Receptor-Based Targeted Treatment of Hepatocellular Carcinoma by Nanoparticulate Systems

Tumor Biol. (2015) 36:55–67 DOI 10.1007/s13277-014-2855-3

REVIEW

Active radar guides missile to its target: receptor-based targeted treatment of hepatocellular carcinoma by nanoparticulate systems

Jing-Jun Yan & Jia-Zhi Liao & Ju-Sheng Lin & Xing-Xing He

Received: 31 July 2014 /Accepted: 13 November 2014 /Published online: 26 November 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Patients with hepatocellular carcinoma (HCC) usu- FR Folate receptor ally present at advanced stages and do not benefit from surgi- PTX Paclitaxel cal resection, so drug therapy should deserve a prominent DOX Doxorubicin place in unresectable HCC treatment. But chemotherapy LA Lactobionic acid agents, such as doxorubicin, cisplatin, and paclitaxel, fre- HCPT 10-Hydroxycamptothecin quently encounter important problems such as low specificity 5-FU 5-Fluorouracil and non-selective biodistribution. Recently, the development RBV Ribavirin of nanotechnology led to significant breakthroughs to over- siRNA Small interfering RNA come these problems. Decorating the surfaces of shRNA Short hairpin RNA nanoparticulate-based drug carriers with homing devices has miRNA microRNA demonstrated its potential in concentrating chemotherapy RBITC Rhodamine B isothiocyanate agents specifically to HCC cells. In this paper, we reviewed PHA Polyhydroxyalkanoates the current status of active targeting strategies for PhaP PHA granule-binding protein nanoparticulate systems based on various receptors such as LPEI Linear polyethylenimine asialoglycoprotein receptor, transferrin receptor, epidermal PEG Polyethylene glycol growth factor receptor, folate receptor, integrin, and CD44, NIS Sodium iodide symporter which are abundantly expressed on the surfaces of hepato- QD Quantum dot cytes or liver cancer cells. Furthermore, we pointed out their DENA Diethylnitrosamine merits and defects and provided theoretical references for ECM Extracellular matrix further research. CSCs Cancer stem cells HA Hyaluronan GA Glycyrrhetinic acid Keywords Active drug delivery . Nanotechnology . GPC3 Glypican-3 Asialoglycoprotein receptor . EGFR . Transferrin receptor . SSTRs Somatostatin receptors Folate receptor VEGFR Vascular endothelial growth factor

Abbreviations HCC Hepatocellular carcinoma ASGPR Asialoglycoprotein receptor Introduction EGFR Epidermal growth factor receptor TfR Transferrin receptor Hepatocellular carcinoma (HCC) is the fifth most common : : : malignancy worldwide and the second leading cause of J.glycoproteins, fight against cancer aims to extend the survival of this popu- ASGPR plays critical roles in maintaining serum glycoprotein lation. However, these molecules frequently encounter impor- homeostasis [7]. These properties make ASGPR an enticing tant problems such as low specificity and non-selective target for receptor-directed drug delivery to hepatocytes and biodistribution. For example, paclitaxel (PTX) and doxorubi- hepatocarcinoma cells. Incorporation of terminal galactose cin (DOX) are potent anti-cancer drugs. It is unsatisfactory moieties onto the surface of nanoplatforms allows concentrat- that administration of PTX brings various off-target effects ing chemotherapy agent or therapeutic genes into the HCC such as hypersensitivity reactions, myelosuppression, neuro- cells (especially HepG2 cells) [8–10]. The efficiency of rec- toxicity, and so on [2]. DOX is also described to induce ognition and cell internalization showed positive correlation undesired effects, particularly cardiotoxicity [2]. Thus, novel with the ligands density on the surface of nanoparticles [8, 9]. methods which help conquer drug off-target effects and en- Table 2 summarizes the use of ASGPR as a targeting receptor hance efficacy of chemotherapeutic drugs may significantly to guide therapeutics to the liver. improve survival rates of HCC patients. In recent years, nanotechnology has showed unprecedented ASGPR-directed nanoparticles used for delivering expansion across all areas of medicine including oncology. As chemotherapy drugs showninTable1, the properties of various nanoparticle plat- forms usually used for nanomedicine delivery were summarized. ASGPR ligand-conjugated nanoparticles were successfully Having benefited from their unique biological properties, such as used to load various chemotherapy drugs to target HCC both smallsizeandlargesurfacearea-to-volume ratio, nanoparticles in vitro and in vivo. Galactosamine-coupled and lactobionic could provide tiny vehicles to deliver various compounds includ- acid (LA)-grafted nanoparticles which loaded with PTX ing small molecule drugs, DNA, RNA, proteins, and probes with showed enhanced cytotoxicity in HepG2 cell compared with high efficiency [3]. However, several studies reported that after free PTX, while ASGPR-deficient SK-Hep-1 cells, BGC-823 entering the bloodstream, the majority of the conventional nano- cells, and P388 cells exhibited less sensitive to it [11–13]. In particles (not surface-modified) are rapidly taken by liver mac- vivo studies indicated that the galactosamine-conjugated and rophages in addition to other reticuloendothelial organs but not PTX-loaded nanoparticles (Gal-P/NPs) had a specific interac- by hepatocytes or cancer cells which are the major target for tion with the hepatoma induced in nude mice via ligand– therapeutic agents [4]. Due to the enhanced permeability and receptor recognition [14]. retention (EPR) effect, nanoparticles would take advantage of the ASGPR-directed copolymer also holds great potential for irregular architecture of the tumor blood vessels to passively liver-targeted intracellular delivery of DOX [15–18]. Guhagarkar concentrate in the tumor interstitium. But in this case, the efficacy et al. used pullulan as ASGPR ligand to guide polyethylene of nanoparticles is largely mediated through the local release of sebacate (PES)-DOX nanoparticles (PES-DOX NP PUL) for the drug near the cancer cells [5]. However, a better anti-cancer hepatic targeting. This nanomaterial exhibited better serum sta- effect can be hypothesized if the nanomedicine is adsorbed onto bility, lower hemolytic potential, and longer circulating nature the malignant cell surface and further endocytosed into the tumor compared with PES-DOX-NP without pullulan. Lower DOX cell. Just like an active radar guides missile to its target, concentration was also found in the heart which indicated lower nanocarriers equipped with homing devices as a specific recog- cardiotoxicity [15]. Zhou et al. reached similar conclusion using nition mechanism for malignant cells have been studied in lactosylated liposomes encapsulating DOX (Lac-L-DOX) in various cancers including HCC [6]. These so-called homing nude mice bearing HepG2 xenografts [16]. devices (or homing ligands) can be small molecules, peptides, Galactosylated nanomaterials were also used to carry other or antibodies which have high affinity with receptors chemotherapy agents, such as 10-hydroxycamptothecin overexpressed on hepatocytes or HCC cells. Such modified (HCPT) [19], oridonin [20, 21], and 5-fluorouracil (5-FU) nanoparticles could be internalized by malignant cells through [22], which not only exhibited higher anti-tumor activity in receptor-mediated endocytosis, leading to an intracellular drug ASGPR overexpressed liver cancer cells but also lower cyto- release and a greater cytotoxic activity (Fig. 1)[4]. Our review toxicity in ASGPR-deficient cells. In addition, porphyrin- and recapitulates recent development in receptor-based targeted treat- galactosyl-conjugated polymer micelles exhibited higher ment of HCC by nanoparticulate systems (Fig. 2). targeting and photodynamic therapy efficacy in HepG2 cells than in laryngocarcinoma cells Hep2 which lack ASGPR [23].

Asialoglycoprotein receptor ASGPR-directed nanoparticles used for delivering DNA

As an endocytic cell surface receptor, asialoglycoprotein re- The incorporation of galactose moiety onto the surface of ceptor (ASGPR) is abundantly and almost uniquely expressed nanoplatforms could enhance the efficiency of gene Tumor Biol. (2015) 36:55–67 57

Table 1 Summary of types of nanoparticles and their properties

Nanoparticle (size) Properties Imaging Therapeutic References potential potential

Liposome Passive and active targeting; carry multiple drugs at once; tunable drug release in response Yes [5, 102, (20–250 nm) to temperature, pH, or other environmental inputs 103] Polymeric micelles Passive and active targeting; a hydrophobic inner core acts as a drug reservoir especially Yes [104] (20–100 nm) for hydrophobic drugs, whereas the hydrophilic outer shells avoid endocytosis by mononuclear phagocyte system; easy sterilization; controllable drug-loading and release Polymeric NanoGel Passive and active targeting; less susceptible to reticuloendothelial system clearance and Yes [103] (20–220 nm) better penetrate in tissues and cells Quantum dot Passive and active targeting; imaging through tunable autofluorescence; treatment when Yes Yes [3, 105] (2–100 nm) combined with photosensitizing agent Dendrimer Well-controlled synthesis methods; a single dendrimer molecule can carry a therapeutic Yes Yes [5, 105] (5–200 nm) drug, a diagnostic agent, and an active targeting molecule; pH- or enzymatic-sensitive linkages to disrupt dendrimer micelles and trigger the site-specific release of their payload Gold nanoparticle Passive and active targeting; reticuloendothelial system (RES) evasion; multimodal Yes Yes [3, 103, (1–100 nm) imaging: MRI contrast, fluorescence, optical properties; simple changes to the size and 105] shape of gold nanoparticles can give rise to a variety of biomedical applications; optoelectronic properties can be changed to apply for the desired cancer treatment by tuning their aspect ratio transfection in hepatocytes. Lim et al. built a block copolymer copolymer could significantly enhance the gene transfection composed of cationic polymer and polyethylene glycol (PEG) efficiency, almost comparable to commercial Lipofectamine incorporated with galactose moiety to carry DNA and evalu- [24]. Besides, lactose-side chains (Lac-PEG-C)-modified ated the in vitro gene transfection efficiency in HepG2. Re- plasmid/protamine (PRT) complex did not coagulate the sults revealed that this galactose moiety-conjugated erythrocytes and showed very low cytotoxicity. Moreover,

Fig. 1 Schematic representation of the mechanism of tumor- targeted drug delivery for treating hepatocellular carcinoma 58 Tumor Biol. (2015) 36:55–67

Fig. 2 Schematic drawing of hepatocellular carcinoma-specific targeting nanoparticles, dendrimers, etc. It is not one-to one relation between the of cargo-loaded nanoparticles via receptor-mediated endocytosis. nanocarriers and receptors in the figure; for instance, liposome could not Receptors used for active drug delivery systems include only bind folate to target HCC but also bind ligands for ASGPR, TfR, asialoglycoprotein receptor (ASGPR), epidermal growth factor receptor EGFR, etc. Therapeutic molecules (cargos) carried by these nanoparticles (EGFR), transferrin receptor (TfR), integrin, folate receptor (FR), etc. included chemotherapy drugs, DNA, siRNA, microRNA, and so on Types of nanocarriers cover liposomes, polymeric micelles, metal core incubating this modified complex with HepG2 cells devel- They found the transfection efficiency of the peptide was oped extremely enhanced gene expression (nearly 56-fold improved by increasing the number of modified galactose higher expression) compared with complex without Lac- residues on the peptide. They also suggested that the translo- PEG-C [25]. Similarly, galactosylated polycation vehicles cation of the peptide–DNA complex from the endocytic com- were also reported to induce ten-fold higher transfection effi- partments to the cytosol mainly occurred during an early ciency than controlled glycosylated particles but showed no endosome step [26]. preferential transfection efficiency in cell lines lacking It is worth mentioning that ASGPR is abundantly ASGPR [26]. expressed on the surface of normal hepatocytes as well as Niidome et al. studied the endocytosis of galactose- malignant hepatic cells. A problem needs to be solved is that modified alpha-helical peptides which carried plasmid DNA. how to further aim at malignant cells without side effects on

Table 2 Summary of the targeting HCC nanoparticles used asialoglycoprotein receptor (ASGPR)

Cargos carried by nanoparticles Types of ligands Cell lines employed References

DNA Sialoglycoprotein, asialoglycoprotein, galactosamine, HepG2, Huh7 [24–27, 106–108] lactose-side chains, galactose moiety siRNA N-acetylgalactosamine Mice primary hepatocytes, HepG2.2.15 [28, 29, 109] MicroRNA Lac-DOPE HepG2 [34] DOX Galactosamine, Lac-DOPE, pullulan HepG2 [15–18] PTX Lactobionic acid, galactosamine, galactose moiety HepG2 [11–14] Oridonin Galactose moiety, galactosamine HepG2 [20, 21] HCPT Galactosamine HepG2 [19] 5-FU Lactobionic acid HepG2 [22] RBV prodrug Galactosamine HepG2 [110] siRNA small interfering RNA, Lac-DOPE N-lactobionyl-dioleoyl phosphatidylethanolamine, DOX doxorubicin, PTX paclitaxel, HCPT 10- hydroxycamptothecin, 5-FU 5-fluorouracil, RBV ribavirin Tumor Biol. (2015) 36:55–67 59 normal hepatic cells. In vitro experiment indicated that regulating target genes involved in cell proliferation, apopto- Apoptin could selectively induce apoptosis in malignant cells. sis, metastasis, and survival [32]. Using asialoglycoprotein as a ligand for ASGPR, Peng and his Novel liver-specific delivery vehicle for anti-microRNA coworkers constructed a systemic delivery vehicle to load therapy was also designed by making use of ASGPR [33]. Apoptin gene in order to target HCC cells. In vitro, the dual MicroRNA-155 (miR-155) is reported to be an oncogenic hepatocyte-targeting protein–DNA complex Asor-Apoptin in- miRNA frequently overexpressed in HCC. Lipid nanoparti- duced apoptosis in HepG2 cells but not in normal hepatocytes cles conjugating lactosylated gramicidin (Lac-GLN) were L02 and human pulmonary carcinoma A549 cells with prepared for delivery of anti-miR-155 to HCC cells [33]. ASGPR deficiency. Systemic administration of this novel Lac-GLN could effectively load anti-miR-155 to HCC cells complex via the tail vein into mice bearing in situ and even exhibit greater efficiency than Lipofectamine 2000 hepatocarcinoma resulted in specific and efficient distribution in vitro, hence resulting in a 16.1- and 4.1-fold upregulation of of Apoptin in both HCC cells and normal hepatocytes. How- miR-155 target genes C/EBPbeta and FOXP3, respectively. ever, by day 5, in situ hepatocarcinoma showed significant Then, in in vivo test, intravenous injection of Lac-GLN con- regression while surrounding normal hepatocytes remained taining Cy3-anti-miR-155 into mice led to optimized accumu- unchanged [27]. lation of the anti-miR-155 in hepatocytes, resulting in enhanced expression of C/EBPbeta and FOXP3 [33]. ASGPR-directed nanoparticles used for delivering siRNA

Small interfering RNA (siRNA) and short hairpin RNA Epidermal growth factor receptor (shRNA) have emerged as potential therapy for cancer cur- rently. A promising liver-targeting siRNA (silence apolipo- Epidermal growth factor receptor (EGFR) is a member of the protein B) delivery system based on N-acetylgalactosamine protein kinase superfamily and a transmembrane glycoprotein functionalized mixed micellar nanoparticles (Gal-MNP/ receptor for epidermal growth factor (EGF). Binding of the siapoB) was described by Wang and his colleagues. Systemic receptor to ligand induces receptor dimerization and tyrosine administration of Gal-MNP/siapoB to BALB/c mice could autophosphorylation and then triggers uncontrolled cell pro- efficiently deliver siRNA to hepatocytes and successfully liferation, angiogenesis, and inhibition of apoptosis. Since silence the target gene apolipoprotein B expression at both elevated EGFR expression is prevalent in many cancers such transcriptional and protein levels. Furthermore, intravenous as lung cancer, colorectal cancer, and breast cancer and is injection of this nanoparticle did not induce the innate immune often related to poor prognosis, this receptor has been identi- response [28]. Akinc et al. also demonstrated that lipid nano- fied as a relevant target for treatment of solid tumors [34–37]. particles modified with N-acetylgalactosamine appear to be a High EGFR expression is also detected in most HCC patients highly effective strategy for the delivery of siRNA to the liver by immunohistochemistry [38–40], which make it an attrac- [29]. More recently, researchers in Alnylam Pharmaceuticals tive receptor to guide targeting treatment for HCC. Table 3 designed an advancing proprietary technology that conjugates summarizes the EGFR-guided nanocarriers for HCC-targeted a sugar molecule called “GalNAc” to the siRNA molecule to delivery. achieve targeted delivery of RNAi therapeutics to hepatocytes through uptake by ASGPR (Http://www.Alnylam.Com/our- Using ligand or peptide to recognize EGFR approach/delivery-of-rnai-therapeutics/. 2014.10.28) [30]. Alnylam’s Enhanced Stabilization Chemistry (ESC) As a ligand for EGFR, EGF was used to achieve receptor- GalNAc-conjugate technology enables subcutaneous dosing mediated endocytosis. Wolschek et al. described with increased potency, durability, and a wide therapeutic polyethylenimine (PEI)-based DNA complexes, shielded by index and is being employed in several of Alnylam’sgenetic covalent attachment of PEG, and made use of EGF as a ligand medicine programs, including programs in clinical develop- to deliver genes to EGFR-expressing hepatocellular carcino- ment. Besides, they also elucidated the mechanism of its ma cells (Huh7 and HepG2). Following intravenous injection targeted delivery to hepatocytes [30, 31]. of this nanoparticle into human HCC xenograft-bearing SCID mice, highly specific expression of transgene in HCC tumors ASGPR-directed nanoparticles used for delivering microRNA was exhibited [41]. Similar results were concluded by Ogris and his group [42]. Yao et al. also constructed a specific drug The discovery of microRNAs (miRNAs) has opened up new delivery system which could be used to package most hydro- sight on the post-transcriptional regulation of gene expression phobic drugs. This receptor-mediated drug delivery system in living organisms. Accumulating evidence suggested that was developed based on polyhydroxyalkanoates (PHA) and miRNAs could act as oncogenes or tumor suppressors by PHA granule-binding protein (PhaP). EGF was fused to PhaP 60 Tumor Biol. (2015) 36:55–67

Table 3 Summary of HCC-targeting nanoparticles aimed at growth factor receptor (EGFR)

Targeting moieties Types of nanocarriers reported Cargos carried by Cell lines employed References nanoparticles

Anti-EGFR Fab′ Liposome DOX or/and siRNA SMMC-7721, HepG2, Huh7, LM3, [48–50, Hep3B 52] Anti-EGFR antibody Polymer-lipid hybrid nanoparticle DOX SMMC-7721, HepG2, Huh7 [51] Anti-EGFR antibody (9B9 Polymeric nanoparticle DNA SMMC-7721, HepG2, Bel-7404 [111] mA) Anti-EGFR antibody Quantum dots and Fe(3) O(4) – SMMC-7721 [112] (12H23) nanoparticle Anti-EGFR antibody Polymeric nanoparticle Rhodamine B SMMC-7721 [47] EGF PEI-based DNA complex DNA HepG2, Huh7 [41] EGF Hydrophobic polymer Rhodamine B BEL-7402 [43] EGF Cationic PEI polymer DNA Huh7 [42] EGFR-specific peptide Polymeric nanoparticle DNA Huh7 [45, 46] (GE11) EGFR-specific peptide Polypeptide/DNA complex DNA Huh7, Bel-7402 [44] (GE7)

EGF epidermal growth factor, Fab′ fragments, siRNA small interfering RNA, PEI polyethylenimine to pull the nanoparticles to the target cells. This nanoparticle Using monoclonal antibody to recognize EGFR could be absorbed by HCC cell (BEL-7402) in vitro and in vivo. When Rhodamine B isothiocyanate (RBITC) was Several studies also indicated that EGFR monoclonal used as a delivery model drug, the nanoparticle system was antibody-conjugated nanoparticles have great potential appli- clearly visible in the targeted cells and organs under fluores- cations in liver cancer therapy [47–49]. Targeted liposome– cence microscopy [43]. polycation–DNA (LPD complex) conjugated with anti-EGFR Except EGF, peptide is another choice selected for EGFR Fab′ was constructed for siRNA delivery. These modified recognition. Lee et al. used an oligopeptide which recognizes liposomes could efficiently bind and deliver to SMMC-7721 EGFR to improve delivery efficiency of wild-type p53 gene hepatoma cells, resulting in reinforced gene silencing com- transfer system in HCC. They found that the gene transfer pared to untargeted LPD [50]. Intravenous injection of the efficiency in Huh7 and HepG2 cells peaked at day 5 at about modified LPD into mice which bear orthotopic HCC resulted 50 %, while the normal liver cell line (L02) with weak EGFR in higher accumulation and more potent gene silencing activ- expression showed less than 1 % transfer efficiency through- ity in HCC compared with non-targeted control [48]. out. In addition, using this vector system, transfection of wild- Polymer-lipid hybrid nanoparticles conjugated with anti- type p53 into Huh-7 cells that had mutated p53 resulted in EGFR and loaded with DOX (PLNP-Mal-EGFR) also increased p53 protein expression and significant growth inhi- showed higher accumulation in SMMC-7721 and could en- bition of cancer cells. Similar phenomenon was found in nude hance anti-tumor function against HCC in vivo compared with mice bearing HCC xenograft [44]. free DOX and non-targeted nanoparticles [51]. Targeted LPD Klutz and his group constructed two kinds of nano-gene conjugated with anti-EGFR Fab′, co-delivering DOX and vectors to target HCC. The first one named linear ribonucleotide reductase M2 (RRM2) siRNA (DOX-RRM2- polyethylenimine (LPEI)-PEG-GE11 was based on LPEI, TLPD), was further produced for the treatment of HCC. Both PEG, and used synthetic peptide GE11 as an EGFR-specific in vitro and in vivo experiments indicated that DOX-RRM2- ligand to deliver a sodium iodide symporter (NIS)-expressing TLPD could efficiently and specifically deliver DOX and plasmid to Huh7 cells [45]. Another biodegradable RRM2 siRNA to HCC cells, and enhanced therapeutic effects nanocarrier used pseudodendritic oligoamines (G2-HD-OEI) were shown compared with single-drug loaded or non- for systemic NIS gene delivery [46]. They transfected NIS targeted controls [49]. cDNA into HCC cells using the two kinds of vectors to It is worth mentioning that anti-EGFR immunoliposomes evaluate the efficiency and tumor specificity and found that loaded with DOX (anti-EGFR ILs-DOX) have already been using G2-HD-OEI resulted in a 44-fold increase in iodide successfully applied for phase 1 clinical study [52]. The safety, uptake in vitro as compared with the control group, while a tolerability, and efficacy of this pharmacon were evaluated in 22-fold increase using LPEI-PEG-GE11 than using the con- 26 patients with EGFR-overexpressing solid tumors, includ- trol polyplexes without EGFR-targeting [46]. ing one HCC patient [52]. Up to 50 mg doxorubicin per m2 Tumor Biol. (2015) 36:55–67 61 was well tolerated using this anti-EGFR ILs-DOX, and further which conjugated with different amounts of transferrin was assessment of this nanoparticle at the same dose in phase 2 transfected into HCC cells (Huh7 and SK-Hep-1), and the trials had been warranted [52]. transfection efficiency of p53 was increasing coupling with the increasing transferrin in a dose-dependent manner. Then, in vivo anti-cancer experiments were done on VX2 rabbit hepatocarcinoma model. Interventional injection of Transferrin receptor transferrin-p53-Lipofectamine complex into hepatic artery could enhance the gene transfer efficiency and improved Transferrin receptor (TfR) has also captured much attention in animal survival [61]. Han et al. selected peptide T7 as a tumor targeting. TfR, usually present as a homodimer, is a homing device to guide a co-delivery system to target HCC. glycoprotein which plays important roles in iron regulation This system was developed to co-deliver DOX and therapeu- and cell growth. Iron-bound transferrin (Tf) exhibits high tic gene encoding human tumor necrosis factor-related apo- affinity for TfR. The combination of the ligand with the ptosis-inducing ligand (pORF-hTRAIL). In vitro experiment receptor initiates the endocytosis by which iron-bound-TfR using Bel-7402 cells indicated that T7-modified co-delivery complex gets internalized. When in acidic environment, iron system exhibited higher efficiency in cellular uptake and gene is released and receptors recycle back to the surface [53]. TfR expression than unmodified ones, resulting in more apoptosis is reported to be overexpressed in various cancer cells includ- and growth inhibition of tumor cells. In vivo experiment using ing breast, bladder, lung adenocarcinoma, and especially HCC mice model bearing Bel-7402 xenografts showed more accu- [53–55]. TfR ligand-conjugated nanoparticles had showed mulation of nanoparticles in tumor region, and lower doses of promising prospects to target HCC, for a number of studies co-delivery system played more effective roles in tumor inhi- revealed that these ligands could facilitate the accumulation of bition comparable to free DOX [57]. modified nanoparticles in liver cancer cells and enhance drug efficacy [56–59]. Date was summarized in Table 4. Golla et al. chose apotransferrin and lactoferrin to modify Folate receptor DOX (apodoxonano or lactodoxonano), and these nanoparticles were applied intravenously in rat HCC model induced by Folate receptor (FR) is a secreted protein that either anchors to diethylnitrosamine (DENA). These modified agents not only membrane via a glycosyl-phosphatidylinositol linkage or ex- minimized the cardiotoxicity of DOX but also enhanced its ists as a soluble form. FRs exhibit very limited distribution in bioavailability and efficacy in a target-specific mode [59]. normal tissues but revealed overexpression on malignant cells. Oral administration of apodoxonano and lactodoxonano also For example, lung, gastric, colorectal carcinoma, ovarian, and obtained improved efficacy, bioavailability, and safety com- breast cancer all exhibit high FR expression [62]. FRs could pared with unmodified DOX [60]. Luo et al. also utilized bind folate and its reduced derivatives and transport 5- human serum transferrin (HTf) coupling cisplatin to minimize methyltetrahydrofolate into cells. The ligand folate is a type the side effects of the latter. They found that each HTf could of vitamin B which is a cofactor required for the synthesis of bind over 22 cisplatins, and the apo-HTf-cisplatin polymer purines and pyrimidines. Folate has been extensively used as could specifically deliver cisplatin to HepG2 cells in vitro and homing ligand in cancer-targeting treatment due to its small then stimulate apoptosis [56]. size, non-toxic, non-immunogenic nature, price cheapness, Likewise, transferrin receptor was used to facilitate the and easy conjugation to nanocarriers [62]. In fact, the employ- therapeutic gene transfection in HCC. p53-Lipofectamine ment of folate to guide nanomaterials which carried drugs or

Table 4 Summary of the transferrin receptor (TfR)-based nanoparticles to target HCC

Targeting moieties Types of nanocarriers reported Cargos carried by nanoparticles Cell lines or animal models employed References

Apotransferrin and lactoferrin Protein nanoparticle DOX DENA-treated rats [59, 60] Tf Solid lipid nanoparticle DNA HepG2 [83] Tf Au-HMP – J5 [58] TfR-specific peptide Polyamidoamine dendrimer DOX Bel-7402 [57] Tf liposome DNA Huh7, SK-Hep-1 [61] Tf QD – HepG2 [113] Tf Protein nanoparticle Cisplatin HepG2, BEL-7402 [56] Tf liposome DNA VX2 rabbit hepatocarcinoma [114]

DOX doxorubicin, Tf Transferrin, TfR transferrin receptor, QD quantum dot, Au-HMP sodium hexametaphosphate (HMP)-stabilized gold nanoparticles 62 Tumor Biol. (2015) 36:55–67 nucleic acid (e.g., DOX, gemcitabine, docetaxel, survivin the nanoparticle administration [66]. Compared to non- gene, BCL-2 siRNA) has led to a selective accumulation into specific delivery, the folate-targeted delivery of BCL-2 siRNA HCC cells [63–67]. Table 5 summarizes the folate-directed induced more significant gene suppression, resulted in more nanoparticles for HCC-targeted delivery of therapeutics. cancer cell apoptosis, and in addition improved the therapeutic Polymeric micelles which loaded with DOX and attached efficacy of the DOX [66]. with folate showed high intake by Bel-7402 cells [68]. In vivo tumor targeting efficacy was evaluated in rats and rabbits with liver cancer. In comparison with free DOX, folate-conjugated Integrin DOX showed enhanced anti-cancer efficacy and could specifically target FR-expressing tumors, which resulted in inten- Integrins are a large family of heterodimeric transmembrane sive bioavailability of DOX [63]. Furthermore, the relative glycoproteins consisting of α and αβ subunits. In total, 24 tumor size of the folate-conjugated DOX group was decreased different subtypes of integrins have been discovered to date, two- and four-fold compared with the free DOX group in the and almost half of them bind to various extracellular matrix rat and rabbit models, respectively [63]. In addition, the re- (ECM) proteins through the tripeptide motif Arg-Gly-Asp lease of DOX from the nanoparticles was reported to be PH- (RGD) and play important roles in cell signaling, cell–cell sensitive. When at neutral or alkalescent pH, the release was adhesion, cell matrix adhesion, and apoptosis [2]. Some sub- slow and sustained, while in the weak acidic environment, the types of integrins are described to be overexpression on cancer release was much faster [67–69]. cells or neovascular endothelial cells and play pivotal roles in Co-delivery systems conjugated with folate were also de- tumor angiogenesis and metastasis. For example, the αvβ3 signed to achieve magnifying treatment effect in HCC [65, integrin is highly expressed on melanoma, glioblastoma, ovar- 66]. Folate-modified multifunctional nanoassembly (FNA) ian, breast, and prostate tumor cells [62]. Alterant expression loading both docetaxel and iSur-pDNA (survivin) was report- and distribution of integrins have also been reported in HCC ed to be safer and more efficient strategy for the treatment of [70]. Among all integrins with altered expression, the overex- advanced HCC [65]. Cytotoxicity of FNAs against BEL-7402 pression of integrin α6β1 in HCC was further highlighted by was much higher than either docetaxel or non-docetaxel FNAs several studies [70]. (nFNAs) loading only iSur-pDNA and was also superior to In vitro and in vivo studies suggested that RGD-modified the combined treatment with free docetaxel and nFNAs [65]. nanoparticles are promising drug carriers for HCC active In vivo study on mouse HCC xenograft model using the FNAs targeting therapy (Table 5). Cellular uptake analysis indicated also showed better anti-tumor efficacy and low systemic tox- that RGD-modified micelles could markedly increase the icity [65]. Likewise, co-delivery of BCL-2 siRNA and DOX DOX concentration in BEL-7402 cells but not in human was constructed to achieve synergistic effect of RNA interfer- cervical cancer cell HeLa. In addition, DOX-loaded RGD- ence and anti-tumor drug [66]. High transfection efficiency modified micelles could specifically enhance the cytotoxicity and ideally controlled release of agents were exhibited after against BEL-7402 compared with naked DOX and DOX-

Table 5 Summary of nanoparticles targeting folate receptor, integrin, and CD44 expressed on hepatocellular carcinoma cells

Receptors Targeting moieties Types of nanocarriers reported Cargos carried by nanoparticles Cell lines employed References

Folate receptor Folate Polymeric micelle DOX Bel-7402 [68, 69] Folate Polymeric nanoparticle DOX Hep3B [63] Folate FNA Docetaxel and DNA BEL-7402 [65] Folate Amphiphilic copolymer of PCL and PEI siRNA and DOX Bel-7402 [66] Folate Micellar nanoparticles PDTC and DOX HepG2 [67] Folate Gold nanorod – HepG2 [115] Folate Magnetite nanoparticle Gemcitabine PLC/PRF/5 [64] Integrin RGD peptide Polymeric micelle DOX BEL-7402 [71] RGD peptide – DOX MH134 [72] RGD peptide Superparamagnetic iron oxide nanoparticle siRNA Bel-7402 [73] CD44 HA HGA nanoparticle PTX HepG2 [76] Anti-CD44 antibody Liposome DOX HepG2 [77]

DOX doxorubicin, FNA folate-modified multifunctional nanoassembly, PCL poly(epsilon-caprolactone), PEI poly(ethylene imine), siRNA small interfering RNA, PDTC pyrrolidine dithiocarbamate, RGD tripeptide motif Arg-Gly-Asp, DOX doxorubicin, siRNA small interfering RNA, PTX paclitaxel, HA hyaluronic acid, HGA glycyrrhetinic acid-graft-hyaluronic acid conjugate Tumor Biol. (2015) 36:55–67 63 loaded nanoparticles without RGD ligand [71]. A better anti- simplex virus-truncated thymidine kinase (HSV-ttk) and tumor effect was also observed in orthotopic mouse hepatoma renilla luciferase (Rluc) and red fluorescent protein (RFP). model. Intravenous administration to mice with the DOX- Then, adopting optical bioluminescence imaging of Fluc, they RGD nanoparticles could inhibit the growth of hepatoma could monitor the growth status of tumor and track specific more effectively than free DOX (mean tumor volumes 24 targeting of the liposomal nanoparticle by Rluc imaging. vs. 67 mm3,respectively)[72]. Integrin-targeted vectors were also reported to deliver siRNA to HCC cells. The RGD-modified nanoparticles Others showed higher efficiency in transferring survivin siRNA into Bel-7402 cells. More significant gene suppression at both the Except receptors mentioned above, many other receptors have survivin mRNA and protein expression levels was exhibited also been explored for developing HCC-targeting treatment, compared with a non-targeted delivery system, and as a result, such as lipoprotein (low-density lipoprotein (LDL)) receptors the tumor growth in the nude mice hepatoma model was more [78–82], mannose receptor (MR) [83–85], glypican-3 (GPC3) significantly inhibited [73]. [86], 5-HT receptor [87], somatostatin receptor (SSTR) [88], and vascular endothelial growth factor receptor (VEGFR) [89]. For instance, Chang et al. developed hematoporphyrin CD44 (HP)-modified DOX-loaded nanoparticles (HP-NPs) to improve the therapeutic effect of photodynamic therapy (PDT) in In recent years, studies have demonstrated that cancer stem treating liver cancer. HP used here is not only a ligand for LDL cells (CSCs) are related to cell growth and metastasis in receptors on the HCC cells but also a well-known photosen- various cancers, including HCC. Therapeutic targeting CSCs sitizer for PDT [78]. In addition, GA [90–93] and HCC- may provide a novel strategy in liver cancer treatment. CD44, binding peptide SP94 [94–98] modified nanomaterial as well whose principle ligand is hyaluronan (HA), is one of the draw much attention for their affinity toward HCC cells characterized marker for CSCs [74]. The interaction between through unknown target molecules expressed by HCC cells CD44 and HA could induce various intracellular signaling but not by normal tissues. pathways. In addition, HA is a biodegradable, biocompatible, Moreover, advances in nanotechnology and personalized non-toxic, non-immunogenic, and non-inflammatory linear medicine have already made nanoparticle-based theranostics a polysaccharide, which has been used for various medical research hotspot, and theranostic agents have also been devel- applications such as arthritis treatment, ocular surgery, tissue oped to target HCC [99, 100]. Liu et al. developed a multi- augmentation, and so on [75].ThesepropertiesmakeCD44 functional pH-sensitive polymeric nanoparticle system for and its ligand the promising constituent of nanocarriers in simultaneous tumor magnetic resonance imaging (MRI) and targeted drug delivery system (Table 5). therapy. The anti-HCC drug sorafenib was encapsulated in- Zhang et al. [76] designed a glycyrrhetinic acid-graft- side the nanoparticles, and the biotinylated VEGFR antibodies hyaluronic acid (HGA) nanoparticle to load PTX to aim at were linked to the surface of nanoparticles to form the target HCC cells. These HGA nanoparticles showed high drug- pH-sensitive theranostic nanoparticles (TPTN). The in vitro loading capacity. Besides, after treated with PTX-loaded and in vivo studies showed that this TPTN possessed dual- HGA nanoparticles, more significant cytotoxicity to HepG2 loading drugs and imaging agents, active targeting, and pH- cells than B16F10 cells was exhibited due to the simultaneous triggered drug release properties in one platform with good overexpression of HA and glycyrrhizic acid (GA) receptors on biocompatibility [99]. HepG2 cells. In vivo imaging analysis revealed that the fluorescence signals of DiR-labeled HGA nanoparticles in tumor and liver were 2.88- and 1.83-fold stronger than that of the Conclusion control, respectively. Wang et al. [77] designed a CD44 antibody-mediated lipo- Non-selective cytotoxicity and side effects are pivotal barriers for somal nanoparticle loaded with triple fusion (TF) plasmids to chemotherapy drug used for advanced HCC. Drug delivery monitor and evaluate targeting efficacy by non-invasive mo- systems based on nanomaterials surface functionalized with lecular imaging. They injected HepG2 cells into the liver of homing devices provide novel opportunities to overcome the NOD/SCID mice to model in situ liver cancer. These HepG2 above limitations. The ideal receptor chosen to recognize HCC- cells carried a double fusion (DF) reporter gene consisting of targeted delivery system should be almost uniquely expressed on firefly luciferase (Fluc) and green fluorescent protein (GFP) liver cancer cells. Among receptors mentioned above, ASGPR is [77]. Then, they constructed an anti-CD44 antibody-mediated expressed abundantly in liver tissue; however, no study revealed liposomal nanoparticle to specifically target HCC. This nano- its differential expression between normal hepatocytes and hep- particle was loaded with a TF gene containing the herpes atoma cells. Folate and hyaluronan, which recognize folate 64 Tumor Biol. (2015) 36:55–67 receptor and CD44, respectively, are non-protein substances and 11. Zhang X, Yu P, Han Y, Li Y, Li C. Hydrotropic polymeric mixed hence showed favorable biocompatibility and non-immunoge- micelles based on functional hyperbranched polyglycerol copolymers as hepatoma-targeting drug delivery system. J Pharm Sci. nicity. But the expression level of FR in HCC is controversial 2013;102:145–53. [101], and more work is needed for further confirmation. In 12. 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