remove blood stasis and their active components on tumor cells and the TME.

CHMs	Active components	Tumor cells					TME		Blood circulation	References
		Apoptosis induction	Proliferation inhibition	Autophagy regulation	Anti-multidrug resistance	Migration and invasion inhibition	Anti-angiogenesis	Tregs	Platelet aggregation
Panax notoginseng (san qi)	Notoginsenoside R1, R7, and Ft1	√	√			√			√	[218–221]
Rhizome chuanxiong (chuan xiong)	Ligustrazine	√	√	√	√	√	√		√	[222,223]
Salvia miltiorrhiza (dan shen)	Tanshinone I and IIA	√	√	√		√	√		√	[224–226]
	Cryptotanshinone	√	√			√	√		√	[226,227]
	Rosmarinic acid	√	√	√		√	√		√	[228–230]
	Danshensu	√	√			√				[231]
	Salvianolic acid	√	√				√		√	[232,233]
	Dihydrotanshinone	√	√	√		√	√		√	[234–236]
Crocus sativus (xi hong hua)	Crocin and Crocetin	√	√						√	[237–239]
Erigeron breviscapus (deng zhan xi xin)	Scutellarin	√	√			√	√		√	[240,241]
Flos carthami (hong hua)	Hydroxysafflor yellow A	√	√				√		√	[242,243]
Rhodiola rosea (hong jing tian)	Salidroside	√	√			√	√		√	[244,245]
Cortex moutan radicis (mu dan pi)	Paeonol	√	√						√	[246,247]
Ardisia japonica (ai di cha)	Bergenin		√			√				[248]
Peach kernel (tao ren)	Amygdalin	√	√			√			√	[249,250]
	Naringenin	√	√		√			√	√	[9,251,252]
Rhizoma curcumae (e zhu)	Curcumin	√	√	√	√	√	√	√	√	[9,253–256]
	Curcumol	√	√	√		√	√			[257,258]

The possible mechanisms of their antitumor effects are mainly through the following aspects. First is the direct cytotoxic and antimutagenic effects, which can inhibit tumor cell proliferation and induce differentiation. Second is the down regulation of vascular endothelial growth factor and its receptor, leading to inhibition, promotion, and normalization of angiogenesis. Angiogenesis is a tightly regulated process integral to tumor growth, involving endothelial cell growth, differentiation, and migration [211]. The mechanisms of CHMs exhibiting anti-angiogenesis effects include the following models: (1) direct inhibition of vascular endothelial cell proliferation and migration; (2) inhibiting MMP activity; (3) inhibiting signal transduction of tumor angiogenic factors; (4) promoting tumor angiogenesis inhibitor expression [212–214]. Third is to weaken platelet aggregation; decreased platelet activation and platelet aggregation inhibits tumor progression [215]. Attenuated platelet aggregation and blood stasis prevent cancer cells from staying, adhering, aggregating, and planting in the blood. By avoiding of the shear forces or attack of the immune system in the blood stream, less than 0.01% of tumor cells are needed to cause successful hematogenous metastasis by increasing tumor cell emboli arrest in microcirculation. Platelet aggregation is also proposed to protect tumor cells from immunological assault in circulation [216]. Therefore, phytochemicals that promote blood circulation and remove blood stasis were used to reduce metastasis and affect microcirculation.

However, there are several shortcomings in tumor treatment by targeting the blood microenvironment alone: (1) if only a single target is inhibited, the rest of the signaling pathway will compensate and reduce the curative effect; (2) tumor cells may be dormant, preventing eradication [217]. Therefore, the promoting blood circulation and removing blood stasis treatment needs to be combined with other drugs to cure cancer.

3.4. CHMs that resolve phlegm and eliminate dampness and their active components

There are 21 kinds of resolving phlegm and eliminating dampness herbs and decoction pieces recorded in ChP (2015 edition, volume I). Of these, 15 are reported to exhibit anti-cancer effect, 6 are used as extraction, and 9 as both extraction and active components.

Phlegm coagulation is a main pathological factor of tumors. While dampness evil invades the body, phlegm and dampness gather together, leading to stagnation of qi and blood stasis, followed by accumulation in tumors which improves their invasive and metastasis abilities [259]. The relative lack of oxygen, high glucose absorption rate, glycolysis rate, and accumulation of acid metabolites or other abnormal intercellular components in tumors are manifestations of phlegm caused by the abnormal metabolism of body fluids and accumulation of dampness. The active components of the crude drugs resolving phlegm and eliminating dampness and their effects on tumors are summarized in Table 4.

Table 4.

Mechanism of the CHMs that resolve phlegm and eliminate dampness and their active components on tumor cells and the TME.

CHMs	Active components	Tumor cells					TME	References
		Apoptosis induction	Proliferation inhibition	Autophagy regulation	Anti-multidrug resistance	Migration and invasion inhibition	Anti-angiogenesis
Acorus tatarinowii schott (shi chang pu)	Alpha-asarone	√	√	√	√	√		[263,264]
Rhizoma alismatis (ze xie)	Alisol B and Alisol B23-acetate	√	√	√	√	√		[265,266]
Angelica dahurica (bai zhi)	Imperatorin and Isoimperatorin		√		√	√	√	[267–270]
Fructus citri sarcodactylis (fo shou)	Bergapten	√	√			√		[271,272]
	Diosmetin	√	√			√	√	[273,274]
	Diosmin	√	√					[275]
	Hesperidin and Hesperetin	√	√		√	√	√	[276–278]
Dried tangerine peel (chen pi)	Nobiletin	√	√		√	√	√	[279–281]
	Tangeretin	√	√		√	√		[282–284]
Magnolia bark (hou pu)	Magnolol and Honokiol	√	√	√	√	√	√	[285,286]
Pipernigrum (hu jiao)	Piperine, Citronellal, and Piperlongumine	√	√	√	√	√	√	[287–290]

The underlying mechanism includes regulating immunity, inhibiting tumor growth and angiogenesis, and inducing tumor cell apoptosis. Furthermore, resolving phlegm and eliminating dampness CHMs can effectively inhibit tumor metastasis by: (1) regulating expression of cell adhesion molecules (such as E-cadherin and vimentin) [260]; (2) inhibiting expression of MMP-2 and −9 proteases to delay extracellular matrix degradation [261]; (3) regulating abnormal neovascularization; (4) reversing the epithelial mesenchymal transition of tumor cells through the Wnt/β-catenin signaling pathway [262], ERK pathway, and TGF-β pathway to inhibit tumor metastasis.

3.5. CHMs that strengthen vital energy and consolidate body resistance and their active components

There are 53 kinds of strengthening vital energy and consolidating body resistance herbs and decoction pieces recorded in ChP (2015 edition, volume I). Of these, 47 are reported to exhibit anti-cancer effect, 2 are used as crude materials, 20 as extraction, and 25 as both extraction and active components.

According to TCM, after radiotherapy, chemotherapy or surgery, the human body is in a state of qi and blood deficiency. If we continue to use the “attack” method, the burden on the immune system will increase. Therefore, the treatment should change to maximize the recovery of bone marrow and immune function, that is, reconstruction of positive qi [61]. Chinese medical plants can regulate the immune response of hosts by elevating the proportion of effector T cells (T lymphocytes) to Tregs, inducing interferon gamma (IFN-γ) production in effector T cell, the phagocytic function of macrophages, the activity of natural killer cells (NK cells), promoting spleen dendritic cell (DC) differentiation, reducing myeloid-derived suppressor cells (MDSCs), decreasing TGF-β and Interleukin (IL)-10 levels, prevention of both IL-2 consumption and IL-2 expansion, regulating the balance of immune cell differentiation, and enhancing immune ability [291,292]. The active components of the crude drugs strengthening vital energy and consolidating body resistance and their effects on tumors are summarized in Table 5.

Table 5.

Mechanism of the CHMs that strengthen vital energy and consolidate body resistance and their active components on tumor cells and the TME.

CHMs	Active components	Tumor cells						TME							References
		Apoptosis induction	Proliferation inhibition	Autophagy regulation	Anti-multidrug resistance	Migration and invasion inhibition	CSCs population reduction	Anti-angiogenesis	TAFs	DCs	TAMs	NK cells	T cells	MDSCs
Panax ginseng (ren shen)	Ginsenoside Rg3	√	√	√	√	√	√	√		√		√	√	√	[218,293–297]
	Ginsenoside Rg1, Rh2, Rg5, Rh4, Rk3, Rb2, Rb1, Rd, F2, and CK	√	√	√		√	√	√		√	√				[218,293–302]
Radix polygalae (Yuan zhi)	Onjisaponin B			√											[303]
Ganoderma lucidum (lingzhi)	Ganoderic acid	√	√	√								√	√	√	[304,305]
Semen ziziphi spinosae (suan zao ren)	Jujuboside A/B			√											[306]
Dioscorea opposite (shan yao)	Diosgenin	√	√		√	√	√								[307]
Fructus ligustri lucidi (nv zhen zi)	Oleuropein	√	√			√			√						[308]
	Verbascoside	√	√			√									[309,310]
Fructus schisandrae chinensis (wu wei zi)	Kadsuphilactone B	√													[311]
	Deoxyschizandrin		√								√				[312]
	Schisandrin B	√	√		√	√		√							[313]
Atractylodes macrocephalae (bai zhu)	Atractylone, Atractylenolides I and III	√	√				√		√			√	√		[314]
	β-Eudesmol	√	√					√							[315]
	β-Elemene	√	√		√	√		√		√	√	√	√		[316]
Ophiopogon japonicus (mai dong)	Ophiopogonin D	√	√	√		√					√				[317]
	Ophiopogonin B	√	√	√		√									[318]
Herba epimedium (yin yang huo)	Icariin	√	√			√		√			√	√	√	√	[319,320]
	Baohuoside I	√	√										√		[321,322]
Fructus psoraleae (bu gu zhi)	Psoralen and Isopsoralen	√	√						√	√	√	√	√		[323,324]
	Bakuchiol	√	√			√									[325]
	Isobavachalcone	√	√			√									[326]
Acanthopanax senticosus (ci wu jia)	Sesamin	√	√						√		√	√			[327,328]
	Puerarin	√	√					√	√		√				[329–331]
Radix astragali (huang qi)	Astragaloside I/II/III	√		√				√					√		[332]
	Astragaloside IV	√	√	√	√	√			√		√		√		[333,334]
Dendrobii caulis (shi hu)	Gigantol	√				√	√								[335,336]
	Dendrofalconerol A	√				√									[337]
	Erianin	√	√	√		√		√							[338–341]
Radix angelicae sinensis (dang gui)	Imperatorin, Decursin, and Imperatorol	√	√	√	√			√							[342–344]
	Ferulic acid	√				√		√	√	√		√[	√[		[345–347]

3.6. Active components that suppress aerobic glycolysis in tumor cells

Metabolic reprogramming, especially switching to aerobic glycolysis, is a hallmark property of the cancer cells [348,349]. Aerobic glycolysis, also refers to the Warburg effect, could (1) provide tumor cells with the energy they need for survival. Although less adenosine triphosphate (ATP) is produced for per mole glucose through aerobic glycolysis (18~19-fold less than that of glucose metabolism under sufficient oxygen in normal cells), the speed of ATP generation is faster for per unit time, compared to oxidative metabolism of glucose [350]; (2) convert glucose in tumor cells to pyruvate and lactate even in the presence of oxygen. The lactate accumulation in the extracellular space results in an acidic TME (pH<6.8). The acidification of TME enhances chemotherapy resistance, induces epithelial-to-mesenchymal transition and promotes tumor migration and metastasis [351]; and (3) provides a large amount of glycolytic intermediates for the synthesis and metabolism of the tumor cells, including nucleotides, lipids and nonessential amino acids [352].

Acceleration of the glycolysis is triggered by transcription factor hypoxia-inducible factor-1α (HIF-1α) through PI3K/Akt/mTOR pathway, which associates with increased glucose transport through glucose transporters (GLUT-1 and GLUT-3) and upregulated glycolytic enzymes, such as hexokinase (HK), pyruvate dehydrogenase kinase 1 (PDK-1), lactate dehydrogenase A (LDH-A) and phosphofructokinase (PFK). Although the internal mechanism of aerobic glycolysis is still elusive, glycolysis suppression has been considered as a potential strategy for metastasis inhibition and anti-tumor treatment. The clinical application of current glycolysis inhibitors (such as 2-deoxyglucose and 3-bromopyruvic acid) are limited due to the serious systemic adverse effects [353]. Therefore, discovering natural products with high safety targeting glycolysis pathway is highly appreciated. The active components from CHMs for cancer cells aerobic glycolysis inhibition and their mechanism are summarized in Table 6.

Table 6.

The active components from CHMs for cancer aerobic glycolysis inhibition and their mechanism.

CHMs	Active components	Mechanism	Targets	Cancer model	Routes	References
Glycyrrhiza glabra (guang guo gan cao)	Glabridin	Inhibition of glucose transport and lactate formation	Downregulation of the GLUT-1 and LDH-A	In vitro MDA-MB-231 cells	\	[354]
Pulsatilla chinensis (bai tou weng)	Quercetin	Inhibition of glucose transport and lactate formation	Downregulation of the GLUT-1, LDH-A and pyruvate kinase M2 (PKM2)	In vivo Balb/c mice xenografted breast cancer (MCF-7 cells) In vitro MCF-7 and MDA-MB-231 cells	Intraperitoneal injection	[355]
	Oleanolic acid	Blockade of HIF-1α-mediated aerobic glycolysis	Reduction of the nuclear abundance of yes-associated protein	In vitro MKN-45 and SGC-7901 cells	\	[356]
Salvia miltiorrhiza (dan shen)	Rosmarinic acid	Suppression of glucose consumption and lactate generation	Inhibition of the expression of HIF-1α	In vitro HCT-8, HCT-116, Ls174-T, and Lovo cells	\	[357]
Birch bark (hua shu pi)	Betulinic acid	Inhibition of lactate production, glucose uptake and extracellular acidification rate	Suppression cancer glycolysis via caveolin-1/NF-κB/c-Myc pathway and upregulation of glucose-regulated protein 78	In vivo Mammary-tumor-prone transgenic mice/ Balb/c mice lung colonization breast cancer (MDA-MB-231 cells) In vitro MDA-MB-231, BT-549 and MCF-7 cells	Intraperitoneal injection	[349,358]
Cortex pseudolaricis (tu jing pi)	Pseudolaric acid B	Inhibition of glucose uptake, and ATP generation	p53 mutation or inactivation	In vitro H1299 cells	\	[359]
Rabdosia rubescens (dong ling cao)	Oridonin	Inhibiting glucose uptake and reducing lactate export	Downregulation of the protein levels of GLUT1 and monocarboxylate transporter 1 (MCT1)	In vivo Balb/c mice xenografted colorectal cancer (SW480 cells) In vitro p53-mutated colorectal cancer cells	Intraperitoneal injection	[360]
Radix Dichroae (chang shan)	Halofuginone	Inhibition of glycolysis and lipid biosynthesis	Inhibition of protein kinase B/mammalian target of rapamycin complex 1 (Akt/mTORC1) pathway	In vivo Balb/c nude mice xenografted colorectal cancer (HCT116 cells) In vitro SW480, HCT116, SW620, HT29 and DLD-1 cells	Intraperitoneal injection	[361]
Radix liquiritiae (gan cao)	Licochalcone A	Inhibition of the glucose consumption and lactate production	Inhibition of HK2 expression and blockade of the Akt signaling pathway	In vivo Balb/c nude mice xenografted gastric cancer (MKN45 cells) In vitro MKN45 and SGC7901 cells	Intraperitoneal injection	[362]
Salvia miltiorrhiza (dan shen)	Tanshinone IIA	Dysregulation of gluconeogenesis	Suppression of the expression of lactate dehydrogenase B chains and malate dehydrogenase 1	In vitro CRL-1739 cells	\	[363]
		Inhibition of glycolysis	Downregulation of glucose-6-phosphate isomerase (GPI)
Cortex fraxini (qin pi)	Esculetin	Inhibition of the rates of glucose consumption and lactate production	Alteration of the conformation of glycolysis-related enzymes, phosphoglycerate kinase 2 (PGK2), glycerol-3-phosphate dehydrogenase (GPD2), and GPI	In vivo Balb/c mice xenografted liver cancer (H22 cells) In vitro HepG2 cells	Intraperitoneal injection	[364]

4. Surface functionalization strategies of liposomes for monoconstituents of CHMs to treat cancer

A wide spectrum of bioactivities has been found in CHM-derived monoconstituents, and most have also shown anti-tumor efficacy through multiple mechanisms, including inhibition of angiogenesis, tumor progression, invasion and metastasis. Although they have important roles in different anti-tumor processes as previously discussed, there have been limited anticancer applications of these plant-derived medicines (alkaloids, saponins, flavonoids, organic acids, and so on) [4,365]. This is primarily due to the following. First, their low hydrophilicity (terpenoids such as oridonin, oleanolic acid and andrographolide; polyphenols such as curcumin, resveratrol, quercetin, silymarin, and puerarin; alkaloids such as tetrahydropalmatine and tetrandrine) results in low cellular uptake and low chemical stability. Second, some active components (saponins, such as saikosaponin, ginsenoside Rg1) undergo a series of reactions in animals to be transformed into water-soluble metabolites, which are then excreted in urine and bile, and some drugs are also excreted in their original form. They generally show low protein binding in blood and clearance by the reticuloendothelial system (RES) due to their hydrophilicity, related to the number and location of glycosides [366,367]. Therefore, they are rapidly eliminated, and these poor pharmacokinetic behaviors limit their effectiveness. In addition to the negative properties, side effects also play a vital role in impeding their clinical use. For example, ophiopogon, saponin, and ginsenosides cause hemolysis phenomena [368,369]. Some alkaloids produce neurotoxicity and hepatorenal toxicity [365]. Furthermore, high-frequency administration of some bioactive drugs is necessary to maintain their efficacies (such as triptolide and podophyllotoxin), which leads to low compliance and even cumulative toxicity in patients [370]. To overcome these limitations, the use of drug delivery systems has been used. In TCM, these approaches are used to guide the clinical application to expand from supporting the healthy energy and combating poison with poison to “supporting the vital qi in the disease area, and targeted combating poison with poison” during treatment development [371,372]. Nowadays, various types of drug carriers have been developed for TCM which target delivery to cancer cells or animal xenografts, including polymeric nanoparticles, iron oxide nanoparticles, liposomes, micelles, and dendrimers [373].

Liposomes have a vesicular structure composed of lipid bilayers and were discovered by Bangham et al. in 1965 [374]. Liposomal delivery is an ideal dosage form for formulations available in the market for clinical use of antitumor therapeutic compounds, such as Doxil^® (PEGylated liposomal doxorubicin; Centocor Ortho Biotech Inc., USA), DaunoXome^® (non-PEGylated liposomal daunorubicin; Diatos, France), and Myocet^® (non-PEGylated liposomal doxorubicin; Sopherion Therapeutics, USA) [375]. Liposomes can encapsulate both hydrophobic and hydrophilic agents within the lipid bilayer and inner water phase, respectively. They are characterized by high biocompatibility, elevated encapsulation efficiency, and easy modified for optimization of their properties, such as increased blood circulation time and receptor-mediated site-specific distribution [4,376,377]. Furthermore, liposomes can reduce the incidence of adverse reactions, by reducing local drug amount distributed in normal tissues [378]. Although growing numbers of investigations of TCM-loaded liposomes have proven to produce a strong anticancer response, there is still much room for improvement of anti-tumor liposomal TCM treatment via injection, and significant advances are continuously achieved.

Here we focus on targeting delivery of monoconstituents of CHMs to cancer cells and overcoming the barriers of the TME, including current strategies for achieving these goals by functionalized liposomal surfaces, taking advantages of physiological and biochemical differences between cancerous tissue and normal tissue (Figure 2). External stimuli-driven tumor targeting and drug release, such as magnetism, heating, and laser, fall outside the scope of this review.

Figure 2.

Overview of tumor-specific targeting strategies of liposomes for delivery of monoconstituents of CHMs (Abbreviation: EPR effect, enhanced permeability and retention effect; PEG, polyethylene glycol; TPGS, D-alpha tocopheryl polyethylene glycol 1000 succinate; DQA, dequalinium; Tf/Lf, transferrin/lactoferrin; FA, folic acid; HA, hyaluronic acid; EGCG, epigallocatechin 3-gallate; GA, glycyrrhetinic acid).

4.1. Long-circulating liposomes

Conventional liposomes are often composed of cholesterol and soy phosphatidylcholine in specific proportions and are not modified with other moieties [365]. These vesicles always show as significant interaction with the RES, mainly residing in the liver and the spleen, due to the opsonization as foreign particles, and are rapidly cleared from the blood circulation before accumulating in tumors [379]. A widely used strategy to achieve RES-avoidance is to graft the vesicle surface with polyethylene glycol (PEG), taking advantage of steric repulsion to create a long circulating “stealth” effect. PEG creates a hydrated outer shell, which also shields the nanoparticles from recognition and clearance by the RES. PEG grafting results in an extended drug half-life and improved tissue distribution [148,380,381]. The increased blood circulation time of small-sized liposomes (40–200 nm) achieved by PEG-lipid incorporation in bilayers enhances drug accumulation in tumors, where leaky vascular structure is found. This strategy maximizes the enhanced permeability and retention (EPR) effect. This strategy is called passive targeting, which improves the therapeutic effect of entrapped agents. Various other polymers have been reported to improve blood circulation time of liposome, but PEG remains the primary choice thus far [382–385].

PEG2000 (molecular weight of 2000) is the most frequently used PEG polymer to avoid RES uptake [386,387]. PEG is usually covalently attached to a lipid (PEG-lipid), such as distearoylphosphatidylethanolamine (DSPE), which can be incorporated into the lipid bilayers through hydrophobic interaction. Due to the amphiphilicity of the PEG-lipid, the surface area available for PEGylation is quite limited, with a maximum of 7±2 mol% to maintain membrane integrity [388]. Huang’s group formulated a membrane-core structured Lipid-Calcium-Phosphate (LCP) nanoparticle with a surface that can be modified with various amounts of PEGylation, up to 20 %. It was found that a high density of PEG is required to achieve the steric stabilization and thus create the “stealth” property for particles, according to the concentration and type of PEG-lipids [386]. The long-circulating liposomes with active components of CHMs overcome their disadvantages in physicochemical properties and their applications are shown in Table 7.

Table 7.

Long-circulating liposomes with active components of CHMs overcome their disadvantages in physicochemical properties and their application

Entrapped components	Disadvantages	Drug loading	Cancer model	Main results	Reference
β-Elemene	Phlebitis, fever, pain and induced bleeding are caused by solubilizing agents after intravenous injection	Ethanol injection method	In vivo Kunming mice	In vivo The size of PEGylated liposomes was 221.4 nm and the EE% was 92.7%. The AUC and half-life of PEGylated liposomes were both enhanced, being 1.5- and 6.0-fold larger than those of conventional β-elemene liposomes and free β-elemene, respectively.	[389]
Brucine	The therapeutic amount of brucine is close to the toxic amount	Ethanol injection and ammonium sulphate gradient methods	\	Different molecular weights of PEG were screened for their modification on the liposomes surface. The size of PEGylated liposomes was 120 nm with the EE% at 93.7%. The liposomes had a better sustained-released effect than free brucine and good stability in 4 °C.	[390]
Berberine	Hypotension and vasodilation, and a quick and wide distribution to major organs after solution injection	pH gradient-film dispersion method	In vivo Balb/c nude mice xenografted gastric tumor (SGC-7901 cells)	In vivo PEG-modified berberine liposomes significantly increased circulation retention compared to berberine solution and proved effective and safe in suppressing the 45.8 % tumor growth in nude mice.	[377,391,392]
Oxymatrine	Short half-life (133 min) in human body after intramuscular injection	Thin-film dispersion, reversed-phase evaporation, pH gradient and ammonium-sulfate gradient methods	\	Oxymatrine liposomes could be produced with EE% at 57.2% by ammonium-sulfate gradient method. The ammonium sulfate concentration, incubation time, and drug-to-lipid ratio greatly influenced the EE%, while the incubation temperature had almost no effect on EE%.	[393]
Oridonin	Low water solubility	Ethanol injection method followed by freeze-drying	In vivo SD rats	In vivo The levels of stealth liposomal oridonin in the blood were increased, with MRT about 2- and 6-fold longer than that of conventional liposomes and free oridonin solution, respectively.	[394]
Artemisinin	Low water solubility	Ethanol injection method	In vitro MCF-7 cells	In vitro The cytotoxicity effect of pegylated liposomal artemisinin was greater (IC50=1.58 μg/mL) than that of liposomal artemisinin.	[395]
Glycyrrhetinic acid (GA)	Low water solubility	Film-dispersion method	In vivo SD rats	GA-loaded PEGylated liposomes had a 2.75-fold larger AUC, a 1.7-fold longer MRT, and a 0.4-fold lower CL, compared to GA solution.	[396]
Oleanolic acid (OA) and Doxorubicin (DOX)	Low aqueous solubility and low permeability across the intestinal mucosa	Ethanol injection method	In vivo Balb/c nude mice xenografted liver tumor (HepG2 cells) In vitro) HepG2 cells	In vivo OA-loaded liposomes formulation inhibited tumor growth and OA had a protective effect to attenuate DOX toxicity.) In vivo) The IC50 value for codelivery of OA and DOX in liposomes is significantly lower than OA-loaded liposomes alone.	[397]
Honokiol	Poor stability and water solubility	Ethanol injection method followed by freeze-drying	In vivo BALB/c nude mice xenografted NSCLC tumor (HCC827 and H1975 cells)	In vivo Liposomal honokiol showed antitumor activities in four xenografted models, including inoculating HCC827 (gefitinib-sensitive) and H1975 (gefitinib-resistant) cells.	[398]
Plumbagin (PLB)	Severe side effects caused by solubilizing agents in injection	Film hydration method	In vivo C57BL/6J mice bearing melanoma (B16F1)	In vivo The long circulating liposomes exhibited a 36.4-fold increased plasma and significantly less CL in comparison to free PLB. The PEGylated liposomes significantly delayed tumor growth with median survival value at 27 days.	[399]
Quercetin (Que) and Adriamycin (ADM)	Low aqueous solubility, low bioavailability, and instability during processing and storage	Film-ultrasound technique with ammonium sulfate gradient method	In vivo BALB/c nude mice xenografted breast cancer (MCF-7/ADR) In vitro ADM-resistant cell strains (HL-6/ADR and MCF-7/ADR)	In vivo Liposomes enveloping QUE and ADM extended the half-life and increased the blood concentration of ADM, with the highest inhibition rate of tumor growth. In vitro The combination of ADM and QUE can enhance cell toxicity to ADM-resistant cells, achieving 4.81- and 3.21-fold higher in HL-6/ADR and MCF-7/ADR, respectively.	[400]
Crocin	Water solubility, and instability during processing and storage	Film-ultrasound technique with extrusion	In vivo BALB/c mice xenografted colorectal cancer (CT26 cells) In vitro CT26 cells	In vivo Liposomal crocin (50 and 100 mg/kg) significantly prolonged survival time compared with crocin and free doxorubicin, in a dose-dependent manner. In vitro PEGylated liposomes with higher cholesterol retained crocin more effectively and thus showed higher IC50 values in C26 cells compared to crocin alone.	[401]
Silibinin and Glycyrrhizic acid	Poor solubility in water and oil and low bioavailability	Film hydration method with HEPES buffer and sonication	In vitro HepG2 and fibroblast cell lines	In vitro The IC50 for pegylated liposomal silibinin and glycyrrhizic acid was 10-times and 2.3-times lower than that of free silibinin and glycyrrhizic acid, in the HepG2 and fibroblast cells, respectively.	[402]
Resveratrol (Res) and DOX	Low water solubility	Film hydration method	In vitro NT8e cells	In vitro The formulation exhibited a superior effect on the apoptosis proteins and cell cycle but had a higher IC50 value than that of free DOX.	[403]
Celastrol	Highly hydrophobic nature	Film hydration method and extrusion	In vitro Vertebral-cancer of the prostate (VCaP) cells	In vitro The lipid composition influenced the EE%, serum stability and drug release of celastrol from PEGylated liposomes.	[404]
Ursolic acid	Low water solubility	Film hydration method and homogenization	In vivo Nude mice xenografted breast tumor (MCF-7 cells)	In vivo Magnetic resonance parameters of tumor-bearing animals showed a possible antiangiogenic effect induced by ursolic acid-loaded liposomes	[405]
Caffeic acid, Carvacrol, pterostilbene, Caffeic acid phenethyl ester (CAPE), Carvacrol, N-(3-oxo-dodecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), Thymol, and Resveratrol	Poor water solubility or instability	Film hydration method	In vivo Balb/c nude mice xenografted head and neck squamous-cell carcinoma (14C-tumor cells)	In vivo These compounds were entrapped in liposomes with better stability. Liposomal 3-oxo-C12-HSL and resveratrol inhibited tumor growth significantly as compared to control group.	[406]
Betulinic acid (BA)	Poor water solubility	Ethanol injection technique	In vivo Kunming mice xenografted cervical cancer (U14 cells) In vitro HepG2 cells and HeLa cells	In vivo The PEGylated BA-loaded liposomes with sustained release profile can effectively accumulate in tumor tissues. The tumor inhibitory rate of PEGylated BA was 1.2-fold stronger than that of BA liposomes.	[407]

Abbreviations: AUC, the area under the plasma concentration-time curve; MRT, mean residence time; NSCLC, non-small cell lung cancer; CL, clearance; IC₅₀, half inhibit concentration

4.2. Active-targeting liposomes

Active-targeting liposomes exert their targeting capabilities through a series of affinity-based interactions (such as that between ligands and receptors) on the diseased cells and release drugs specifically into the target sites, with minimum deposition at healthy tissues or with less off-target effects [365,408,409]. Active- and passive-targeting liposomes are transported to tumor sites in the same manner. Only after arriving in the tumor tissue are ligands able to work, and it was found that some PEGylated liposomes were unable to release drugs at the tumor site. Moreover, with an increasing number of injections administered, PEGylated materials are prone to be cleared from the blood, which is called the accelerated blood clearance (ABC) phenomenon [410–412]. This means that long blood circulation times and efficient tumor target binding are both required for appreciable tumor accumulation of ligand-targeted liposomes [385]. Active targeting ligands could be attached directly to the liposomal surface, together with PEG-lipids, or conjugated to the distal end of PEG chains and incorporated into liposome membrane as ligand-PEG-lipids [413–416].

There are two issue to consider when taking advantage of a specific ligand to functionalize liposomes. First, it is important to optimize the ligand density on the liposome surface by relevant surface engineering techniques to properly form a targeted liposome system. High ligand density in a feasible range could promote target binding; however, issues such as aggregation may be caused by increasing ligand density over the optimal density [417]. Second, as the binding affinity for a tumor-related ligand rises, liposomes would bind tightly to the cancer cells they first encounter near blood vessels. This might block the tumor penetration of vesicles into cancer regions far away. Then subsequent vesicles are unable to reach free receptors and are thus forced back out of the tumor. It thus diminishes effective targeting [418,419]. In other words, exceedingly strong affinity ligands may impair tumor penetration and fail to increase tumor drug accumulation. Even if with increased tumor accumulation, anti-tumor efficacy is not necessarily improved if drug accumulation only takes places near blood vessels [385]. Various liposomal active-targeting ligands for active components of CHMs for solid tumor therapy are shown in Table 8.

Table 8.

Various liposomal active targeting ligands for active components of CHMs targeted to cancer cells and the TME

Targeting ligands	Targeting site	Entrapped components	Drug loading	Cancer model	Main results	Reference
Over-expressed receptors on the surface of cancer cells
β-Galactose	Asialoglycoprotein receptors	Glycyrrhetinic acid (GA)	Film dispersion method	In vivo Kunming mice In vitro HepG2 cells	In vivo Galactosylated GA-liposomes (Gal-GA-LP) showed long-circulating profile in Kunming mice with the MRT of Gal-GA-LP being 1.48- and 1.3-fold increasement when compared with GA solution and nontargeting GA-loaded liposomes, respectively. In vitro Gal-GA-LP showed 1.4-fold higher drug concentration in cells than that of GA-liposomes.	[420]
		Oridonin (ORI)	Ethanol injection method	In vivo Kunming mice	In vivo The MRT of ORI-loaded liposomes modified with galactose (NOH-ORI-LP) was 5.56-fold longer than that of ORI solution in mice, with lower clearance from liver.	[421]
Lactose		Matrine (MA)	Reversed-phase evaporation method	In vivo Kunming mice In vitro HepG2 cells	In vivo The accumulation of MA in targeting liposomes in the mice liver was 2.7 times higher than in the spleen, 3 times higher than in the lung, 6.6 times higher than in the kidney, and 8.5 times higher than in the heart. In vitro Inhibitory rate of targeting liposomes of MA was 1.6- and 1.83-fold higher than those of conventional MA liposomes and MA solution at 0.5 mg/mL concentration, respectively.	[422]
Transferrin (Tf)	Transferrin receptors	Tetrandrine and Vincristine	Film dispersion method	In vivo Glioma-bearing ICR mice model In vitro C6 cells, C6/ADR cells and murine brain microvascular endothelial cells (BMVECs)	In vivo Tf targeting liposomes improved accumulation in brain tumor tissue with fluorescent signal maintained up to 48 h and animals exhibited prolonged survival time (42.67 ± 3.56 days) compared with the saline group. In vitro The targeting liposomes showed significantly prolonged circulation time and increased accumulation in tumors.	[423]
Lactoferrin (Lf)		Honokiol and Daunorubicin	Film dispersion plus ammonium sulfate gradient methods	In vivo Glioma-bearing ICR mice model In vitro C6 cells and BMVECs	In vivo The targeting liposomes improved accumulation in brain tumor tissue indicated by fluorescence probe and conferred prolonged survival time. In vitro Lf modified daunorubicin plus honokiol liposomes enhanced drug transportation across the blood-brain barrier, inhibited C6 cells invasion, and destroyed vasculogenic mimicry channels.	[424]
Folic acid	Folate receptors (FR)	Curcumin (Cur)	Film dispersion method	In vitro KB, Hela, and A549 cells	In vitro FR-positive cells endocytosed more FR-targeting liposomes than nontargeted liposomal Cur. Targeted liposomes more effectively inhibited cellular proliferation and caused higher dose- and time-dependent apoptosis. KB cells were more sensitive to FR-targeting liposomal Cur than Hela cells.	[425]
		Baicalin (Bai)	Film hydration and homogenization	In vitro HeLa cells	In vitro Cytotoxicity and cellular uptake of Bai-loaded FR-targeting liposomes were higher than that of non-targeted liposomes.	[426]
		Ursolic acid (UA)	Film hydration and extrusion	In vivo Balb/c nude mice xenografted oral cancer (KB cells) In vitro KB cells	In vivo Compared with passive targeting liposomes, FA-PEG modified UA liposomes significantly inhibit oral tumor growth. In vitro The EE% of UA was more than 85%. FR targeting UA PEGylated liposomes showed improved tumor cell uptake, proliferation inhibition, and apoptotic effects as compared with nontargeting PEGylated liposomes.	[427]
Glycyrrhetinic acid (GA)	GA receptors	Ginsenoside Rh2	Film hydration method	In vitro SMMC-7721 cells	In vitro IC50 of GA-modified liposomes in SMMC-7721 cells increased by 0.5- and 0.6-fold compared with Rh2 solution and Rh2-loaded liposomes, respectively.	[428]
		Baicalin	Film hydration and ethanol injection methods	\	The size of GA modified baicalin liposomes was 150 nm with EE% at 41.8%.	[429]
		Brucine	Ethanol injection method	\	The formulation of GA modified brucine liposomes was optimized with a lipid to cholesterol (w/w) ratio of 6, lipid to drug (w/w) ratio of 15, and lipid to GA (w/w) ratio of 35 (g/g). The size of targeted liposomes was 147.2 nm and the EE% of brucine was 82.8%.	[430]
		Wogonin	Reversed-phase evaporation method	In vivo ICR mice xenografted liver cancer (HepG2 cells) In vitro HepG2, L-02 and LX-2 cells	In vivo GA modified liposomes rapidly and highly accumulated in the tumor and liver shortly after administration. The tumor inhibitory ratio of targeted liposomes was 1.7-fold higher than that of nontargeted liposomes. In vitro GA-modified liposomes showed 1.46-fold higher growth inhibition than that of non-targeted liposomes in HepG2 cells.	[428]
		Cur	Ethanol injection method	In vitro HepG-2 and H22 cells	In vivo GA modified liposomes loaded with Cur inhibited tumor growth, reduced tumor microvascular density and regulated the expression of Caspases3 and VEGF proteins in H22 tumor tissues. In vitro GA modified liposomes induced more HepG2 cellular apoptosis and tumor growth inhibition.	[431]
Epigallocatechin 3-gallate (EGCG)	67LR	Doxarubicin	Film hydration method	In vivo C57BL/6 mice melanoma (B16F10 cells)	In vivo Targeting liposomes showed superior anti-tumor effect without observed side effects.	[432]
Targeting cytoplasmic organelles
Mitochondrion	Dequalinium (DQA)	Resveratrol	Film dispersion and extrusion	In vivo Balb/c nude mice xenografted resistant lung cancer (A549/cDDP cells) In vitro A549 cells and A549/cDDP cells	In vivo The combination administration of targeting resveratrol liposomes with vinorelbine liposomes exhibited superior prohibition of tumor growth. In vitro The increased mitochondrial uptake and solubility of resveratrol in targeting liposomes promoted inhibitory cell activity by triggering cytochrome C release.	[433]
		Berberine	Film dispersion and extrusion	In vivo Balb/c nude mice xenografted breast cancer (MCF-7 dissociated cancer stem cells) In vitro MCF-7 cells	In vivo The synergistic effect of targeting berberine liposomes and paclitaxel liposomes exhibited the strongest anti-tumor effect. In vitro Targeting berberine liposomes (1, 5, and 10 μM) could synergistically enhance the toxicity of paclitaxel liposomes in a dose-dependent manner, while achieving selective mitochondrial accumulation.	[434]
	4-Carboxybutyl triphenylphosphonium bromide (TPP) or DQA	Resveratrol	Film dispersion and extrusion	In vitro B16F10 cells	In vitro TPP and DQA conjugated with DSPE-PEG liposomes carrying resveratrol caused better cellular uptake and selective accumulation in mitochondria.	[435]
Targeting the TME
Hyaluronic acid (HA)	CD44 and the receptor for hyaluronan-mediated motility	Curcumin (Cur)	Reversed-phase evaporation method	In vitro HepG2 and A549 cells	In vitro The binding rate of HA-lipid to Cur-liposomes was 72%. The IC50 values were found to be 0.054, 0.032 and 0.021 mol/ml for free Cur, nontargeted Cur liposomes and modified Cur liposomes, respectively, after incubation with A549 cells overexpressing the CD44 receptor.	[436]
		Matrine	Film dispersion, reversed-phase evaporation and ethanol injection methods	In vivo Balb/c mice xenografted liver cancer (H22 cells)	In vivo The inhibition rate of HA-modified MA liposomes on H22 hepatocarcinoma was similar to that of cyclophosphamide, and 2.0- and 1.4- fold higher than that of MA solution and MA conventional liposomes, respectively.	[437]
Surface modification with antibodies
Anti-CD44 antibody	CD44 receptors	Timosaponin AIII (TAIII)	Film evaporation and ultrasonic methods	In vivo Balb/c nude mice xenografted liver cancer (HepG2 cells) In vitro HepG2 cells	In vivo CD44 targeting liposomes showed significantly inhibition on tumor growth, which was 7.2- and 1.3- fold greater than that of free drug and non-targeted liposomes, respectively. In vitro Targeting liposomes were more toxic to tumor cells with lower IC50 than non-targeting liposomes.	[438]
Trastuzumab	Human epidermal growth factor receptor-2 (HER2)	Curcumin and Resveratrol	Film evaporation and extrusion method	In vitro JIMT1 an MCF-7 cells	In vitro The immunoliposomes of curcumin and resveratrol showed notably higher cytotoxicity, compared to free drugs. The finding was more prominent for curcumin than resveratrol.	[439]
Surface modification with peptides
T7 (HAIYPRH) peptide	Transferrin receptors	Quercetin	Film hydration method	In vivo Balb/c nude mice with orthotopic lung cancer (A549-Luc cells) In vitro A549-Luc cells	In vivo T7 modified liposomes significantly delayed tumor growth and enhanced the lifespan of mice following pulmonary administration. In vitro T7 modified liposomes showed higher cellular uptake and significantly higher tumor-spheroid growth inhibition.	[440]
NGR (Asn-Gly-Arg)	CD13	Triptolide	Film hydration method	In vitro Human umbilical vein endothelial cells (HUVEC)	In vitro Triptolide-loaded liposomes modified with NGR showed stronger cellular toxicity with lowest IC50 on HUVECs at 11 μM.	[441]
		Brucine	Ammonium sulfate gradient method	In vivo Balb/c nude mice xenografted fibrosarcoma (HT1080 cells) In vitro HT1080 cells and HUVEC	In vivo The highest fluorescence signals in tumors were found in animals treated with NGR-modified liposomes with HSPC and SPC together than that with HSPC or SPC used alone. In vitro The composition of lipids did not exhibit significant influence on EE% of brucine. The cellular uptake of NGR-modified liposomes was about 1.5-fold higher than that of non-targeted liposomes.	[442]
RGD (Arg-Gly-Asp)	Integrins	Tetrandrine and Vinorelbine	Film dispersion followed by ammonium sulfate gradient method	In vivo Glioma-bearing ICR mice model (Doxorubicin-resistant C6 cells) In vitro C6 cells and BMVECs	In vivo RGD modified vinorelbine and tetrandrine liposomes prolonged elimination half-life compared with free drug, and extended survival times. Strong fluorescent signals of targeting liposomes were observed in tumors within 24 h after administration, while the signals of free drugs was in the liver. In vitro Targeted liposomes significantly enhanced transportation across the BBB and uptake in tumor cells.	[443]
		Matrine	Film dispersion followed by pH-gradient method	In vitro Bcap-37, HT-29 and A375 cells	In vitro RGD-PEG-DSPE was employed to obtain matrine liposomes with an EE% of 83.13%. Compared with free matrine, targeting liposomes induced stronger inhibition of proliferation and induced more apoptosis in tumor cells.	[444]
		Shikonin (SHK)	Film hydration method	In vitro MDA-MB-231 cells	In vitro RGD modified SHK-loaded liposomes with EE% of 94.9% greatly induced cellular apoptosis by increasing the ratio of Bcl-2-associated X protein (Bax)/Bcl-2, and inhibited cellular proliferation, migration and invasion, as compared with nontargeted SHK-loaded liposomes.	[445]
Octaarginine (R8)	Syndecan 4	Dihydroartemisinin and Epirubicin	Film dispersion method	In vitro A549 cells	In vivo R8 modified liposomes increased the selective drug accumulation at tumor sites and showed a targeting capable of reducing tumor volume. In vitro R8 modified liposomes exhibited powerful cytotoxicity on A549 cells, effectively suppressed vasculogenic mimicry channels, and regulated metastasis related proteins.	[446]
R8GD (RGD and R8)	Integrins	Emodin	Film dispersion method	In vivo Balb/c nude mice xenografted breast cancer (MDA-MB-435S cells) In vitro MDA-MB-435S cells	In vivo Strong fluorescence signals of R8GD modified liposomes were found in tumors within 24 h after administration. R8GD modified daunorubicin liposomes significantly inhibited tumor growth with the combination of R8GD modified emodin liposomes. Their efficacy was 3-fold stronger than that of nontargeting liposomes combination. In vitro The cellular uptake was increased for targeting liposomes.	[447]
HIV protein transactivator of transcription (TAT) together with HA	Syndecan 4	Curcumin and Celecoxib	Film hydration method	In vivo Balb/c mice with orthotopic breast cancer (4T1 cells) In vitro RAW264.7, HUVEC and 4T1 cells	In vivo NF-κB essential modulator-binding domain (NBD) fused with TAT peptide modified liposomes co-delivering curcumin and celecoxib were coated with HA (HA/TN-CCLP). HA/TN-CCLP could prolong circulation time, increase tumor accumulation, and block lung metastasis. In vitro HA/TN-CCLP improved cytotoxicity, inhibited migration, and enhanced anti-inflammation effect, when compared to nontargeted liposomes.	[448]
tLyp-1 (sequence CGNKRTR)	Neuropilin-1	Ginsenoside CK and Parthenolide	Film hydration method	In vivo Balb/c nude mice xenografted lung cancer (A549 cells) In vitro A549 cells	In vivo Targeting liposomes with hydrophilic PEG shell demonstrated stronger antitumor effect than free drugs combination without observed toxicity. In vitro The cellular uptake of targeted liposomes was through micropinocytosis and the liposomes were escaped from lysosome in a time-dependent manner.	[449]
Surface modification with polymers
D-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS)	\	Luteolin	Film-dispersion method	In vivo Balb/c nude mice xenografted lung cancer (A549 cells) In vitro A549 cells	In vivo TPGS coated liposomes loaded with luteolin were significantly accumulated in tumor sites. The modified liposomes exhibited higher tumor inhibition rate to 51.7% than that of free luteolin. In vitro TPGS coated liposomes improved cytotoxicity in A549 cells by increasing the Bax/Bcl-2 ratio.	[450]
	\	Ginsenoside compound K	Film hydration method	In vivo Balb/c nude mice xenografted lung cancer (A549 cells) In vitro A549 cells	In vivo Targeted liposomes delivered ginsenoside compound K into tumor sites, enhanced its permeability and retained it in tumor cells. In vitro The EE% of targeted liposomes was 98.4%, and the size was (119.3±1.4) nm at the ratio of 7:3 (phospholipid: TPGS). The IC50 of targeted liposomes was significantly reduced to 25% of that of free drugs.	[451]

4.2.1. Targeting specific receptors over-expressed on the surface of cancer cells

4.2.1.1. Glycolipids

Liposomes with different glycosyl combining on their surfaces can be differentially distributed in vivo. Liposomes carrying mannose residues are enriched in sinusoids, involving Kupffer cells, endothelial cells, and stellate cells in liver. Galactosylated liposome-entrapped materials were largely distributed to hepatocytes [376,452]. To exhibit significant therapeutic efficacies, various glycolipids with low molecular weight were used to modify the liposomal surface by conjugating the sugar moieties to lipids. Galactosylated liposomes are bound specifically to asialoglycoprotein receptors (ASGPRs) to achieve hepatocyte-specific drug delivery after entering systemic circulation because (1) ASGPRs are specifically over-expressed in hepatocytes; (2) because of the rich blood flow and discontinuous endothelium of the liver, particles can easily access hepatocytes [452,453]. However, interactions between glycolipids and plasma lipoproteins or tissue lipids after intravenous injection may interfere with the integrity of liposomes by removing glycolipids from bilayers, and thus lead to reduced tumor cell selectivity [454]. Thus, cholesterol was used to introduce the galactosyl moiety to liposome surface as the hydrophobic anchor. Cholesten-5-yloxy-N-(4-((1-imino-2-L-D-thiogalactosylethyl)amino)butyl)formamide (Gal-C4-Chol), cholesten-5-yloxy-N-(4-((1-imino-2-L-D-thiomannosylethyl) amino)butyl) formamide (Man-C4-Chol), and cholesten-5-yloxy-N-(4-((1-imino-2-L-L-thiofucosylethyl)amino)-butyl)formamide (Fuc-C4-Chol) are synthesized as different efficient glycosylating agents with cell-specific hepatic targeting potential [455]. Furthermore, when coating the galactosylated liposomes with PEG to obtain the long-circulating effect, the molecular weight of PEG is important because longer PEG chains might retard asialoglycoprotein receptor-mediated uptake of galactosylated liposomes by steric hindrance [456].

4.2.1.2. Transferrin family

The blood-brain barrier (BBB) protects the brain as a highly selective barrier. Nearly all large-molecules and 98% of small-molecule drugs are unable to be transported across the barrier in the active transport manner [423,457]. Transferrin (Tf), an iron transporter, regulates free iron levels in biological fluids. Transferrin receptors (TfR1 and TfR2 or CD77) are lowly expressed in most normal cells, and overexpressed in tumor cells (~100 fold) due to increased iron demand of cancer cells, such as liver cancer cells, brain glioma cells, or endothelial cells of the BBB, thus Tf could target the transferrin receptor and exhibit tumor-specific cell binding [458]. In addition to Tf, lactoferrin (Lf) also belongs to the transferrin family. Drug carriers with Lf modifications cross the BBB via receptor-mediated endocytosis [424]. In fact, Lf is also reported to inhibit the cell cycle in G0/G1 and G2 phases of U87MG cells through downregulation of cyclin D1 and D4 [459].

4.2.1.3. Folic acid

Folic acid (FA, folate or vitamin B9) is a crucial vitamin for nucleotide synthesis in all living cells [385]. Living cells take in FA through folate receptors (FRs)-mediated endocytosis by stimulating the clathrin-independent pathway, which is a characteristic used for macromolecular drug delivery. FRs are highly expressed on lung, ovarian, kidney, breast, brain, endometrial, and colon cancer cells. Limited expression of FRs on most normal tissues, but overexpression on cancer cells are observed. Further, FR density increases with the deterioration of tumor grade or stage [460]. As such, it is suggested that FA can be used as a potential targeting molecule for active drug delivery to tumor cells [425,461]. FA was also reported to be conjugated with chitosan by an acylation reaction to obtain folic acid-chitosan, which could form a coating liposomal system by electrostatic interactions on the surface of the negatively charged nano-liposomes with better stability and sustained release of curcumin [462].

4.2.1.4. Glycyrrhetinic acid (GA)

GA, a pentacyclic triterpeneglycoside, is the hydrolysis product of glycyrrhizin. In addition to its effective anticancer effects against hepatocellular carcinoma, GA was also reported to specifically target hepatocytes where the GA receptors (GA-R) are abundantly expressed on hepatocyte membranes [463,464]. Both GA-R and glycyrrhizin receptor (GL-R) could achieve active hepatic targeting of drug delivery. However, GA-R is more effective because GA binding sites outnumber GL binding sites [465]. Recently, growing interest has developed on carriers modified with GA as hepatocyte-targeted delivery systems [428–431].

4.2.1.5. Epigallocatechin 3-gallate (EGCG)

EGCG, a polyphenol from green tea, has been reported to reduce the expression of MMP-2 and MMP-9, involving in tumor growth, angiogenesis, and metastasis. Moreover, it selectively binds the 67-kDa laminin receptor (67LR) [466], which is more highly expressed on tumor cells, such as breast, bile duct, colorectal cancers, and melanoma cells, than on normal cells [467]. Therefore, EGCG-PEG-modified liposomes loaded with doxorubicin took advantage of not only the apoptosis inducing effect of EGCG, but also the tumor-targeting of EGCG together with the PEG shielding to synergistically combine with the topisomerase II inhibition induced by doxorubicin. The dual-effect liposomes showed outstanding antitumor efficacy on melanoma [432].

4.2.1.6. Anisamide derivatives

Anisamide and its derivatives modified liposomes were proven to be highly selectively targeting to sigma receptors, leading to stronger tumor growth retardation, compared to non-targeted liposomes [468]. Sigma receptors, subdivided into sigma-1 and sigma-2 receptors, are over-expressed membrane proteins on TAFs and tumor cells, such as melanoma, breast tumors, non-small cell lung carcinoma, hepatocellular carcinoma and prostate cancer, and their expression is much higher compared to normal cells [469,470]. Aminoethyl anisamide (AEAA) is a targeting ligand for sigma receptor with high affinity (K_d=9 nM) employed for phytochemicals delivery in other lipidic formulations (emulsions and micelles). Puerarin in DSPE-PEG-AEAA modified nanoemulsions were shown to significantly facilitate chemotherapeutic effect of paclitaxel and activate immune microenvironment, compared to the control group [330]. AEAA targeting nanocarriers loaded with celastrol and mitoxantrone were also demonstrated the increased drug accumulation in tumors, compared to free drugs [471]. Icaritin was co-delivered with doxorubicin in micelles with the composition of poly lactic-co-glycolic acid (PLGA)-PEG-AEAA (PLGA-PEG-AEAA). The AEAA modified fluorescence micelles accumulated 4-fold higher than non-targeted micelles in tumor sites, with much less amount in normal livers [472].

4.2.2. Targeting specific receptors on desired organelles

Mitochondria are membrane-enclosed organelles described as “cellular power plants”. They are an ideal antitumor target due to their involvement in at least six cancer hallmarks, including deregulated cellular energetics, cell apoptosis resistance, expanded invasion and metastasis, immortal replication, genomic instability, and tumor-promoting inflammation [473]. Moreover, tumor cell mitochondria are distinct from their normal counterparts in structures and functions, showing increased reactive oxygen species production, higher mitochondrial membrane potential, and decreased oxidative phosphorylation [474–476]. Given these features, tumor cells require high energy levels for proliferation and are more susceptible to mitochondrial perturbation than healthy cells [433,477]. Consequently, mitochondria-targeting therapies emerge as an attractive means to selectively eliminate cancers [478]. TPP, rhodamine B, DQA, and mitochondrial targeting signal peptides (MTSs) were used to pack or modify onto lipid carriers (mitochondriotropic molecules) to target mitochondria [477,479,480]. Among these molecules, delocalized cationic 4-carboxybutyl triphenylphosphonium bromide facilitates mitochondrial uptake by interacting with the highly negatively charged mitochondrial membrane in cancer cells, leading to its improved accumulation or access to the mitochondria [481]. DQA, a cationic amphiphile, promotes specific mitochondrial accumulation driven by the transmembrane electrical potential [434,482].

4.2.3. Targeting specific receptors on cells in the TME

Tumors consist not only of neoplastic cells, but also of stromal cells (such as tumor-associated fibroblasts, mesenchymal cells, pericytes, occasionally adipocytes, blood and lymphatic vasculature) and immune cells that maintain the TME. The ECM and secreted extracellular molecules also bear tumor development [483–485].

Among ECM components, hyaluronic acid (HA) is the main one characterizing fibrotic process. HA is a natural acidic polysaccharide synthesized by three transmembrane HA synthases (HAS1, HAS2, and HAS3) that is non-toxic, biodegradable, and possesses a strong affinity for CD44 and the receptor for hyaluronan-mediated motility (RHAMM) [486,487]. CD44 is a non-kinase transmembrane cell-surface glycoprotein with endogenous limited expression in healthy cells and highly expressed as one of the most common cancer stem cell surface markers in various cancer types, including breast, prostate, pancreas, colon, and hepatocellular cancer. Therefore, CD44 is a potential receptor for targeting drug delivery systems, which is involved in tumor invasion and metastasis [488–491]. HA can be covalently linked to the polar headgroup of the lipid, or coats cationic liposomes via electrostatic interaction, in order to achieve CD44-receptor targeting drug delivery. A negatively charged HA-based prodrug can also be formed to insert into the lipid bilayers [492].

4.3. Others

4.3.1. Liposomal surface modifications with antibodies

Monoclonal or polyclonal antibodies conjugated on the surface of liposomes that recognize and bind to specific antigens on the surface of targeted tumor cells are called immunoliposomes. They are able to distinguish the target cell and enhance the selective targeting effects of liposomes [376].

Various tumor cell surface markers were reported as potential targets for selective targeting of therapeutic agents, such as CD147, CD133 and CD44 [438]. Antibody targeted therapies are effective for the following main reasons: (1) The over-expressed epitope is found on the cancer cell; (2) The antibody could get better access to the tumor sites than to healthy sites, due to the longer circulation time and EPR effect. Anti-CD44 antibodies modified liposomes were characterized with enhanced cellular uptake through CD44 receptor-mediated endocytosis, and increased selective targeting effect [493,494].

Human epidermal growth factor receptor-2 (HER2) is a member of epidermal growth factor receptor family. It is a transmembrane receptor tyrosine kinase, generally over-expressed in 25–30% of ovarian and breast cancer patients and normally expressed at low levels in adult cells. It is commonly targeted for breast cancer therapy. Trastuzumab (Herceptin^®, Genentech, Inc., USA) was the first humanized monoclonal antibody that binds the extracellular domain of the HER2 receptor for clinical breast cancer therapy [495,496]. Trastuzumab coupled HER2-targeted immunoliposomes loaded with curcumin and resveratrol showed notably higher cytotoxicity in HER2 positive human breast cancer cells than that of non-targeting liposomes or free drugs [439].

4.3.2. Liposomal surface modifications with peptides

Peptides (a short amino acid chain) for surface functionalization are non-covalently or covalently bonded to liposome surface through the MAL linkage bond, sulfanyl bond, peptide bond, or disulfide bond [382]. They are divided into cell-targeting peptides and cell-penetrating peptides, having receptor-specific and non-specific binding and internalization, respectively.

4.3.2.1. Cell-targeting peptides (CTPs)

T7 (HAIYPRH) peptide specifically targets a small cavity on the transferrin receptor surface without interfering transferrin binding to transferrin receptor and is then transported inside the cell through endocytosis [440,497].

RGD (Arg-Gly-Asp) or NGR (Asn-Gly-Arg) are ligands for targeting angiogenic blood vessels, which have several proteins that are absent or expressed at much lower levels in established blood vessels. Targeting the tumor vasculature has some advantages, as compared to targets on the cancer cell surface. First, liposomes do not have to penetrate the tumor tissue to find and reach antigens on tumor cell surface, while they have better access to antigens on the tumor vasculature. Second, abnormal rapid proliferation of cancer cells required sufficient nutrients and oxygen for further growth. Therefore, killing of a relatively small number of vascular endothelial cells can destroy a large number of cancer cells supported by these blood vessels. Third, as compared to cancer cells, vascular endothelial cells are more genetically stable and less susceptible to develop drug resistance [385].

RGD (Arg-Gly-Asp) selectively binds to cell surface integrin α_vβ₃/α_vβ₅. Integrins are transmembrane glycoprotein receptors that play an essential role in the attachment between a cell and its extracellular matrix [498]. They are overexpressed in cancers (glioma cells, melanoma cells, lung cancer cells, breast cancer cells, and so on) and can enhance disease progression partially through angiogenesis and metastasis, making them worthy molecules for targeted drug delivery into tumor tissues [499,500]. Liposomes modified with RGD have clear benefits as anticancer drug delivery systems over non-targeted liposomes [501]. The ability of NGR peptides to target the tumor vasculature and improve therapeutic outcomes has also been demonstrated. NGR can specifically bind to CD13, which is a transmembrane metalloproteinase, involving in cell migration and angiogenesis. CD13 is not activated in normal vascular endothelial cells but is highly expressed in tumor vasculature and some tumor cells [441,502]. Therefore, the NGR peptide is a potential ligand to target the tumor vascular antigen CD13 with good selectivity and low immunogenicity [503]. The effect of phosphatidylcholine (PC) composition with different T_m used to prepare targeted liposomes has significant effects on the desirable properties of resulting liposomes. PCs with different T_ms, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC, T_m=41 °C), hydrogenated soy phosphatidylcholine (HSPC, T_m=53 °C), and soy phosphatidylcholine (SPC, T_m blow 0 °C) were investigated and found that the decrease of T_m of lipids facilitated drug release, and more fluidic liposomes exhibited improved cellular uptake [442].

4.3.2.2. Cell-penetrating peptides (CPPs)

CPPs are positively charged short peptides (5–30 amino acids) capable of penetrating cells across the cell membrane through endocytosis [373]. They allow a wide array of biomolecules, from peptides and proteins to nucleic acids, to rapidly and efficiently traverse cytoplasmic membranes.

For polyarginines, a popular cell penetration tool, the optimum chain length is eight arginine units (R₈) [446]. The positively charged R₈ peptide makes the coated particles positively charged for internalization and mediates efficient cellular uptake micropinocytosis, improving intracellular gene transportation and expression [504]. R₈ with positive charges was also combined with RGD in modified emodin as a tandem peptide (R₈GD) to treat breast cancer. The targeting mechanism of R₈GD includes receptor-mediated drug targeting and electrostatic incorporation [505]. The R₈GD-modified emodin liposomes with small particle size and high EE% exhibited a distinct antitumor effect together with R₈GD modified daunorubicin-loaded liposomes [447].

HIV protein transactivator of transcription (TAT, YGRKKRRQRRR, residues 47–57 of HIV-1 Tat protein) readily passes through the plasma membrane of uninfected mammalian cells [448]. Full-length TAT could bind the extensively expressed cellular heparan sulfate proteoglycans (HSPGs), integrins and chemokine (C-X-C motif) receptor 4 (CXCR4). HSPG syndecan 4, a potential receptor, enhances the uptake of TAT through energy-dependent micropinocytosis. Recently, TAT was reported to form transient pores and translocate across the membrane by diffusing on these pores, without observed cell death due to leakage [506,507]. After delivery to the cells, endosomal entrapment leads to most of CPP cargos eventually undergo degradation in lysosome or distribute back to cell membrane for recycling and then ejection from the cell, which is called “endosomal escape problem”. It could be solved to some extent by using endosomolytic agents, reversible covalent binding and high-affinity non-covalent binding [508].

The tumor-homing peptide tLyp-1 contains the sequence motif (R/K)XX(R/K). It mediates tissue penetration and tumor targeting through neuropilin-1 dependent C-end rule internalization and overcome the barriers caused by dysfunctional tumor vasculature and high interstitial pressure. The liposomes coated with tLyp-1 initially target the receptor specifically highly expressed on the tumor cell surface, internalize through micropinocytosis and then escape from lysosome in a time-dependent manner [449,509,510].

4.3.3. Surface modifications with polymers

D-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS) is an amphiphilic PEG derivative of vitamin E succinate, with hydrophilic-liphophilic balance value between 15 and 19. Therefore, it is an ideal emulsifier and solubilizer for hydrophobic drugs (67 times higher than polyvinyl alcohol) and greatly enhanced cellular uptake, in vitro cancer cell cytotoxicity [450,511], and prolonged long-circulation pharmacokinetic behaviors [148]. Moreover, TPGS also inhibited multidrug resistance by inhibiting the P-glycoprotein pump [451].

5. Conclusions

This article studied the antitumor effects of active ingredients of CHMs, counteracting toxin with toxin, heat-clearing and detoxifying, promoting blood circulation and removing blood stasis, resolving phlegm and eliminating dampness, and strengthening healthy energy and consolidating body resistance. We find that the active components function to inhibit proliferation, inhibit invasion and migration, induce apoptosis, reverse multidrug resistance, block angiogenesis, suppress aerobic glycolysis and improve immunity. Despite treatment potential, the undesirable physicochemical properties (poor permeability, instability, highly hydrophilicity or hydrophobicity, and toxicity) and unwanted pharmacokinetic profiles (short half-life in blood and low bioavailability) limit the clinical studies of CHMs. Various targeting liposomes have shown immense advantages in the delivery of active components of CHMs, involving improved physicochemical characteristics and pharmacokinetic profiles, enhanced therapeutic efficacies and reduced side effects. This review could be regarded as a useful and informative reference for these CHM components.

6. Limitations and future perspectives

TCMs are the oldest alternative and complementary medicines and there are rising interests on the investigation of CHMs. Using modern technology, the mechanism of some active components of CHMs were determined to some degree at the cellular, molecular, and pharmacological level. However, there are still some issues to consider.

First, there are only a few studies on the effect of single or combined use of monoconstituents on immunology regulation. The clinical application of CHMs are very complex as the act not only through a single factor, but also through multiple targets and based on a holistic approach, pointing to the entire human body. Current research is primarily focused on individual monoconstituent or the combination of active ingredients from CHMs with chemotherapeutic drugs and their bioactions on tumor cells and non-immune tumor microenvironment. The anti-tumor characteristics of plant-derived medicines, including inducing apoptosis, reversing multidrug resistance, regulating angiogenesis, killing TAFs, and inhibiting cell metastasis and invasion, make the combination with chemotherapeutic drugs reasonable and effective. In fact, the improved tumor immune microenvironment caused by phytochemicals is attracting and proved to enhance therapeutically effect with combination of chemo drugs or checkpoint inhibitors. Huang’s group reported that celastrol, a pentacyclic triterpene from Tripterygium wilfordii functioning in counteracting toxin with toxin, induced ICD with mitoxantrone synergistically to remodel immune-suppressive TME with a robust immune memory response in melanoma [471]. In order to avoid using the highly toxicity of CHMs, icaritin from herbs warming kidney-yang, were studied and the combination of icaritin with doxorubicin caused synergistic ICD induction through mitophagy and apoptosis, leading to improved hepatocellular carcinoma treatment [472]. Puerarin could not only downregulate intra-tumoral reactive oxygen species and deactivate TAFs, but also serves as an adjuvant therapy in nanoparticles for programmed cell death-ligand 1 (PD-L1) monoclonal antibody in triple-negative breast cancer model [330]. In fact, the combination of phytochemicals themselves (quercetin and alantolactone) was proved to induce synergistic ICD at low doses in CT26 and 4T1 cells. The micelles loaded with optimal ratio of quercetin and alantolactone stimulated the host immune response and inhibited murine orthotopic colorectal and breast tumor growth [148]. Besides using monoconstituents of CHMs, the components of TCMs could be studied at a relatively complex level, in order to make good use of the resources of TCMs, such as the classical CHM pairs (Pinellia-Aconitum, Scutellaria barbata-Oldenlandia diffusa, Rhizoma coptidis-Fructus evodiae). The effective extractions of Chinese medicine (such as essential oil) could also be potential agents for tumor treatment because their efficacy has already been clinically proven, and the herbs have relatively simple compositions.

Second, as for the targeting preparation of TCMs, the stability, metabolism, synthesis and quality evaluation need to be improved, especially for more complex combinations of ingredients of CHMs, to exhibit synergistic effects for tumor cells and for specific targeting to the TME. Liposomes can realize the co-delivery of medicinal ingredients with different pharmacological effects and physicochemical properties. In fact, by encapsulating all drug combinations in liposomes, the pharmacokinetic behavior of multiple drugs in vivo can be controlled while prolonging their circulation half-life. Multi drugs encapsulation in liposomes modified with targeting ligands makes liposomes versatile carriers, which is suitable for the application of herbal compound recipes in TCM [512,513]. There are several routes of administration for liposomes, and this review selects the route of systematic administration. Compared with the oral administration route, especially for lipophilic compounds from CHMs, the transportation of drugs from liposomal to biological membranes via intravenous injection are different from oral drug delivery [514]. The characteristics of intravenous injection of liposomes with functionalized surfaces different from other routes of administration are maintaining drugs combination at certain ratios, controlling drugs release, permitting selective delivery of drugs to targeting tissue [515–517]. Although the review demonstrated that targeting liposomal nanocarriers are effective and favorable delivery systems for CHMs to treat cancers, it is still at the exploratory stage.

Further investigations are necessary for anti-tumor mechanisms of some potential monoconstituents, especially the identification of subcellular targets and regulation of immunity through modulating immune-suppressive tumor microenvironment and priming immune responses effects. Site-specific and selective liposomal delivery enhances the accumulated drug concentrations at the site of action and comes with superior efficacy and safety in cancer treatments by reducing the cost and duration of therapy.