Theranostics and Precision Medicine for the Management of Hepatocellular Carcinoma, Volume 3: Translational and Clinical Outcomes

Theranostics and Precision Medicine for the Management of Hepatocellular Carcinoma: Translational and Clinical Outcomes, Volume Three provides comprehensive information about ongoing research and clinical data on liver cancer. The book presents detailed descriptions about diagnostics and therapeutic options for easy understanding, with a focus on precision medicine approaches to improve treatment outcomes. This updated volume discusses topics such as clinical and safety assessment of HCC patients, liver transplantation as a therapeutic option, immunotherapy interventions, and image-based surveillance. In addition, it discusses immunohistology of HCC-enabled precision medicine and artificial intelligence for hepatocellular carcinomas.

This is a valuable resource for cancer researchers, oncologists, graduate students, hepathologists and members of biomedical research who need to understand more about liver cancer to apply in their research work or clinical setting.

• Provides best practices for the management of hepatocellular carcinoma in the clinical setting
• Discusses emerging treatment approaches based on artificial intelligence and precision medicine tools and techniques
• Brings updated information on international clinical trials for the treatment of HCC

• Language: English
• Publisher: Academic Press
• Release date: Apr 15, 2022
• ISBN: 9780323992848

Book preview

Preface

Ganji Purnachandra Nagaraju and Sarfraz Ahmad

Hepatocellular carcinoma (HCC) is a major form of primary liver cancer that has emerged as one of the most frequently diagnosed human malignancies worldwide. It predominantly targets individuals with underlying conditions, such as cirrhosis and hepatitis B and hepatitis C infections. Despite the widespread occurrences of HCC, its underlying mechanisms that lead to tumor progression are still unclear. In contrast to various other cancers, systemic therapies for HCC, including chemotherapy and radiotherapy, are not effective enough. Currently, the only viable options for managing advanced HCC are surgical resection and transplantation. Therefore it is crucial to investigate pathways and factors that lead to HCC tumor suppression, thereby advancing novel therapeutic and management options for HCC.

In the current series, we have three different volumes. Volume 1 discusses the biology, pathophysiology, and progression of HCC. Volume 2 discusses multiple signaling and molecular mechanisms/targets associated with HCC progression and metastasis, including renowned and novel biomarkers, as well as diagnostic approaches for advanced HCC. Volume 3 includes a detailed discussion about the translational and clinical outcomes as it relates to the application of nanoparticles, small molecules, phytochemicals, and precision medicine for HCC treatment and management.

Nanoparticles are essential players in contemporary treatment strategies as its diverse application spans from magnetic resonance imaging contrast enhancers to drug delivery agents into the tumor. Chapters 1–3 discuss the elaborate role of nanoparticles and small molecules in HCC theranostic applications. Next, the application of phytochemicals, including curcumin and resveratrol, in liver neoplasm is discussed. Further, nanoparticles formulated from phytochemicals or phyto-nano-formulations and their applicability in HCC treatment are elucidated. Multiple immune checkpoint inhibitors for HCC are outlined.

Further, this volume examines recent developments in the field of immunotherapy for managing HCC with updated clinical trials. It scrutinizes precision medicine approaches in detail applied toward HCC treatment. The expanding role of extracellular vesicles in modulating explicit aspects of HCC advancement, angiogenesis, and metastasis is discussed. The structure, function, tumorigenesis, and prognostic value of cathepsin B are explained briefly. Moreover, recent updates and developments in chemotherapy, which is the most substantial treatment option for advanced HCC, have been evaluated. Finally, recent advances in the medical treatment strategies, perspectives on the therapeutic importance of mRNAs, and pharmacogenomics for HCC are scrutinized.

The aim of this series is to illustrate the biology, pathophysiology, diagnosis, therapeutic targets, and clinical significance of HCC. Understanding these roles could provide important steps forward in evaluating disease progression and potential therapeutic strategies. This series will provide novel ideas to researchers and scholars as well as innovative future perspectives in the field of research and clinical applications.

Chapter 1

Nanoparticles for diagnosis and treatment of hepatocellular carcinoma

Sheik Aliya and Yun Suk Huh,    Department of Biological Engineering, NanoBio High-Tech Materials Research Center, Inha University, Incheon, Republic of Korea

Abstract

Primary liver cancer universally continues to be one of the leading causes of health problem. Hepatocellular carcinoma (HCC), is a prominent form of primary liver cancer, is also the major cause of cancer-related death worldwide due to lack of proper diagnosis at the early stage of the disease and scarce of appropriate treatment tools. Thus there is high demand for a novel therapeutic approach to treat HCC. Nanotechnology is presently a fast-growing field which offers infinite prospects to design nanoscale products with imaging, diagnosis, and treatment purpose. Forty-five papers were included in the review, most of them contributed latest research related to nanoparticle-based targeted therapy for HCC cancer. Nanoparticles with significant therapeutic potential in clinical trials have been discussed and some of the major limitations are also addressed.

Keywords

Hepatocellular carcinoma; liver cancer; nanoparticles; drug delivery

Abbreviations

ATO Arsenic trioxide

Au-NPs Gold NPs

CMCNP-GL O-carboxymethyl chitosan NP modified with glycyrrhizin

CUR Curcumin

DSN Diosin

DT Doxorubicin-Transdrug

FDA Food and drug administration

GA Glycyrrhetinic acid

HCC Hepatocellular carcinoma

MSN Mesoporous silica nanoparticles

NPs Nanoparticles

PEAL-NPs mPEG-PLGA-PLL copolymer-based NPs

RNAi RNA interference

SLNs Solid lipid nanoparticles

SPIONs Superparamagnetic ironoxide nanoparticles

TiO-NPs Titanium oxide NPs

Introduction

Hepatocellular carcinoma (HCC), a primary liver cancer, statistically stands in the third position causing major cancer-related death worldwide. Conventional HCC treatment includes curative and palliative care, but it is not effective as the cancer is detected at the advanced stage. Surgical resection is one of the best options considered. While it is limited to patients with metastatic tumors and high chances of recurrent have decreased the survival rate of HCC patient. Chemotherapy is one of the best therapeutic modalities, but the main drawback is the lack of target specificity which leads to damage of the normal cells and concurrent administration leads to the development of drug resistance. Another big challenge with HCC patients is 80%–90% develop liver cirrhosis, which burdens the patient with two diseases [1]. The major reasons for failure in therapy are poor absorption, insolubility of the drug, high fluctuations in blood due to lack of bioavailability, its rapid metabolism and elimination. Thus there is demand for an effective therapeutic strategy to combat advanced or recurrent HCC by developing a drug carrier system.

Cancer Nanotechnology is rapidly growing toward the development of anticancer nanoagents which promises to improve the therapeutic approach against dreadful disease cancer. Nanoparticles (NPs) (nanosize particles of range 1–100 nm) due to their unique properties have wide application promisingly in tumor diagnosis and therapy. Nanosize makes it advantageous as it effectively enters the cell promising it a potential drug delivery system. Peter Speiser, a pioneer who introduced NPs to the world, developed it for vaccination drives, designed to release antigen in a slow pace leading to enhanced immune response than the conventional ones. The group discovered lysosomotropic effect of NPs, the nanocapsules induce drugs into the cells without intracellular accumulation. The properties such as low drug systemic toxicity and targeting specific tumors mark the NPs to have the promising application [2]. Active pharmaceutical ingredients with poor pharmacokinetics and biodistribution are delivered through NPs with low cost, less risk, marginal toxicity, and with high efficiency. With multifunctionality and advanced targeting strategy, NPs are reported to have significant applications in nanomedicine as contrast agents in bioimaging and drug carriers to target tumors [3]. Clinically, to diagnose HCC, ultrasound is the common imaging method applied. However, the sensitivity of this method is limited to 60%–80% only, as it cannot distinguish between normal tissues and lesion ones. Lesions less than 5 mm are not visualized. The imaging modalities include photoacoustic imaging and fluorescence imaging has its own limitation. Recently, photothermal therapy has gained a lot of interest in cancer treatment for its nominal invasiveness. Infrared laser irradiation is used along with photoabsorbers to burn tumor tissue with high specificity. NPs with strong NIR region optical absorbance are currently used in photothermal therapy, they include gold NPs (Au-NPs), carbon nanotubes, Prussian blue NPs, polypyrrole NPs, and copper NPs. Among them, the potential photoacoustic/photothermal imaging agent used for HCC diagnosis and treatment is polypyrrole NPs tagged with SP94 (SFSIIHTPILPL; HCC target peptide). These uniformly tagged NPs exhibit low cytotoxicity, high specificity for HCC cells, high stability, and efficient photothermal conversion proves it to be a potential candidate [4,5]. Normally, NPs injected intravenously are taken up by the liver by opsonization through size-dependent passive targeting process. Studies have been performed on the intracellular transport mechanism of NPs in cells. It has been observed that intracellular trafficking of NPs and its uptake mechanism are different in different cell lines [6]. Active targeting is an effective approach which can be achieved by decorating or glycosylating NPs with a ligand which has high specificity to asialoglycoprotein receptors (highly expressed on hepatoma cells). Notably, HCC patients are reported to exhibit different clinical symptoms mainly because of difference in properties of hepatoma cells [7]. Thus recently a lot of research is done in search of effective NPs to cure HCC. The NPs which are successfully used in the treatment of HCC are represented in Fig. 1.1. Multifunctional NPs are designed or constructed with a combination of drugs to enhance therapeutic effect and a combination of drug with imaging agent to enhance both therapeutic and diagnosis of liver disease [8]. Therefore this review highlights the application of different NPs-based diagnosis and targeted therapy against HCC.

Figure 1.1 Different types of nanoparticles used in the treatment of hepatocellular carcinoma.

Chitosan nanoparticles

Chitosan, a derivative of chitin (present in the shells of crustaceans and cell wall of fungi), a biodegradable biocompatible polymer, has been approved for drug delivery and tissue engineering (wound dressing) by the US Food and drug administration (US-FDA). Since it is regarded as safe for human consumption, its modified form is exploited in a wide range of potential biomedical applications. NPs derived from chitosan and its derivatives have been used for cancer treatment, heart and lung diseases, drug delivery to infected liver, brain, and ocular cells. These NPs carry surface positive charges, mucoadhesive properties and have been reported to be exhibiting low toxicity in both in vitro and in vivo, make it an efficient human drug delivery system [9]. A substantial number of experimental studies have been done to understand the interaction between NPs with different surface charges and the cell membrane. It has been reported that electrostatic interaction during phagocytosis mechanism mainly increased the uptake of charged chitosan NPs by macrophages compared to the neutral particles. While in no-phagocytic normal human liver cell line L02 and hepatoma cell line SMMC-7721, it was observed that positively charged chitosan NPs were taken up in large amounts into the cells compared to the negatively charged ones due to the force (attractive/repulsive) between charged NPs and the negatively charged cancer cell membrane [10]. In another study paclitaxel, a potent anticancer drug loaded in O-carboxymethyl chitosan NP modified with glycyrrhizin (CMCNP-GL) was used to evaluate HCC targeting therapy. The NPs synthesized possessed desired surface charge, small particle size, and high stability facilitated both in vitro and in vivo cellular uptake efficiently. CMCNP-GL was reported to significantly facilitate targeted delivery of paclitaxel into hepatoma carcinoma cells. Accumulation of paclitaxel-enhanced hepatic and systemic toxicity compared to the control makes CMCNP-GL a potential HCC targeting drug carrier [11].

Mesoporous silica nanoparticles

Mesoporous silica nanoparticles (MSNs) have attracted great attention due to remarkable chemical stability, excellent biocompatibility, rigid framework, easily modifiable surface chemistry, large surface area, unique morphological characteristics, high pore volume with well-defined structure, and especially high drug loading capacity [12–14]. Earlier the MSN synthesized had very small pore which limited its application for drug delivery. Recently MSN with large pore and novel structures are developed which improved its efficacy for therapeutic application and also reduced toxicity levels [13]. Two types of mesoporous silica NPs synthesized are short rod and long rod forms. Short less spherical rods tend to accumulate in liver and get easily eliminated through urine and feces whereas the long ones accumulate in spleen with less elimination potential.

The phytochemicals with antiinflammatory, antitumor, antiangiogenesis, and many other pharmacological activities are identified and studied extensively. However, due to water insolubility and low bioavailability limits their clinical efficacy. The above limitations can be addressed by hosting the bioactive phytoconstitutents in the MSN. Pharmacological studies have been done on those constituents which have specific binding sites on the surface of hepatocytes of the rats and HCC cells. Phytochemicals isolated from the roots of Glycyrrhiza glabra L. such as with glycyrrhetinic acid (GA) and glycyrrhizin, and curcumin (CUR) derived from Curcuma longa were covalently decorated uniformly on MSN surface with high loading capacity. The in vitro experiment showed that MSN-GA-CUR significantly enhanced cellular uptake toward HCC (HepG2) cells through GA receptor-mediated endocytosis mechanism and thus increased cytotoxicity of HCC. The study provides a promising nanoplatform for HCC targeting [12].

Iron oxide nanoparticles

Iron oxide NPs’ diameter ranges from 5 nm to 100 nm exhibits highly unique properties which make it feasible for biomedical application. First, they are approved by FDA. Iron oxide NPs have high magnetization value, the so-called superparamagnetic NPs make it an eligible candidate for magnetic resonance imaging (MRI). The paramagnetic property in the presence of magnetic field guides the NPs to the location and drug is released on heated up by magnetic field. Earlier, a magnetic-targeted carrier of size 1–2 µm was designed by a pharmaceutical company to deliver drug (doxorubicin) to cancerous liver cells. The drug-loaded NPs were injected near the liver tumor and powerful magnetic field was applied. This helped in localized targeted retention with extravasation of the NPs into the surrounding tissue [15]. There were no drastic side effects reported because the magnetic field induced drug release from the NPs into the tumor, but when the effect was turned off, the NPs were trapped in the tumor causing very less drug to be circulated throughout the body. Till now phase III clinical trials were done, further study was not done as these NPs increased the survival rate of patients with less curative effect. Further these NPs were later used by labeling them with β-emitters (yttrium-90 and rhenium-188) reported to be very effective against liver tumors [16]. Wilson et al.’s clinical study monitored transcatheter targeted delivery of drug doxorubicin conjugated to magnetic NPs in hepatic cells using intraprocedural MRI. The treated liver tumor volume drastically reduced compared to the normal control ones [17].

The iron NPs advantageous property includes biocompatibility and high conjugation ability with drug biomolecules makes these particles to be used in drug targeting, drug delivery, and local hyperthermia. However, the major limitation is opsonization, which can be overcome by surface modification of iron oxide NPs [18]. Superparamagnetic ironoxide nanoparticles (SPIONs) are synthetic maghemite or magnetite particles of core diameter ranging from 10 nm to 100 nm. Particle size plays a major limitation factor in internalization of NPs in cells. Small size NPs (less than 50 nm) penetrate efficiently than large-size NPs and are easily eliminated through the renal system and Kupffer cells of the liver. Large size evades penetration into cells, increases the blood circulation time period and gradually these particles are prominently taken up by phagocytes (macrophages) in the reticuloendothelial system of liver and other tissues such as bone marrow, spleen, and lymph [19]. SPIONs are coated with polyethylene glycol or any other biocompatible polymers which provide base for therapeutic agents to conjugate and mainly to improve distribution profile of the particles in the blood. SPIONs coated with Pluronics/oleic acid with 193 nm hydrodynamic diameter as observed to be accumulated in the liver of the rat and mainly in the tumors compared to the normal cells [20]. Similar results were reported with experiment using radioactive 59Fe-NPs. In the presence of magnetic field, 114 times more activity of NPs was observed in the tumor region than control without the magnetic field [16]. These particles with magnetic property have opened wide range of horizons as agents of drug delivery and MRI.

Liposomal nanoparticles

Liposomes have been used as drug delivery system for a long period of time owing to their unique properties such as biocompatibility, biodegradability, low cytotoxicity, and immune response, encapsulates both hydrophilic and hydrophobic drugs and even drug combinations. Nanoscale liposomal vesicles are promising candidates for systematic drug delivery systems. The main advantage of liposomal NPs is they encapsulate potential anticancerous natural water insoluble drug and delivers to the targeted site due receptor-specific modification of the surface of the NPs. These NPs have recently emerged as promising nanocarriers to deliver drug combination as evidenced by reports of more clinical trials [21]. The physiochemical properties were evaluated such as size factor, drug release profile, biocompatibility, zeta potential, encapsulation capacity and efficiency, and mainly synchronous release of drug at the targeted site. A comparative study on the effect of dual drug, monodrug, and free drug cocktail on HepG2 cells (in vitro cytotoxicity assay) and HCC xenograft mouse tumor models (in vivo analysis). Curcumin delivered through liposomal NPs neutralize the toxic effect of chemo drug cisplatin. Curcumin proved to regulate the Sp1 and also p-ERK1/2 protein expression through ROS generation which indirectly enhanced the antitumor efficiency of cisplatin. In vivo experiments were done to optimize the synergistic interaction of both the drugs for liver tumors. In another phase III clinical trial on liposomal formulation with drug CPX-351 encapsulated along with two drugs cytarabine and daunorubicin have reported improved therapeutic outcomes compared to the free drug cocktail given. Liposomes have been reported to control the release of drugs and its systemic accumulation in tumor cells without affecting normal cells proves it to be a promising candidate for codelivery of poorly insoluble drugs both in vitro and in vivo [22]. Diosin (DSN), a flavonoid with anticancer potential against colon and HCC did not show effective therapeutic potential due to poor solubility. The authors developed a new liposomal NPs loaded with DSN showed efficient drug dissolution and improved cell permeation into intestine, designating liposomal NPs a potential drug carrier and delivery system [4]. Viroonchatapan et al. designed a core/shell structure, magnetoliposomes, in which iron oxide magnetic core is surrounded by liposome. The core with dextran-iron oxide NPs incorporated into liposomes containing calcein (fluorescent marker) was targeted to mouse liver with the help of an extracorporeal magnet. These structures are very small compared to the albumin microsphere making it a potential drug carrier and targeting the drug delivery with high efficiency [23].

Solid lipid nanoparticles

Solid lipid nanoparticles (SLNs) are made up of biodegradable solid lipids with the mean diameter (photon correlation spectroscopy) ranging from 50 nm to 1000 nm. SLNs of size 120 nm were synthesized at elevated temperature (65°C) at high pressure by homogenization method [24]. SLNs are mainly administered intravenously and are disseminated more in the liver and kidneys [25]. SLNs (medication carriers) was introduced in 1991 as a variable substitute to traditional colloidal carriers (liposomes). They have very remarkable properties which appeal them to be potential particles to enhance drug delivery. The properties are small size, large surface area, and high drug loading capacity. The specific property includes physical stability, exceptional tolerability in any formulations and administration routes whether oral, visual, dermal, rectal or pulmonar, controlled release, the communication of stages at the interface to deliver the drug efficiently and mainly liable degradation with nontoxic byproducts [26]. A new NP prepared with galactosylated dioleoylphosphatidyl ethanolamine abbreviated as tSLN (targeted SLN) with docetaxel-loaded targeted to hepatoma (BEL7402 cell lines) was designed. The NPs exhibited encapsulation efficiency of about more than 90%, low initial burst effect, and nearly 29 days of slow and sustained release of drug. The in vitro cytotoxic activity was greater for tSLN compared to Taxotere and also nontargeted SLN (nSLN). Similar results were also reported in in vivo murine hepatoma model. The effect was mainly due to larger cellular uptake and accumulation of tSLN loaded with drug in tumors. Altogether, tSLN did not show any detrimental effect on normal cells. The results clearly demonstrate that tSLNs loaded with docetaxel are very efficient in the treatment of advanced and metastatic HCC [27]. A lipid NP formulation of small interfering RNA (siRNA), TKM-080301, in preclinical evaluation targeted knocks down of polo-like kinase 1 (PLK1) which are overexpressed in HCC. This resulted in downregulation of RNA induced silencing complex and inhibition of cell proliferation. This proves to be a novel approach to combat solid tumors by targeting PLK1 [28].

Gold nanoparticles

For centuries, gold has been used in medicinal formulations. Now a days Au-NPs have been widely designed for biomedicine application such as molecular imaging, target specific drug delivery and cancer treatment. A chemotherapy strategy in which Au-NPs (1 nm in diameter) conjugates with chemo drug has proved to be very efficient to control the proliferation of HCC. The size factor makes the particle more compatible to cross the biological cell membrane and even reaches the nucleus to attach to DNA molecules. A study reported that Au-NPs at low concentration did not show cytotoxic activity against HCC cell lines, HepG2 while high concentration showed remarkable anticancer effect. Engineered NPs have been used as active particles for cancer therapy. Study reported that Au-NPs primarily accumulated in liver. But to target the particles to specific organ or target tumor site, surface modification with appropriate functional moiety is done. In one of the experimental study, Au-NPs were capped with PEG or sodium citrate or dendrimers. These particles exhibited marked cytotoxicity at a very low concentration against HepG2 cells and even toxicity effect by DNA damage was also observed. Altogether, the finding suggests that designing and modification of surface factors enhance the therapeutic potential of Au-NPs against HCC. In future more research should be targeted toward development of NPs with high specificity and reduced toxicity or side effects for efficient liver cancer therapy [29].

Titanium oxide nanoparticles

Titanium oxide nanoparticles (TiO-NPs), metallic NP has attracted researchers’ interest for its photodynamic therapy application to kill cancer cells as well as antibiotic resistant bacteria. They have been proved to be excellent nanodrug carriers for targeted drug delivery [30]. Ismail et al. reported that TiO-NPs statistically did not show any cytotoxic activity in HepG2 cell lines due to low cellular accumulation. The authors state that may be due to limited exposure to UV light and unmodified surface results in higher agglomeration [31]. TiO-NPs synthesized by wet chemical method, have been reported to have more biocompatibility and the surface modification with PEG and folic acid ligand, targets the NPs to the surface of cancerous cell. Since TiO-NPs functional efficiency increases when it is complexed or surface modified, TiO2–PEG–FA complex with paclitaxel drug conjugated was studied for its effect on HepG2, human liver cancer cell lines. Cytotoxic studies proved that the complex showed more cytotoxic effect on the cells reporting the potential use of TiO-NPs as efficient drug nanocarriers [30]. Lee et al. worked on defective TiO-NPs for photocatalytic destruction of HCC using long wavelength visible light. It is to be noted that TiO-NPs increase the strength of nanocomposites, the authors synthesized liposome—TiO-NPs composites. HepG2 cells were subjected to different concentration of TiO-NPs composites, irradiated with 300 W xenon lamp (long-wavelength visible light) for different time interval and singlet oxygen and ROS was monitored. The results report high cytotoxic activity proves the potential application of TiO-NPs for photodynamic cancer treatment [32].

RNA interference (RNAi) has been reported to be a potent and specific regulator of expression of gene by silencing it. Lipid NPs with encapsulated RNAi have been proved to be highly effective to treat liver tumors. Clinical trials of RNAi-lipid NP formulation, ALN-VSP targeting VEGF and kinesin spindle protein were examined (Table 1.1). In tumor biopsies, it was detected that siRNA-mediated targeted cleavage of mRNA in liver, leads to downregulation of specific growth promoting genes and ultimately progression of liver cells to damage reports antitumor activity, and complete liver metastasis regression. The biweekly dose injection of ALN-VSP has been reported to be safe and tolerable. This provides proof that RNAi therapeutics opens new path for development of NPs for treatment of cancer. In future, specific multitargeting and more drugs encapsulated lipid NPs can be designed for better prospects in cancer therapy [33].

Table 1.1

Note: Data retrieved from https://www.clinicaltrials.gov

HCC, Hepatocellular carcinoma; NPs, nanoparticles; SPION, superparamagnetic ironoxide nanoparticles.

Pectin-based nanoparticles

Pectin, a homogalactoronan, a linear biopolymer of α-(1,-4)-linked D-polygalacturonic acid units. The pectin NPs synthesized under mild conditions using aqueous medium containing calcium ions and carbonate ions are extensively studied as they demonstrated potential candidates for drug delivery because of their unique properties. They include subcellular size, thermal stability, redispersibility, drug crystallinity, better intestinal absorption, and dissolution. These polymeric NPs penetrate epithelia very diligently and exhibit sustained release of water insoluble encapsulated drugs very efficiently [34]. Further standardization of increasing the drug loading capacity of these galactose-based NPs to exhibit significant in vivo pharmacokinetics are done. Pectin-based NPs encapsulated with anticancer drug have been used to target HCC (HepG2 cells). The polymeric units without any chemical modification played an efficient targeting headgroup role. 5-FU-loaded pectin-based NPs were fabricated as drug delivery system to HepG2 cells with overexpressed asialoglycoprotein receptor (in vitro cytotoxic studies) and mouse model with hepatic tumor (in vivo studies). Both the studies showed that 5-FU-loaded pectin-based NPs exhibited higher cytotoxic activity and constant drug release profile with better tissue distribution of the drug when compared to free 5-FU delivered. This demonstrates the potential ability of pectin-based NPs as targeted drug delivery system for the treatment of HCC [35].

mPEG-PLGA-PLL copolymer-based nanoparticles

mPEG-PLGA-PLL copolymer-based nanoparticles (PEAL-NPs) are made up of FDA-approved triblock copolymer. They are monomethoxy polyethylene glycol (mPEG) which improves stability and increases half-life of NPs, poly (D,L-lactic-coglycolic acid) (PLGA) which show biocompatible and biodegradable properties and poly(L-lysine) (PLL), a stable cationic molecule which can be modified with many different functional groups [36]. PEAL-NPs conjugated along with lactobionic acid and antibody of vascular endothelial growth factor abbreviated as PEAL-LA/VEGFab-NPs have been used effectively to treat HCC. These NPs offer great advantages over other by showing high stability, less toxicity, cost efficiency, biocompatibility, and targeted delivery of drug. Lactobionic acid is the targeting moiety, which binds specifically to asialoglycoprotein receptor which is overexpressed on HCC cell surface. VEGFab is another moiety which is highly specific for human tumors such as HCC. In HCC, microRNA-99a (miR-99a), a tumor suppressor has been reported to be commonly downregulated. To inhibit HCC progression miR-99a expression should be restored. PEAL-LA/VEGFab-NPs have been efficiently used to deliver miR-99a specifically in both in vitro and in vivo and HCC progression was completely repressed [37]. PEAL and 4-O-beta-D-Galactopyranosyl-D-gluconic acid (Gal)-modified PEAL (PEAL-Gal) are used to investigate cellular uptake mechanism and intracellular trafficking of NPs in different hepatoma cell lines such as HepG2, Huh7, and PLC cells. This study is highly necessary to understand the therapeutic efficiency of NPs [38].

Arsenic, a trace metalloid found in earth’s crust exists has highly toxic forms. Arsenic trioxide (ATO) is industrially produced has an exciting application transformation from king of poisons into an anticancer drug. Earlier, ATO has been used in combination of drugs for acute promyelocytic leukemia treatment [39]. PEAL-NPs, a nano drug delivery system, have been successfully used to deliver ATO in the treatment of HCC effects. ATO exhibited antitumor effects by inducing various mechanisms such as initiation of cell cycle arrest at G2/M phase and apoptosis action. ATO induces pyroptosis (cell death mechanism which is triggered by innate immune defense mechanism associated with caspase 1) which activates caspases 3 which cleaves inactive gasdermin D to an active form which is tumor suppressor or pyroptosis executor gasdermin D (molecule induces pore formation). PEAL-NPs conjugated with ATO have been exploited for therapeutic application to treat HCC as it has been proved to induce pyroptosis in HepG2 cells and liver tumors [40].

Nanoparticles in clinical trials to treat hepatocellular carcinoma

Prospective clinical trials and research help in the development and identification of a potential drug that increases our understanding of HCC evaluation and designing strategies for diagnosis and treatment. This would result in the decrease mortality and morbidity related with HCC. Clinical trials are summarized in Table 1.1. Doxorubicin-Transdrug (DT) is a nanoparticle formulation of doxorubicin. Both in vitro and in vivo studies (X/myc bi-transgenic MDR murine model of HCC) have shown that DT is effective to overcome multidrug resistance.

Toxicity

NPs are highly advantageous in biomedical application is mainly due to nano size. But this itself reports being the greatest disadvantageous factor. Due to its small size, the particles are able to penetrate the smallest capillaries and get distributed in the whole body, can pass through the membranes affecting the normal physiological function of the cell. While being excreted it is processed in the liver and filtrated through the kidney. If the size is greater than the particles to be filtered through the glomerulus, then they are accumulated in kidney causing renal failure. Some NPs, such as silver NPs have been reported to show doze (5–100 µg/mL) and size (60 nm) dependent toxicity by creating oxidative stress even in germline stem cells, liver, lung and neuroendocrine cells within 24 hours of treatment. While in 28 days, it was reported to alter the blood cholesterol and plasma alkaline phosphatase indicating liver damage probabilities. Liver histopathological studies of rats treated with silver NP have revealed hepatic cytoplasmic vacuolization and focal necrosis [41,42]. In another study, it was reported that mice treated with silica-coated NPs were distributed in most of the organs in 4 weeks of treatment. The liver mainly takes up the NPs and is found to be distributed in other organs such as spleen, lungs, kidney, and heart [43]. Chen et al. have demonstrated greater toxicological effects on ingestion of copper NPs by histological analysis in mice spleen, kidney, and liver [44]. Wang et al. worked on the effect of TiO-NPs (of size 25–80 nm) on female mice. They observed severe biochemical alterations such as changes in lactate dehydrogenase, aspartate amino transferase, and other liver-related enzymes activity. Nephrotoxicity was observed as an increased level of BUN. And pathological effect on liver of mice treated with high dose of TiO-NPs was observed as swelling and degeneration of cells around central vein, hepatocytes with spotty necrosis, and accumulation of NPs. This leads to necrosis not only in kidney and liver but also in spleen and lung tissue [45]. A comparative study on the effect of different NPs (silver, molybdenum oxide aluminum, iron, and TiO-NPs) on rat cell line BRL3A revealed that silver NPs are highly toxic, molybdenum oxide NPs are moderately toxic whereas iron, aluminum, and TiO-NPs displayed low cytotoxicity [42].

Conclusion

HCC, the deadliest disease affecting millions worldwide. Inefficient therapeutic and diagnostic strategies have led to an increase in the death rate. Recent advances in the field of nanomedicine have allowed construction and synthesis of different types of NPs conjugated with drugs exhibiting improved pharmacokinetic properties. Some have been subjected to clinical trials and had been proved to be very effective. But still, some NPs relatively show toxic effects on prolonged and high-dosage administration. Overall, the enhanced controllability, bioavailability, and target specificity of NPs broaden the spectrum in diagnosis and therapeutic application of HCC.

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