Hepatocellular carcinoma (HCC) is the predominant liver malignancy that accounts for over 80% of liver cancer types. According to global cancer statistics, HCC is one of the most commonly diagnosed cancers and a third leading cause of death worldwide, causing more than 800,000 new deaths in 2020. Patients with HCC barely present apparent symptoms, which leads to diagnosis at late stages and limited therapeutic options are available. HCC usually arises from chronic liver diseases with various etiologies, including viral infection of hepatitis C and hepatitis B, non-alcoholic steatohepatitis, and alcoholic liver disease[4,5]. In Asia and Africa, other risk factors such as aflatoxin B1, the potent mycotoxin taken in through food contamination, also contribute to the generation of HCC[6,7]. On the other hand, HCC development is a cumulative result of epigenetic and genetic factors, which ultimately lead to altered molecular regulation, such as elevated expression of growth factor receptors.
Currently, the treatment strategy for HCC is formulated depending on the patient’s status, liver function, and tumor stage. For patients with early-stage HCC, liver transplantation, local ablative treatment, and surgical resection were widely applied. As for patients diagnosed at late stages, trans-arterial chemoembolization and systemic therapies are the only available treatment options. Systematic treatment with chemotherapeutic reagents such as mitomycin C, doxorubicin, or cisplatin are used. Nevertheless, the poor therapeutic responses and severe side effects caused by non-specific targeting generate multiple drug resistance and a high recurrence rate significantly restrain the application of chemotherapy. On the other hand, patients with poor liver status and other contraindications are not suitable for surgical intervention.
Compared to conventional chemotherapy, targeted therapy that inhibits specific cell regulatory signaling has been demonstrated as a promising approach for HCC management. For example, sorafenib is the approved first-line therapeutic agent for HCC and suppresses HCC progression by inhibiting the function of multiple kinases such as RAF/MEK, VEGFR, and PDGFR signaling[14,15]. It is worth noting that nanotechnology facilitates the precise delivery and accumulation of anticancer into tumor sites through encapsulation with the help of nanomaterials.
Nanomaterials, such as polymeric, metallic, and lipid-based nanoparticles, have attracted remarkable attention in cancer therapy due to their significant advantages as a drug delivery system; this is because of their smaller size, large surface area, and high biosafety and biocompatibility. These nanomaterials achieve selective and effective targeting of cancer cells by functionalization and surface modification, enhancing their ability to encapsulate drugs and diagnostic agents and bind to specific molecules on the cancer cell surface. By conjugating ligands or markers that are highly affined to specific complementary receptors that are highly expressed or solely expressed on the cancer cell membrane, nanomaterials could precisely deliver therapeutic agents to tumor sites and exhibit low toxicity to normal tissues. This review summarized the nanomaterials that target the cancer cell surface for HCC treatment (Table 1).
|Nanomaterial Composition||Type of Targeting||Components That Are Moiety/Stimuli-Responsive Are Being Targeted||Remarks||Ref.|
|Gal-modified selenium NPs||Decorating moieties||Gal||Higher accumulation of DOX in HCC cells and cell apoptosis|||
|Gal-modified noisome||Decorating moieties||Gal/pH sensitive||Delivery of tanshinone IIA to HCC cells|||
|β-d-galactoside-decorated lipid/calcium/phosphate NPs||Decorating moieties||β-d-galactoside||Delivery of VEGF siRNA to HCC cells and suppressed angiogenesis in tumor microenvironment|||
|Gal-decorated TPGS/polydopamine (PDA) material||Decorating moieties||Gal/pH sensitive/photothermal therapy||Accumulation of DOX and NO in HCC cells and reverse of MDR|||
|GPC antibody GC33||IgG||GPC3 mAb||Antibody-dependent cellular cytotoxicity||[47,48]|
|DNA aptamer-incorporated material||Decorating moieties||GPC3||Specific targeting of GPC3-positive HCC cells and delivery of gemcitabine to impede tumor growth|||
|GPC3 mAb-conjugated ADCs||Decorating moieties||GPC3 mAb||Effective targeting of GPC3-positive HCC cells, drug delivery, and synergetic effect with gemcitabine in HCC treatment|||
|GPC3-binding peptide based nanoprobe||Decorating moieties||GPC3 binding peptide||NIR-II imaging, photoacoustic (PA) imaging-guided photothermal therapy (PTT) for HCC|||
|Transferrin-coated polycarbonate-based polymersome||Decorating moieties||Transferrin||Accumulation of DOX in HCC cells, and cytotoxicity|||
|Transferrin-conjugated nanoparticle with albumin shell and polyvinyl alcohol core||Decorating moieties||Transferrin||Delivery of sorafenib and DOX to induce specific death of HCC cells|||
|Transferrin-coated liposomes||Decorating moieties||Transferrin||Delivery of microRNA-221 inhibitors to promote HCC cell apoptosis||[62,63]|
|Fe3O4-ZnO nanomaterial||Decorating moieties||Transferrin receptor antibody (TFR Ab)||Facilitate chemotherapeutic efficiency of DOX and simultaneously boost sensitivity to radiotherapy|||
|Glycyrrhizin (GL)-modified O-Carboxymethyl chitosan NPs||Decorating moieties||GL||Drug accumulation at target site was 10-fold higher than non-modified nanoparticles|||
|Glycyrrhetinic acid (GA)-modified chitosan micelle||Decorating moieties||GA/pH-responsive||Accumulation and pH-responsive of DOX in HCC cells to kill tumor cells|||
|GA-modified phthalocyanine (SiPC)||Decorating moieties||GA||Effectively accumulated in HepG2 tumor and perform|||
|GA-reinforced thiolated polymer||Decorating moieties||GA||Accumulation of 5-fluorouracil in HCC cells and effective suppression of carbon tetrachloride (CCl4)-induced HCC tumor|||
|RGD-modified PHBV nanoparticle||Cell penetrating peptide||Integrin/pH-sensitive||Release and accumulation of paclitaxel in HCC cells to induce cell death|||
|R6-conjugated LRVG polypeptide||Cell penetrating peptide||Intestinal epithelial cells||Promote absorption of Tyroserleutide in the intestinal system|||
|SP94-PS-DOX nanoparticle||Decorating moieties||SP94||Specific targeting and cytotoxicity toward SMMC-7721 xenograft model|||
|SP94-functionalized PLGA-PEG-PEI nanoparticles||Decorating moieties||SP94||Specific delivery of prodrugs and thymidine kinase (TK)-p53-nitroreductase therapeutic gene to HCC|||
2 TARGETING LIGANDS FOR HCC THERAPY
Targeting ligands for nanomaterials modification could be peptides, proteins, sugar residues, aptamers, monoclonal antibodies, and other molecules that present specific affinity to target sites. Here, we introduced several representative ligands widely studied in HCC research.
2.1 Protein Receptors for HCC Targeting
2.1.1 Asialoglycoprotein Receptor (ASGPR)
ASGPR is a transmembrane protein abundant in hepatocytes and is explicitly expressed on the basolateral and sinusoidal hepatocellular surface[20,21]. Whereas under pathological states such as inflammation, ASGPR shifts to the canalicular membrane of liver cells. In liver tissues with cirrhosis, the expression of ASGPR and serum level of asialoglycoprotein are notably elevated. Besides, the ASGPR level is enhanced in patients with both early- and late-stage HCC[24,25]. Therefore, ASGPR could be an ideal therapeutic target for HCC treatment. In normal hepatocytes, ASGPR could uptake and clear desialylated proteins in serum[26,27]. On the other hand, ASGPR participates in the interaction and internalization of extracellular glycoproteins with N-acetyl-galactosamine, galactose (Gal), or lactose residues, which makes ASGPR an ideal target for anti-HCC drug delivery and Gal a suitable ligand for decoration of HCC targeting nanomaterials[28,29].
Various studies have indicated the utilization of the ASGPR-Gal interaction pair in selective hepatic delivery[30,31]. For example, Xia and their colleagues encapsulated doxorubicin (DOX) into a Gal-modified selenium nanoparticle to form a GA-Se@DOX NPS. These nanoparticles are effectively taken up by HCC HepG2 cells through the endocytosis pathway and induce significant cell apoptosis with no apparent damage to major organs. A pH-sensitive noisome modified with Gal remarkably enhanced the selectivity and efficacy of tanshinone IIA delivery to HCC cells, other than other cancer cell types, and this process largely depended on the level of Gal. Huang et al. designed and screened 8 galactoside derivatives to modify a lipid/calcium/phosphate nanoparticle and determined that phenyl β-d-galactoside-decorated nanoparticles presented a superior delivery ability of VEGF siRNA, which subsequently suppressed angiogenesis and improved the tumor microenvironment in an orthotopic HCC mouse model. A multiple functional drug delivery system that loaded nitric oxide (NO) donor and DOX into a Gal-decorated TPGS/PDA shell was designed to overcome multiple-drug resistance (MDR) of HCC cells. This NO-DOX@PDA-TPGS-Gal targets HCC cells through Gal, released DOX and NO under photothermal therapy, which, with TPGS, impeded the P-glycoprotein-related efflux of DOX, hence reversing MDR in HCC cells.
2.1.2 Glypican-3 (GPC3)
GPC3, also known as a heparan-sulfate proteoglycan, is a member of the glypican family expressed in the membrane and plasma of hepatocytes[36,37]. The GPC3 protein consists of a heparan sulfate chain, glycosyl phosphatidyl inositol (GPI), and a core protein and is attached to the cell membrane through the GPI anchor[38,39]. Studies have indicated that successful GPC3 is overexpressed in HCC cells but rarely detected in normal and cirrhotic hepatocytes[40,41]. Hsu et al. reported that GPC3 is overexpressed in over 70% of HCC cells and is correlated with poor prognosis and modulates proliferation, metastasis, oncogenesis, and morphogenesis of HCC cells. GPC3 interacts with Wnt protein and activates the canonical Wnt/β-catenin signaling transduction, which significantly participates in tumorigenesis[43,44]. Above mentioned findings suggest GPC3 protein as a specific biomarker and target diagnosis and treatment of HCC[45,46].
The first reagent targeting GPC3 on HCC cells is a humanized mouse anti-GPC3 monoclonal antibody (mAb) (GC33) that could notably and specifically suppress the growth of GPC3-positive HCC xenograft[47,48]. A gemcitabine-incorporated DNA aptamer that targets GPC3 (G12msi) could specifically bind to and internalized by GPC3-positive HCC cells, significantly suppressing HCC tumor growth. Fu et al. proposed the GPC3 antibody as an antibody-drug conjugates (ADCs) component. They screened large drug libraries to construct two GPC3-specific ADCs, which exhibited a synergetic effect with gemcitabine in HCC treatment both in vitro and in vivo. A pH-sensitive self-assembling GPC3-binding peptide-based nanoprobe was developed for NIR-II imaging and PA imaging-guided PTT and sonodynamic therapy of HCC.
2.1.3 Transferrin Receptor (TFR)
TFR, also known as CD71, is a glycoprotein and the receptor of transferrin, an iron-transporting glycoprotein[52,53]. Transferrin acts as a ligand of TFR and exhibits high affinity, and their combination causes endocytosis of the iron-bound TFR complex, which is followed by the release of iron molecules and recycling of TFR receptors to the cell surface. The liver is a vital organ closely correlated with iron storage and iron metabolism. Abnormal iron metabolism and overexpression of TFR1 mRNA have been observed in HCC cells [55,56]. Moreover, as an essential regulator of cellular iron uptake, TFR1 is regarded as a promising target for efficient HCC therapy[57,58]. Based on the high affinity between transferrin and TFR, transferrin has been widely applied as a ligand for the decoration of nanomaterial for HCC therapy.
A transferrin-coated polycarbonate-based polymersome exhibited specific targeting of TFR-positive SMMC-7221 cells and released DOX to perform cytotoxicity with low toxicity on normal organs. The co-delivery of sorafenib, the first-line medicine for HCC, and DOX by transferrin-conjugated nanoparticles with the albumin shell and polyvinyl alcohol core promoted drug uptake by HepG2 cells, consequently inducing cell death. Besides, transferrin-coated liposomes were used to encapsulate oligonucleotides, such as microRNA-221 inhibitors, to promote HCC cell apoptosis, compared with the non-targeted liposomes[62,63]. Other ligands for TFR targeting have also been widely studied. A Fe3O4@ZnO nanomaterial modified with TFR Ab and loaded with DOX was composited and successfully delivered DOX into HCC cells. This nanocomposite could notably facilitate the chemotherapeutic efficiency of DOX and simultaneously boost sensitivity to radiotherapy.
2.1.4 Glycyrrhetinic Acid Receptor (GAR)
The GAR was first found on the sinusoidal surface of mammalian hepatocytes and interacted with GA and GL with different affinity[65,66]. Evidence has suggested that GAR is one of the most suitable receptors for HCC treatment due to its specificity, biosafety, and pharmacological potential. GL-modified nanoparticles were internalized preferentially by SMMC-7721 cells, with a ratio 10-times higher than that of non-modified nanoparticles. Therefore, GA-modified or GL-modified liposomes, nanoparticles, and micelles modified with GA were considered ideal drug delivery materials for HCC therapy.
A pH-responsive micelle based on GA-modified chitosan-conjugated DOX successfully targets HepG2 cells and releases DOX in the low pH environment. These micelles presented higher toxicity in HepG2 cells than in normal HUVEC cells. Kim et al. designed a novel photosensitizer composed of silicon SiPC and a GA targeting moiety for photodynamic therapy of liver cancer. This material was confirmed to accumulate in HepG2 tumors in a mouse model effectively and had 60 times higher growth inhibitory effects than the control material. A thiolated polymer nanocarrier reinforced with GA encapsulated and specifically delivered 5-fluorouracil to suppress the tumor growth in a CCl4-induced in vivo HCC model.
2.2 Cell-penetrating Peptide-based Nanomaterials for HCC Treatment
RGD is a cell-penetrating cyclic peptide composed of Arg-Gly-Asp amino acids and binds to the cell surface integrin receptors, which are abundant in liver cells[73,74]. RGD was first used to adorn nanoparticles for medication delivery into melanoma cells and other cancer cell types. Later studies discovered that when RGD-conjugated nanomaterials were used on liver cancer cells, the internalization ratio of nanomaterials was 500-fold of the non-conjugated materials, as was manifested by the measured fluorescence intensity. The incubation with excess free RGD blocked the function of RGD-conjugated nanomaterials on liver cancer cells. Furthermore, these RGD-conjugated nanomaterials showed a higher affinity to liver cancer cells than cancer cells originating from other organs, including the colon and cervix. Recently, an ultrasound molecular material that loaded hydroxycamptothecin in lipid nanoparticles and decorated with RGD was used for theranostics of HCC, simultaneously exhibiting photoacoustic/ultrasound imaging and anti-proliferative effects. Wu et al. designed a pH-sensitive RGD-modified PHBV nanoparticle loaded with paclitaxel, which targets and responds to the low pH at the HCC tumor site to precisely release paclitaxel, thus, showing remarkable anticancer effect in vivo.
R6 is another cell-penetrating peptide conjugated with LRVG, a polypeptide that targets intestinal epithelial cells, successfully delivered tyroserleutide, an anti-hepatoma drug, to yield a nanomaterial that could be orally taken and absorbed into the intestinal system. This nanomaterial is then released in HCC cells and affects the endocytic pathway and mitochondria function to suppress tumor growth.
Other peptides such as SP94, FFW, Tv1, and HCC79 were also extensively studied in HCC targeting. For example, SP94-modified polymersome doxorubicin hydrochloride (SP94-PS-DOX) performed much better cytotoxicity toward the SMMC-7721 xenograft model than the non-modified PS nanoparticles. PLGA-PEG-PEI nanoparticles functionalized by SP94 exhibited the excellent delivery ability of prodrugs and TK-p53-nitroreductase therapeutic gene to HCC in vivo, which resulted in remarkable expression of these genes’ subsequent sensitivity to prodrugs. This system realized a synergic suppression of HCC growth.
The research and exploration of advanced nanomedicine have provided a powerful approach for specific targeting in HCC therapy compared with traditional methods. Nanomaterials could easily overcome the flaws of a chemical drug, such as poor solubility, rapid clearance, limited permeability, low stability, and deficient tumor-targeting ability. The chemical drug could be easily conjugated with the nanomaterials or encapsulated to overcome the problems. Nanomaterials must be perfected for precision therapy; studies have focused on selecting more suitable ligands for specific targets on tumor cells and elevating drug accumulation at tumor sites. In this review, we introduced several representative cell surface receptors of HCC cells, including ASGPR, GPC3, TFR, and GAR, and their ligands that are widely applied in the decoration of nanomaterials. Other receptors overexpressed on HCC cells, such as a folic acid receptor, somatostatin receptors, and CD44, are also implied in developing materials for HCC-specific targeting[87,88]. We also summarized several peptide ligands, such as RGD, R6, and SP94, for HCC targeting (Figure 1). Thus, various studies have suggested the advantages of nanomaterials with such targeting ligands for anti-HCC therapy.