Glycyrrhetinic Acid Mediated Drug Delivery Carriers for Hepatocellular

Review pubs.acs.org/molecularpharmaceutics

Glycyrrhetinic Acid Mediated Drug Delivery Carriers for Hepatocellular Carcinoma Therapy Yuee Cai,†,‡ Yingqi Xu,†,‡ Hon Fai Chan,§ Xiaobin Fang,† Chengwei He,† and Meiwan Chen*,† †

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau 999078, China § Department of Biomedical Engineering, Columbia University, New York 10027, United States ABSTRACT: Glycyrrhetinic acid (GA), the main hydrolysate of glycyrrhizic acid extracted from the root of licorice, has been used in hepatocellular carcinoma (HCC) therapy. Particularly, GA as a ligand in HCC therapy has been widely explored in different drug delivery systems, including liposomes, micelles, and nanoparticles. There is considerable interest worldwide with respect to the development of GA-modified drug delivery systems due to the extensive presence of GA receptors on the surface of hepatocyte. Up until now, much work has been focused on developing GA-modified drug delivery systems which bear good liveror hepatocyte-targeted efficiency both in vitro and in vivo. Owing to its contribution in overcoming the limitations of low lipophilicity and poor bioavailability as well as its ability to promote receptor-mediated endocytosis, GA-modified drug delivery systems play an important role in enhancing livertargeting efficacy and thus are focused on the treatment of HCC. Moreover, since GA-modified delivery systems present more favorable pharmacokinetic properties and hepatocyte-targeting effects, they may be a promising formulation for GA in the treatment of HCC. In this review, we will give an overview of GA-modified novel drug delivery systems, paying attention to their efficacy in treating HCC and discussing their mechanism and the treatment effects. KEYWORDS: GA, anti-HCC, liver-targeting, delivery systems

1. INTRODUCTION Liver cancer (including HCC and intrahepatic cholangiocarcinoma) is the second leading cause of cancer death worldwide, and the majority of primary liver cancer is HCC, constituting approximately 70−90% of all cases.1−3 The majority of HCC occurs in the background of chronic liver diseases with a variety of morphologic appearance so it is necessary to pay great attention to the prevention of hepatitis virus transmission to decrease the occurrence of HCC.4 Furthermore, surveillance should be applied to detect early HCC, which plays an important role in the care of high-risk patients.5 Unfortunately, most patients are diagnosed at an advanced stage of HCC with higher death rates.6 It is also critical to improve the efficiency of HCC therapy by focusing on the various therapeutic strategies, including novel therapy of targeting cancer stem cells, molecular targeted therapy, and immunotherapy.7−9 Although various chemotherapeutic agents including doxorubicin, mitoxantrone, gemcitabine, irinotecan, etc. have been applied as single or combinational agents to cure HCC, no improvement of overall survival has been observed, and dismal efficacy of recent therapies such as multimolecular targeted drug sorafenib has been noted for drug resistance and systemic toxicity of severe hepatic dysfunction.7,8 In addition, chemotherapeutic agents also encountered some problems, such as nonselective biodistribution, poor bioavailability and solubility, and low specificity. For example, doxorubicin and paclitaxel are © XXXX American Chemical Society

associated with multiple side effects including cardiotoxicity, neurotoxicity, hypersentitivity reactions, etc.10 Therefore, receptor-based targeted therapy of HCC should be developed to conquer the drug off-targeted effects and improve anti-HCC efficacy of chemotherapeutic agents. 18β-Glycyrrhetinic acid (GA, Figure 1), the hydrolysis product of glycyrrhizin with a pentacyclic triterpeneglycoside extracted from the traditional Chinese medicine licorice (Glycyrrhiza glabra),11 has an active pharmacological effect with poor water solubility as well as increased scarce stability. Meanwhile, its hepatoprotective activity has been leveraged in the treatment of liver diseases in Asia for more than 30 years.12 Interestingly, it is reported that GA has effective anticancer ability against HCC by multiple mechanisms, including cell cycle arrest,13 induction of autophagy and apoptosis,14,15 reduction of immunosuppression,16 and so on. Although various drugs have the ability of inhibiting cancer growth, systematic toxicity and poor prognosis are found in the majority of patients with HCC, which is mainly due to the structure of the drugs themselves and deficiency of effective therapy. In order to obtain potent therapy against HCC with low toxicity Received: September 4, 2015 Revised: November 12, 2015 Accepted: November 30, 2015

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DOI: 10.1021/acs.molpharmaceut.5b00677 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Review

Molecular Pharmaceutics

Figure 1. Illustration of the anti-hepatocellular carcinoma and hepatoprotective mechanisms of GA.

2. BIOLOGICAL ACTIVITIES OF GA IN LIVER In previous studies, GA was shown to contribute to many pharmacological effects, including antioxidative, antiviral, antiinflammatory, antiallergenic, anticancer, and immunomodulatory activities.29 Interestingly, GA exerts a potent hepatoprotective effect against retrorsine-induced liver damage and carbon tetrachloride (CCl4) induced hepatotoxicity, while it also performs anti-HCC activity by inducing cell apoptosis, cell cycle arrest, and so on.29,30 Herein, the mechanisms of the hepatoprotective and anti-HCC effects are discussed in detail (Figure 1). 2.1. Anti-Hepatocellular Carcinoma Effect. As previously reported, GA exerts an active anticancer effect against various cancers, especially HCC. There are multiple mechanisms of the effect of GA against HCC, including inhibition of cell proliferation, invasion and metastasis, apoptosis, cell cycle arrest, and creation of immunopotentiating microenvironment.13,15,16 Herein, the anti-HCC effect of GA is discussed in order to provide a reference for future research on the anticancer effect of GA. GA exerts an active anti-HCC effect in HepG2 cells by inhibiting cell proliferation, inducing apoptosis and arresting cell cycle in the G1-phase in a dose-dependent manner with ID50 of 80 μM. Meanwhile, the mechanism of apoptosis induction caused by GA was found to be related to the activation of caspase-8 and reduction of antiapoptotic proteins (Bcl-2 and Bcl-xL), which resulted in the activation of downstream mitochondrial pathway and caspase-3 leading to apoptosis. Interestingly, GA was also found to inhibit the gap junction via reducing the expression level of connexin 32 and actin. It is reported that GA-induced decrease of gap junction may be independent of its anticancer activity in junctionally proficient HepG2 cells, but may counteract the toxic effect of other combinational drugs against HCC in further research.13,31−33 Additionally, as deficient gap junction in cancer cells allowed cell extravasation leading to cancer metastasis,34 it is necessary to further investigate if GA-induced decrease of gap junction enhances metastases of HepG2 cells with anticancer activity. Furthermore, the activation of hepatic stellate cells (HSCs) was found to inhibit the responses of T-cell to HCC, which decreased T-cell killing and enhanced the invasion as well as metastasis of Hepal-6 cells. Fortunately, GA (20 μM) could reverse the HSC-induced immunosuppression in T-cell

to human body, targeted therapy is applied to obtain better and anti-HCC effect.6 It has been found that some kinds of receptors could mediate the active hepatic-targeting drug delivery systems for the cure of hepatic diseases, including glycyrrhetinic acid receptor (GA-R), asialoglycoprotein receptor (ASGP-R), glycyrrhizin receptor (GL-R), hyaluronan receptor (HA-R), folate receptor (FA-R), epidermal growth factor receptor (EGF-R), transferrin receptor (TF-R), etc.17−21 Nevertheless, there is no study to reveal the differential expression of ASGP-R between hepatoma cells and normal hepatocytes, while the expression level of FA-R in HCC is controversial, and one of the drawbacks of TF-R, HA-R, and EGF-R is the unexpected immunogenicity to protein ligands.22,23 Besides, a previous study confirmed that the number of GA binding sites was far more than that of GL binding sites, which suggested that GA-R could be more effective than GL-R in targeting to HCC.24 In view of the issues related to the different receptors in HCC cells mentioned above, GA-R has been deemed to be a promising receptor to target HCC for its safety, pharmacological potential, and potentially better targeting ability to HCC. Therefore, GA has been used as a ligand for liver targeting due to the expression of GA-R on the sinusoidal surface of mammalian hepatocytes and also has been applied to modify drug delivery system to attain better liver- or hepatocyte-targeted efficiency,24,25 such as GAmodified liposomes,26 GA-modified micelles,27 and GAmodified nanoparticles.28 In addition, liposomes are also used to encapsulate GA for a better pharmacological effect against HCC. Table 1 shows different delivery systems of GA with a brief summary. In this review, we aim to provide a brief summary of the anti-hepatocellular carcinoma and hepatoprotective effect of GA and its application on the active hepatic targeting drug delivery systems, such as liposomes, micelles, and nanoparticles. First, the mechanism of anti-hepatocellular carcinoma effect by GA was enunciated. Second, the recent progress of the active targeted delivery systems constructed with GA was illustrated, which might provide insights to develop a novel targeted drug delivery system for the therapy of HCC in the future. Third, the encapsulation of GA in several types of drug delivery systems was demonstrated to take advantage of its potent anti-hepatocellular activity, offering new solution of using GA in the treatment of HCC. B


nanoparticles

micelles

liposomes

delivery systems

1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP); cholesterol (Chol); glycyrrhetinic acidpoly(ethylene glycol)-Chol glycyrrhetinic acid -modified liposomes

GAPEGCLs

C

glycyrrhetinic acid; chitosan; lactic acid

GCGA

glycyrrhetinic acid modified pullulan

glycyrrhetinic acid modified alginate glycyrrhetinic acid modified alginate/doxorubicin-modified alginate complex

glycyrrhetinic acid modified sulfated chitosan glycyrrhetinic acid conjugated stearic acid grafted chitosan chitosan/poly(ethylene glycol)glycyrrhetinic acid

GASCTS GA-CSSA CTS/ PEGGA

GAALG GAALG/ DOXALG GAP

glycyrrhetinic acid-poly(ethylene glycol)-18β-glycyrrhetinic acid conjugates

GAPEGGA

GAPEGPBLG

glycyrrhetinic acid-poly(ethylene glycol)-b-poly(gammabenzyl L-glutamate)

glycyrrhetinic acid modified liposomes

GA-DXLip

GAWGLip

glycyrrhetinic acid modified liposomes

GA-OXLip

materials

3-succinic-30-stearyl glycyrrhetinic acid modified liposomes

Suc-GALip

abbrev

loaded drugs

status

curcumin

in vitro

in vivo

doxorubicin

doxorubicin

in vitro and in vivo in vivo

5-fluorouracil

in vitro

none doxorubicin

none

doxorubicin

in vitro and in vivo in vitro and in vivo

in vitro and in vivo in vitro and in vivo In vitro

in vitro

in vitro and in vivo in vitro

paclitaxel

doxorubicin

wogonin

plasmid DNA

docetaxel

oxaliplatin

calcein

Table 1. Liver-Targeted Delivery Systems of GA advantages

High surface-to-volume ratio, biocompatibility and unique optical properties with targeted ability; utilization of different materials from gold to natural or biomedical product, which may meet with corresponding requirements in the application of nanoparticles, such as diagnosis by GA-modified Au NPs

desirable core−shell nanosize with the ability to prolong circulation of anticancer drugs; tumor-targeted ability with better anticancer efficacy

increases of drug accumulation in tumor tissue; reduction of drug amount in healthy tissues with lower systemic toxicity; various properties of sustained release, biocompatibility and capability of encapsulation

disadvantages

lack of further studies to confirm their clinical application and excavate the promising application of nanoparticles, such as combination of diagnostics and chemotherapy, multifunctional modification (pH and redox sensitivity, penetrating peptide, etc.)

no further studies to investigate the behavior of micelles in blood circulation and extravasation, which may limiting the clinical applications of micelles; no synergistic therapy with potent anticancer efficacy by multifunctional micelles

insufficient researches for the confirmation of anticancer efficacy in liposomes, such as in xenograft and genetic mouse tumor model

57

67

58

28

56

49

25

48

50

39

45

42

26

41

ref

Molecular Pharmaceutics Review


Review 62

59

70

60

61

response via potentiating an immune microenvironment in tumor by decreasing the expression of α-SMA (a marker of activated HSC), while GA at the same concentration showed no cytotoxicity to hepatic cells. To attenuate the immunosuppression, GA reduced T-cell apoptosis in tumor sites and the population of Regulatory T cells (Treg cells, FoxP3+ cells) while increasing CD4+ and CD8+ cells, which resulted in an immunopotentiating tumor microenvironment against HCC. Meanwhile, it was found that GA could suppress the vascularization and lymphatic metastasis in HCC by downregulating the expression of vascular endothelial growth factor (VEGF), LYVE-1 (a marker for lymphatic metastasis) and MMP-2 protein, indicating the anti-invasion and antimetastasis capability of GA against HCC. Although GA could suppress angiogenesis and potentiate immune microenvironment in HCC, there was no evidence to prove whether the anticancer efficacy of GA was higher than that of classic anticancer drug sorafenib and rapamycin or not. Nevertheless, it may be worthwhile to further investigate the combinational therapy of GA and other anticancer drugs to exert a synergistic anticancer effect by the complex interactions between tumor cells and micrenvironment.16 It was reported that GA could induce apoptosis and necrosis in HCC cells, which was verified by the reduction of cell viability (71% cell survival at the concentration of 40 μM GA), release of LDH, and upregulation of cleaved caspase-3 and Bax. Interestingly, after pretreatment with autophagy inhibitors (chloroquine and bafilomycin A1), the cell viability of GAtreated HepG2 cells was reduced from 69.79% to 46.09% and 23.13%, respectively, indicating that GA exerted a protective autophagic response consistent with its anticancer effect against HCC. GA could also upregulate the formation of acidic vesicular organelles (AVO) and expression of LC3-II protein by activation of the extracellular signal-activated protein kinase (ERK) pathway, resulting in protective autophagic response in HCC cells, which may weaken the anti-HCC effect of GA. For example, GA-modified sulfated chitosan (GA-SCTS) loaded with doxorubicin (DOX) (under 0.7 μg/mL) showed a lower antiproliferative effect than free DOX in HepG2 cells possibly due to the protective autophagy effect of GA, while GA-SCTS loaded with DOX (over 0.7 μg/mL) showed higher antiproliferation than free DOX. Thus, despite of the protective autophagy from GA, we could find balances between protective autophagy and anticancer effect triggered by GA via experiments. In addition, the combination of autophagic inhibitors and GA was also suggested to treat HCC to obtain a better anti-HCC effect with targeting ability to HCC.14,25 2.2. Hepatoprotective Effect. The hepatoprotective effect of GA is contributed by the inhibiton of hepatic apoptosis, necrosis, and antihepatic fibrosis mostly due to its antioxidant effect. In a previous study, GA was confirmed to be a promising inhibitor of bile acid induced necrosis and apoptosis in hepatocytes consistent with an antioxidative effect via the induction of mitochondrial permeability transition (MPT) resulting in activation of caspase-9, release of cytochrome c, and poly(ADP-ribose) polymerase (PARP). Decrease of reactive oxygen species (ROS) by GA may activate the downstream pathways against bile acid induced cytotoxicity in hepatocytes, including c-Jun N-terminal kinase (JNK) signaling pathways and caspases. Thus, GA could suppress caspase-10 possibly targeting to Fas ligand and TRAIL-induced activation leading to a protective effect in hepatocytes.35 In addition, GA could inhibit the expression and activity of cytochrome P450 2E1 to

Au; lipoyl tertiary amines; glycyrrhetinic acid-poly(ethylene glycol)

HACystGA

Au NPs

none

in vitro and in vivo none doxorubicin

in vitro HGA

glycyrrhizic acid conjugated bovine serum albumin glycyrrhetinic acid-graf t-hyaluronic acid reduction cleavable hyaluronic acid-cystamine-glycyrrhetinic acid conjugate GA-BSA

10-hydroxycamptothecin (HCPT) paclitaxel

status loaded drugs

doxorubicin

materials

glycyrrhetinic acid modified recombinant human serum albumin

abbrev

GA-HSA

delivery systems

Table 1. continued

in vitro and in vivo in vitro

advantages

disadvantages

ref


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effect (2.03-fold higher) than DX-Lip and similar pharmacokinetic behavior.42 Besides, wogonin was used as an anticancer drug to be loaded in GA-modified liposomes (GA-WG-Lip), which showed better uptake profiles and lower IC50 value (1.46-fold) compared to WG-Lip.39 Thus, GA-modified liposomes displayed potent targeting ability to hepatocytes with no cytotoxicity and resulted in high cellular uptake of chemotherapeutic anti-HCC drugs for better anticancer efficacy than unmodified liposomes. Apart from chemotherapeutic anti-HCC drugs, GA-modified liposomes were also applied to deliver pDNA. Cationic liposomes, one of the potential vectors used to deliver nonviral gene,43 have multiple merits such as large DNA capacity, less immunogenicity, and nononcogenicity.44 The GA-modified stealth cationic liposomes (GA-PEG-CLs) could entrap pDNA with little cytotoxicity to normal hepatocyte cell (L02) and higher gene transfection efficiency in HCC cell line HepG2, suggesting that GA-PEG-CLs could be used to deliver gene for HCC targeting therapy.45 Despite the targeting property and better anti-HCC effect of GA-modified liposomes demonstrated in vitro, further studies should be conducted to confirm their therapeutic effect, such as performing in vivo studies in both xenograft and genetic mouse tumor model. In addition, some microenvironment-responsive functional groups could be introduced into liposome drug delivery system in the merit of producing safe and effective products, which may further enhance their anti-HCC activity. 3.2. Micelles. Amphiphilic copolymers have the ability to form core−shell nanosize micelles, which can be used as micellar carriers for better drug uptake and prolonged circulation to deliver hydrophobic anticancer drug. Examples include chitosan- and PEG-modified polymers.46,47 On top of the advantages of polymeric micelles mentioned above, the surface of micelles could be modified with GA to achieve livertargeted therapy, which could enhance the anti-HCC effect of anticancer drugs. Different types of micelles were modified with GA for better anticancer efficacy, including triblock copolymers with poly(gamma-benzyl L-glutamate) (PBLG), PEG, poly(lactic-co-glycolic acid) (PLGA), and chitosan.48−51 Detailed information on GA-modified micelles is discussed in the following. First, GA-modified PEG-b-PBLG (GA-PEG-PBLG) micelles, a promising liver-targeted drug carrier, was prepared to load DOX with 7.5% drug loading efficiency and a pH-dependent release profile. DOX-loaded GA-PEG-PBLG micelles showed time- and dosage-dependent cytotoxicity and delivered 4.9-fold higher concentration of DOX compared to free DOX.50 Second, GA-SCTS was designed and synthesized to deliver DOX, which showed 2.18-fold increase in the DOX uptake in HepG2 cells compared to Chang liver cells, indicating a potent effect of liver cancer targeting by GA-SCTS.25 GA was also conjugated with stearic acid grafted chitosan (GA-CS-SA), resulting in favorable drug carrier properties such as relatively low critical micelle concentration of 17.49 μg/mL with spherical morphology.49 Third, GA-PEG-GA triblock copolymers were synthesized and used to form self-assembled micelles by thin film hydration method. Paclitaxel (PTX) was encapsulated in the core of GA-PEG-GA micelles with improved micelle binding to liver cells due to the specific binding site of GA to liver cells. Therefore, GA-PEG-GA micelles could be a potential strategy for the treatment of HCC.48 Furthermore, poly(L-histidine) (PHIS) was applied as the pH-sensitive moiety in the copolymer of GA-decorated

decrease serum aminotransferase activity as well as lipid peroxidation in a dose-dependent manner against CCl4-induced hepatoxicity with reduced xenobiotic toxicity via downregulating the metabolic activation. The depletion of glutathione (GSH) by CCl4 was attenuated by GA to conjugate with trichloromethyl radicals by CCl4, which could inhibit the formation of trichloromethyl radicals leading to reduction of hepatotoxicity. However, the pretreatment of GA itself could not increase the cytosolic level of GSH or the activity of glutathione-S-transferase.36 Furthermore, in CCl4-induced liver fibrosis mice, GA exerted hepatoprotective action of relieving liver fibrosis, inflammatory cell infiltration, and hydrophobic degeneration next to the central vein or focal necrosis. The main mechanism of the hepatoprotective effect from GA was to inhibit the lipid peroxidation and increase the activity of antioxidative enzymes such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX2) leading to scavenging of free radicals, which was mediated through the promotion of NF-E2-related factor 2 (Nrf2) nuclear transcription and the Nrf2 gene expression. The high level of MDA (an end product of lipid peroxidation) induced by CCl4 in liver was downregulated by GA, indicating its reversal ability to CCl4-induced liver fibrosis, which was consistent with the ability of inhibiting membrane lipid peroxidation. Additionally, target gene expression of Nrf2 was investigated in the protection of liver fibrosis including the extracellular SOD (EC-SOD) mRNA, GPX2 mRNA, and CAT mRNA. Due to the mechanisms mentioned above, GA acted as a potent oxygen free radical scavenger to exert its hepatoprotective action against CCl4-induced liver fibrosis mice.37

3. LIVER-TARGETED DELIVERY SYSTEM 3.1. Liposomes. Liposomes, spherical bilayer vesicles, are formed spontaneously with phospholipids dispersed in water, which have been applied in drug delivery systems to offer good biocompatibility, sustained release potential, and ability to encapsulate bioactive drugs.38 Besides, the application of liposomes results in increasing drug accumulation in tumor tissue and reduced drug amount in healthy tissues with lower systemic toxicity. In order to improve the anticancer effect of therapeutic drugs in HCC, GA was used to modify the surface of liposomes to achieve targeted delivery in tumor tissues.39 An amphiphilic derivative of glycyrrhetinic acid, named 3-succinic30-stearyl glycyrrhetinic acid (Suc-GA), was synthesized and used as a targeting molecule binding to hepatocytes via specific binding of GA-R on the HCC cell surface, which was introduced onto the surface of liposomes with a certain molar ratio by ethanol injection method.40 Calcein was encapsulated in Suc-GA-modified liposomes with higher cellular uptake via specific receptor-mediated endocytosis in HCC. This was considered a promising way of targeting to hepatocytes.41 Additionally, GA-modified and oxaliplatin-loaded liposomes (GA-OX-liposomes) were produced by the film-dispersion method with spherical morphology and entrapment efficiency of higher than 94%. GA-OX-liposomes showed sustained release of OX and delivered most of the OX to liver, demonstrating the liver-targeted effect of GA in GA-OXliposomes. Moreover, histology studies showed that GA-OXliposomes displayed no toxicity to epithelial cells.26 Similar to GA-OX-liposomes, docetaxel was encapsulated in GA-modified liposomes (GA-DX-Lip) by the same method with 2.28-fold higher uptake in hepatocytes compared to nonparenchymal cells. GA-DX-Lip also showed a better antitumor inhibition E


Review


PEG, which could significantly inhibit tumor growth in H22 cell bearing mice (Figure 2).56

PEG-PLGA (GA-PEG-PHIS-PLGA, GA-PPP), which was employed to encapsulate a model anticancer drug andrographolide (AGP) (AGP/GA-PPP) with a pH-responsive drug release profile and enhancement of selective uptake of AGP/ GA-PPP micelles by tumor targeting property, which improved its anticancer efficacy remarkably.51 An increasing interest was focused on star-shaped polymers which have higher drug loading capacity (three or more times) than linear polymers.52 Zhang et al.53 combined the advantages of GA and star polymers as vector to design a novel livertargeted delivery system. This copolymer, four-armed starshaped poly(ethylene glycol)-b-poly(epsilon-caprolactone) block copolymer (sPCL-b-PEG-GA), was composed of a core of pentaerythritol (PTOL), the inner hydrophobic segments of PCL and external segments of PEG, and more importantly, terminal GA as targeting ligand. The design and synthesis of this star-shaped polymer shed light on novel approaches to improve the targeting efficacy of liver drugs, such as anti-HCC drugs. Although it has been reported that GA-modified micelles are capable of improving the anti-HCC effect of drugs, more studies should be conducted to confirm the liver-targeted affinity of GA-modified micelles. No further study was taken to investigate the behavior of micelles in blood circulation and extravasation as well as the synergistic therapy with potent anticancer efficacy by multifunctional micelles loaded with more than one anticancer drug, which may limit the clinical applications of micelles. Besides, functional moiety introduced into micelles, including pH- and redox-sensitive moiety, may induce stimulus-responsive drug release in response to the tumor microenvironment for controllable drug release, combining the advantages of higher drug accumulation in tumor sites as well as tumor-targeted property of GA. All of these modifications on micelles should meet the properties of safety and efficacy in the clinical application. 3.3. Nanoparticles. Nanoparticles (NPs), displaying outstanding chemical and physical properties such as good biocompatibility, unique optical properties, and high surfaceto-volume ratio, have a broad prospect for clinical application as a novel delivery system for hydrophobic drug. Targeting ligand may guide drug conjugated NPs to specific binding site, leading to better therapeutic efficacy with reduced drug toxicity. Due to the limitation of low level of drug loading, developing NPs with high content of targeting ligand is explored. To achieve desired drug concentration, NPs modified with high density of targeted ligand were pursued by research groups, which were also more likely to induce fast clearance by mononuclear phagocytic system (MPS) and negatively affect the dispersity.54 GAmodified nanoparticles were prepared to enhance anti-HCC activity from multiple materials, including chitosan,55 PEG,56 pullulan,57 alginate,58 hyaluronic acid (HA),59 bovine serum albumin (BSA),60 human serum albumin (HSA),61 Au,62 and CdTe/ZnS quantum dot,63 and applied to deliver anticancer drugs. First, GA was introduced into nanoparticles at the site of the C(30)-carboxyl group or C(3)-hydroxyl group to obtain GAmodified chitosan/PEG nanoparticles (CTS/PEG-GA) by an ionic gelation method. No significant difference of livertargeted ability between them was observed.64 In addition, DOX was encapsulated in CTS/PEG-GA and the DOX accumulation of DOX-loaded CTS/PEG-GA was approximately 2.6-fold higher in liver than that of DOX-loaded CTS/

Figure 2. Liver-targeted delivery of DOX using glycyrrhetinic acid modified chitosan/poly(ethylene glycol) (CTS/PEG-GA) nanoparticles. (A) Illustration of the preparation of the CTS/PEG-GA nanoparticles. (B) Fluorescence histograms of QGY-7703 cells treated with CTS/PEG nanoparticles (b, red) and CTS/PEG-GA nanoparticles (c, green) for 2 h, and background of QGY-7703 cells (a, violet). (C) The images of the mice administrated with CTS/PEG nanoparticles and CTS/PEG-GA nanoparticles at 15, 90, and 180 min. (D) Cytotoxicity for QGY-7703 cells incubated with free DOX and DOX-loaded CTS/PEG-GA nanoparticles for 72 h. (E) Inhibition of tumor growth by injecting physiological saline (control), free DOX, or DOX-loaded CTS/PEG-GA nanoparticles (n = 4). Reprinted with permission from ref 56. Copyright 2010 Elsevier.

Furthermore, polymer−drug conjugates were reported to possess high drug loading efficiency, resistance against recrystallization, and temporally controlled drug release.65 For example, 5-fluorouracil (5-FU) was conjugated with GAmodified chitosan (GCGA) synthesized by lactic acid, GA, and chitosan for liver-targeted therapy. GCGA/5-FU nanoparticles were prepared with better affinity to liver cells and improved cytotoxicity in liver cancer.66 GA-modified alginate (ALG) was also adopted to load DOX (DOX/GA-ALG) for targeting treatment of liver cancer, which showed 2.8-fold higher DOX concentration in the liver than DOX/CHO-ALG without any myocardial necrosis or cells swelling in the heart and liver cells.58 Besides, a liver-targeted drug delivery system with pH sensitivity was developed by selfassembly of GA-modified ALG and DOX-modified ALG (GAF


Review

Molecular Pharmaceutics ALG/DOX-ALG) via the dialysis method, which could trigger DOX release in response to the endosomal/lysosomal environment and showed 1.6-fold and 1.2-fold higher inhibition rate in tumor growth than free DOX and DOX-ALG, respectively.67 Similarly, GA-modified pullulan nanoparticles (GAP) were utilized to deliver curcumin with better stability and solubility of curcumin, showing pH-sensitive release profile and higher cellular uptake and cytotoxicity in HepG2 cells.57 GA was also conjugated with BSA to encapsulate 10hydroxycamptothecin (HCPT), taking advantage of BSA conjugates for its improved half-life of drug and stability, ease of administration and controlled drug release, and decrease of systematic toxicity.68 GA-BSA/HCPT nanoparticles could enhance the anticancer effect in HCC cells with 2-fold and 10-fold lower IC50 compared with HCPT/BSA and free HCPT, respectively. This showed that GA-BSA could be a potent antiHCC drug carrier for HCC therapy.60 Interestingly, GAmodified HSA nanoparticles were also investigated and encapsulated with DOX to improve the therapeutic efficacy of liver tumors and reduce systematic toxicity. Higher accumulation of DOX after the administration of DOX-loaded GA-HSA was detected in liver cells compared to DOX-loaded HSA.61 To enhance the targeting ability of nanoparticles to HCC, dual ligand-modified nanoparticles containing GA and HA were formulated. GA was grafted with amphiphilic HA derivatives69 to self-assemble into nanoparticles to achieve higher targeting ability to liver cells and better entrapment efficiency (92.02%). The affinity of PTX-loaded HGA nanoparticles to liver cells was evaluated in cells overexpressing GA and HA receptors (HepG2 and B16F10) and normal fibroblast cells. Higher cellular uptake of PTX-loaded HGA nanoparticles were detected in HepG2 and B16F10 compared to normal fibroblast cells, demonstrating that HGA nanoparticles may serve as a double targeting drug carrier to liver cells with improved targeting potential against HCC.70 Moreover, a reduction cleavable linkage was introduced into HGA to form a dual targeted and reduction-responsive nanoparticle for DOX delivery to HepG2 cells, which displayed improvements in intracellular release and nuclear delivery of DOX and lower IC50 (over 1.6-fold) compared to free DOX and insensitive nanoparticles (Figure 3).59 In addition, a pH-responsive, reversible tumor targeting of GA was investigated based on the use of gold nanoparticles (Au NPs) with the ability of amphiphilic self-assembly. Au NPs could shield GA from the external environment in normal tissues, while deshielding GA at extracellular pH of tumors. This could become a promising drug delivery system with reversible tumor targeting potential to decrease the absorption by BSA and cellular uptake in normal tissues (Figure 4).62 Based on these GA-modified Au NPs, diagnosis or photothermal therapy could be applied against HCC cells with the ability of reversible shielding or deshielding of GA in further studies. Multiple types of GA-modified nanoparticles were formulated with properties such as pH-sensitive release, reductionsensitive release, or ability of shielding/deshielding targets, using self-assembled/dissembled polymers, polymer−drug conjugates, dual target site conjugates, etc. This could not only achieve liver-targeted therapy for HCC but also improve the bioavailability and pharmacological activities of anticancer drugs. However, more studies should be focused on the antitumor effect in vivo, which played an important role in the

Figure 3. Intracellular delivery of DOX using dual targeted stimulusresponsive nanoparticles based on a hyaluronic acid−glycyrrhetinic acid conjugate (HA-Cyst-GA). (A) Scheme of self-assembly, GA/ CD44 receptor mediated endocytosis, and redox-sensitive disassembly of HA-Cyst-GA nanoparticles in HCC. (B) Flow cytometry analysis of HepG2 cells treated with coumarin-6 loaded HA-Cyst-GA nanoparticles with or without endocytosis competitive inhibitors (HA or GA) for 4 h. (C) NIR fluorescence imaging of H22 tumor-bearing mice treated with DiR labeled HA-Cyst-GA nanoparticles. Reprinted with permission from ref 59. Copyright 2015 Elsevier.

establishment of clinical trials. In addition, further research should be devoted to the exploration of promising applications of nanoparticles, such as combination of diagnostics and chemotherapy, multifunctional modification on the nanoparticles with penetrating peptide, pH and redox sensitivity, etc. In view of the liver-targeted delivery system in present studies, GA plays as the target in the liver-targeted drug delivery system including liposomes, micelles, and nanoparticles. Multiple functional moieties are applied to modify these anticancer drug loaded delivery systems with higher drug accumulation in tumor sites and enhanced anticancer efficacy than free drugs in vitro or in vivo. Nevertheless, it may take a long time to confirm the safety and efficacy of these promising liver-targeted delivery systems on patients. For example, the first Food and Drug Administration (FDA) approved nanodrug, Doxil, composed of doxorubicin-loaded oligolamellar liposomes showed superior advantages against cancers compared to free doxorubicin, such as higher drug accumulation in tumor sites due to enhanced permeability and retention (EPR) effect and reduced side effects of cardiotoxicity, which has improved the compliance and quality of life of patients in the efforts of 17 years.71 Moreover, although these formulations show higher tumor accumulation compared to G


Review


activity was demonstrated.77 As mentioned above in the antiHCC section, GA could inhibit HCC growth by creating an immunopotentiating microenvironment so that GAL might be more effective to suppress HCC growth via enhancing immunological activity. Despite better bioavailability and pharmacological activities of GA-loaded formulations mentioned above, future research should be taken to further probe the anti-HCC effect of GA, including developing other promising delivery systems and combination of GA with other drugs for HCC therapy etc.

5. CONCLUSION GA, the main constituent extracted from licorice root, possesses potent biological activities including anti-HCC and hepatoprotective effects against hepatic diseases. More and more research has been studied to make full use of GA, including chemical conjugation for tumor targeting therapy and physical encapsulation of GA for better bioavailability. On the one hand, it has been found that GA is capable of binding to hepatocytes via the specific site of GA-R on the surface of HCC cells, which provides a promising approach for the targeted drug delivery systems via chemical conjugation. Therefore, GA has been widely used as a ligand in hepatic-targeted drug delivery against hepatic diseases, especially HCC. Multiple novel liver-targeted drug delivery systems including liposomes, micelles, and nanoparticles, based on the mediation of GA-R with controlled release profile, have been developed with the aim of prolonging circulation, releasing drugs in a stimulusresponsive manner, increasing drug accumulation in tumor sites, and reducing side effects. On the other hand, GA has been physically encapsulated in nanoparticles as a chemotherapeutic drug with better bioavailability to enhance the effect of antiHCC, which could be combined with its tumor-targeted property to exert a dual-functional effect. However, further studies should be devoted to figuring out the mechanisms of the tumor-targeted ability of GA, such as looking into the differential expression of GA-R in different cells or the other receptors for GA. Meanwhile, the reality and possibility of these liver-targeted delivery systems applied in the human body should receive much attention in future research. In summary, GA not only produces an anti-HCC effect but also has the excellent ability of liver-targeting, which suggests that the GAmodified novel delivery systems possess favorable pharmacokinetic properties and may be a promising approach for using GA in HCC therapy.

Figure 4. Shieldable tumor-targeted and pH-responsive gold nanoparticles. (A) Scheme of GA ligand’s shield at normal tissue (pH 7.4) and exposure at mildly acidic environment in tumor (pH 6.8), and the mechanism of pH-responsive disassembly and assembly of Au NPs. (B) Fluorescent intensity of pyrene-loaded Au NP assembly at various pH values at the same concentration. (C) Cytotoxicity of Au NPs with a series of concentrations (0.5, 1.0, 2.0 nM) for 24 h. Reprinted with permission from ref 62. Copyright 2014 American Chemical Society.

free drugs, it is reported that less than 5% of the total administered formulations could arrive at tumor sites due to the blood circulation and extravasation, indicating that the advantages of ligand−receptor interaction may not occur in clinical application as they were presented in the present studies.72 Thus, despite that it is promising to investigate multifunctional drug delivery systems with liver-targeted property and stimulus-responsive drug release, the reality and possibility of these designs applied in the human body should be considered. Best efforts will be taken to overcome these obstacles occurring in the clinical applications (blood circulation and extravasation) to fulfill the ideal therapeutic effects of liver-targeted drug delivery systems by GA.

4. ENCAPSULATION OF GA IN DRUG DELIVERY CARRIERS Given the poor water solubility and bioavailability of GA, drug delivery systems were applied to encapsulate GA with the aim of improving the bioavailability and reducing systematic toxicity of GA.73 For example, GA was encapsulated in nanoparticles prepared from PLGA, HSA, dithiothreitol, and acetone, which showed about 9-fold higher cytotoxicity in HepG2 cells than free GA.74 In an effort to inhibit the proliferation of human hepatic stellate cells, liposomes containing TSN, GA, and Sal B (GTSliposomes) were successfully prepared by Lin et al, which not only played a part in enhancing the combination therapy effects but also displayed a sustained release profile to some extent and inhibited the proliferation of hepatic stellate cells (HSC).75 Furthermore, a film dispersion method was applied to form liposomes loaded with GA76 by soybean phospholipid and cholesterol. Good encapsulation efficiency of 83.46% was achieved, and significant improvement of immunological

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AUTHOR INFORMATION

Corresponding Author

*Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, China. E-mail: [email protected]. Author Contributions ‡

Y.C. and Y.X. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Macao Science and Technology Development Fund (062/2013/A2), the Research Fund of the University of Macau (MYRG2014-00033-ICMSQRCM, MYRG2014-00051-ICMS-QRCM, MYRG2015H


Review


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00171-ICMS-QRCM), and the National Natural Science Foundation of China (81403120).

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Glycyrrhetinic Acid Mediated Drug Delivery Carriers for Hepatocellular

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