Targeting and noninvasive treatment of hepatocellular carcinoma in situ by ZnO nanorod-mediated concurrent chemoradiotherapy

Abstract

Nanomaterials have emerged as radiosensitizers to improve therapeutic efficiency. The aim of the current study was to investigate concurrent chemoradiotherapy mediated by ZnO nanorods for noninvasive treatment of hepatocellular carcinoma (HCC) in situ. Transferrin receptor antibody (TfR Ab) functionalized ZnO nanorods, loaded with doxorubicin (Dox), denoted as TfR Ab/Dox/ZnO nanocomposites, were prepared to act as a targeted multifunctional drug delivery system (DDS). The synergistic anticancer effects on HCC were evaluated in vitro and in a murine orthotopic model using a cell viability assay, apoptosis detection, histopathologic examination, and serum biochemistry tests. Our observations demonstrated that the TfR Ab/Dox/ZnO nanocomposites could play the targeted role of the drug carrier to deliver Dox into the HCC SMMC-7721 cells to enhance its chemotherapeutic efficiency. Besides, with the addition of short term and low dose X-ray irradiation, the ZnO nanorods showed excellent radiosensitizer properties, further attacking the cancer cells. Thus, apoptosis was synergistically induced by the concurrent chemoradiotherapy, resulting in a distinct improvement in anticancer activity. Therefore, ZnO nanorods could mediate concurrent chemoradiotherapy for the noninvasive treatment of HCC.

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Introduction

The clinical treatment of hepatocellular carcinoma (HCC), the fifth most common cancer and the third leading cause of cancer-related deaths worldwide, particularly in Asia and Africa, is still challenging.^1,2 The majority of patients usually present at advanced stages when diagnosed and are only candidates for palliative treatment modalities, such as percutaneous ethanol injection, percutaneous microwave coagulation therapy, percutaneous radiofrequency ablation, transcatheter arterial chemoembolization, chemotherapy, and radiotherapy.^3,4 The response rate is disappointing due to either intolerable invasive treatment or the unbearable side effects. Consequently, the overall prognosis of patients with advanced HCC is dismal. Thus, there is an urgent need to improve HCC treatments, especially noninvasive therapeutic strategies, which are easy for patients to accept and doctors to operate.

Recently, the development of biomedical nanotechnology has led to significant breakthroughs in theranostics for cancer.^5–13 Semiconductor nanomaterials, including ZnO, have emerged as ideal multimodal nanomedicine platforms, acting as drug carriers to increase intracellular therapeutic agents and photosensitizers in photodynamic therapy (PDT) of cancer.^2,14,15 However, the effective excitation wavelength of the photosensitizers is located in the ultraviolet (UV) region, which cannot penetrate deeply into human tissues. Thus, PDT is limited to superficial tumors, facing the dilemma of the clinical application for deep-seated cancer. Despite some efforts to develop near-infrared-induced PDT by employing some special photosensitizers and upconversion materials, these still failed to make satisfactory progress.¹⁶ Since the photoelectrochemical reactions observed upon excitation with UV are also promoted under X-ray irradiation, a semiconductor is one of the candidates for radioactive ray-to-electric and/or chemical energy conversion device materials.^17,18 For example, X-ray induced photoelectrochemistry on TiO₂ by this principle has been well investigated.¹⁷ Therefore we proposed to optimize this reaction between X-ray excitation and ZnO nanorods as a new radiotherapy (RT) strategy for HCC. In comparison, RT is virtually immune from the restriction of penetration depth.¹⁶ Unfortunately, RT for liver tumors has not been successful for HCC because X-ray irradiation can induce severe hepatic toxicities at dosages below curative ones, named radiation-induced liver disease (RILD), although technological advances have provided improvements for the application of radiation therapy.¹⁹ Nanoparticles as adjuncts to radiotherapy, radiosensitizers, effectively provide dose enhancement and therefore increase therapeutic efficiency under quite weak and/or short X-ray irradiation.^20–23 Recently, the potential radiosensitization of gold and silver nanoparticles using clinical megavoltage (MV) photon beams has been investigated.^24–26

Since single modalities have not always been sufficiently effective, combinations of cancer therapy modalities are attractive, which increases attention to improve the outcome of treatment. Chemoradiotherapy, the combination of chemotherapy (CT) and radiotherapy, has become a standard treatment option to improve anticancer activity. Doxorubicin (Dox) has been routinely used as a chemotherapeutic agent for HCC, but non-selective cytotoxicities and side effects are the barriers.² Tumor selective and targeting drug delivery systems (DDS) based on nanobiomaterials of surface functionalized homing devices provide opportunities to overcome the above limitations. Coincidentally, the ZnO nanomaterials could be used as drug carriers for loading Dox, which was well illustrated in our previous studies.^2,14 As is well known, the transferrin receptor (TfR) is highly and stably expressed on the tumor cell surface.^27–31 Consequently, TfR could be a specific target in tumor biotherapeutics. Anti-TfR antibody (TfR Ab) combined with a chemotherapeutic agent has been used as a targeted cancer therapeutic strategy.^32–34

To this end, TfR Ab functionalized ZnO nanorods, loaded with the chemotherapeutic drug, Dox, were prepared in this study. They were denoted as TfR Ab/Dox/ZnO nanocomposites, in which ZnO could not only be applied as the targeted drug carrier to deliver Dox into the cancer cells, but also as the radiosensitizer in radiotherapy. To the best of our knowledge, our report is the first to demonstrate the potential of ZnO nanorods as a promising new class of radiosensitizers. The novel strategy of concurrent chemoradiotherapy for HCC could bring less physical and mental burden to patients due to the noninvasive nature of radiotherapy and targeted drug delivery. The in vitro and in vivo anti-tumor properties of concurrent chemoradiotherapy were investigated in a murine HCC orthotopic model.

Materials and methods

Chemicals

Zn(CH₃COO)₂·2H₂O, diethanolamine (DEA), and NaOH were purchased from Shanghai Chemical Reagents Co. Dox was acquired from Zhejiang Hisun Pharmaceuticals, China. 3-Aminopropyltrimethoxysilane (APTMS), 2-(4-morpholino)ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma (St. Louis, MO). RPMI-1640 culture medium, fetal bovine serum (FBS), and culture plates were purchased from Gibco (Gibco/BRL, Carlsbad, CA). All other reagents were of analytical grades.

Preparation of TfR Ab/Dox/ZnO nanocomposites

ZnO nanorods were synthesized according to our previous study,¹⁴ and TfR Ab/ZnO nanocomposites were prepared by covalently bonding the TfR Ab as described in the literature.³⁵ The scheme for this preparation is presented in Fig. 1A. Briefly, ZnO nanorods were prepared by a solid state reaction at room temperature using Zn(CH₃COO)₂·2H₂O, DEA, and NaOH. Amino groups were introduced onto the surface of the obtained ZnO nanorods by using APTMS. Next, G3.5 carboxyl-terminated polyamidoamine (PAMAM) functionalized ZnO (PAMAM–ZnO) was prepared by using EDC and NHS. Then, TfR Ab was labeled on ZnO after activating the carboxyl groups of PAMAM by using EDC and NHS. In the following experiments, a 2 mL aqueous solution of Dox (2 mg mL⁻¹) was added to a 1 mL aqueous suspension of the obtained TfR Ab/ZnO bioconjugate (10 mg mL⁻¹). The reaction mixture was kept in the dark overnight to construct the TfR Ab/Dox/ZnO nanocomplexes. When the above DDS was collected by centrifugation, unbounded Dox in the supernatant was calculated by measuring the absorbance at 490 nm, allowing the estimation of the drug encapsulation and loading efficiency.

Fig. 1 Scheme of ZnO nanorods functioned with TfR Ab as the targeted delivery Dox (A), TEM image of the ZnO nanorods (B), and the histograms of length (C) and width (D) distribution of the nanorods.

Drug release

The drug release properties of TfR Ab/Dox/ZnO were investigated at pH 7.4 (pH of physiological blood), 6.3 (pH of the environment around the tumor) and 5.2 (approximate pH in endosomes or lysosomes) according to our previous study.¹⁴ In brief, TfR Ab/Dox/ZnO were dispersed in PBS (pH 7.4, 5 mL) and transferred into a dialysis bag (molecular weight cut-off 3000 Da), which was then immersed in 95 mL PBS at pH 7.4, 6.3, or 5.2, and continuously agitated with a stirrer at 50 g and 37 °C. At predetermined time intervals, 2 mL of the external medium was collected and replaced with the same fresh PBS. The amount of released Dox in the medium was then determined by high-performance liquid chromatography (LC-310; Skyray Instrument, Nanjing, China).

Cell internalization studies

The human hepatocarcinoma SMMC-7721 cells, obtained from Shanghai Institute of Cells, Chinese Academy of Sciences, were cultured in a RPMI 1640 medium supplemented with 10% FBS, 100 U mL⁻¹ penicillin and 100 to μg mL⁻¹ streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. The cells were treated with different treatments, Dox alone, Dox/ZnO, and TfR Ab/Dox/ZnO for 6 hours. The cells without treatment were used as control. Dox cellular uptake was analyzed by a FACSCalibur™ flow cytometer (BD Biosciences, San Jose, CA) after the cells were washed and resuspended in PBS.

Cell viability assay

Cell viability was assessed using MTT assays.^36,37 Briefly, after the cells had been treated with different treatments, MTT solutions were added and incubated for an additional 4 hours. Then DMSO was added to solubilize the formazan crystal, and the optical density (OD) at 492 nm was recorded by a multiwell spectrophotometer reader (Thermo Labsystems, Vantaa, Finland). Cell viability (%) was calculated as follows: optical density_{[492 nm in test cells]}/optical density_{[492 nm in control cells]} × 100.

Reactive oxygen species (ROS) measurement

In this method, the fluorogenic substrate DCFH-DA (2′,7′-dichlorofluorescin diacetate), a cell permeable dye, can be oxidized to the highly fluorescent DCF (7′-dichlorofluorescein) by ROS and can therefore be used to monitor the intracellular generation of ROS. After 6 h incubation with ZnO nanorods, SMMC-7721 cells were treated with or without X-ray irradiation, followed by the addition of the DCFH-DA ROS probe into the cells for 20 minutes. Then, the stained cells were observed for the production of ROS at 485 nm and 535 nm excitation and emission wavelengths, respectively, using an Olympus IX51 inverted microscope.

Apoptosis assay

Quantification of apoptotic cells was determined with an Annexin V-FITC/PI detection kit (BD Pharmingen, USA) according to the manufacturer’s instructions. Cells were collected after the different treatments, washed twice with cold PBS and resuspended in 100 to μL binding buffer, followed by staining with 5 μL Annexin V-FITC and 10 μL propidium iodide solution at room temperature in the dark for 15 minutes. Analyses were then performed by a FACSCalibur™ flow cytometer.

Orthotopic HCC models

Four-week old BALB/c nude mice, weighing 16–22 g, from the Shanghai National Center for Laboratory Animals, were maintained in a specific pathogen-free facility. All the animal experiments were conducted under protocols approved by the animal ethics committee of the Medical School, Southeast University. The human hepatocarcinoma SMMC-7721 cells were first subcutaneously injected into mice to generate tumors. Then, the obtained tumors were excised and cut into pieces. Similar sized tumoroids were transplanted into the liver of new mice to establish the orthotopic xenograft liver tumor models. One day after the surgery, the mice were intravenously administered via tail veins every three days for four times with 200 mL of saline as control, ZnO, Dox alone, Dox/ZnO, or TfR/Dox/ZnO (with equivalent Dox dose of 5 mg kg⁻¹), with or without X-ray irradiation. X-ray irradiation was accordingly performed with 4 fractions of radiation at 2 Gy to the whole liver, following every intravenous injection, and other organs were protected by lead shielding. On the 14th day, the mice were killed, and the livers as well as the tumors were taken out. Then the rate of tumor inhibition was calculated. To evaluate the anticancer efficiency, two diameters of the tumor were measured with digital calipers, and then the tumor size was calculated using the following formulae: V = 1/2 × a × b², where V is the tumor volume, a is the longest diameter, and b is the shortest diameter. RTV (%) = V_test/V_control × 100, where RTV is relative tumor volume at the end of experiment, V_control represents the volume in control group, and V_test represents the volume after the different treatments.

Histopathologic examination of tumor and liver tissue

After the mice were sacrificed, the livers were quickly removed and fixed in 4% paraformaldehyde, dehydrated in a graded series of alcohol, and then embedded in paraffin. Tissue sections (4 to μm thick) were prepared and stained with hematoxylin and eosin. Thereafter, the sections were examined using an Olympus IX51 microscope (400×; Olympus Corporation, Tokyo, Japan).

Serum biochemistry tests

The blood was collected before the mice were sacrificed, and serum biochemistry tests of indicators for hepatic function, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were performed according to the manufacturer’s instructions. AFP (alpha fetoprotein) values were measured by an electrochemiluminescence immunoassay on a COBAS 6000 system E601 (Elecsys module) immunoassay.

Statistical analysis

Data are presented as the mean ± standard deviation. An F-test was performed using the Statistical Package for Social Science version 19.0 (SPSS Inc., Chicago, IL, USA) for significance, and P ＜ 0.05 was considered to be statistically significant.

Results and discussion

ZnO nanorods synthesized, TfR Ab modified, and drug loaded

The general TEM images of the obtained ZnO are shown in Fig. 1B. As can be seen from the TEM image, the synthesized ZnO are nanorod-shaped, with a mean size of about 20 nm in width and 50 nm in length. The ZnO nanorods had dimensions suitable for a drug carrier increasing drug accumulation in tumor cells after endocytosis and escaping rapid renal excretion, as well as avoiding components of the reticular endothelial system.^2,38 Moreover, as well illustrated in the literature by Chai et al.,³⁵ carboxylic group functionalized ZnO nanorods were obtained via a reaction between the –COOH of G3.5 PAMAM and the –NH₂ by using EDC and NHS after the amino groups were introduced onto the surface of the ZnO nanorods by using APTMS. Activated by EDC and NHS of the carboxylic groups (–COOH) on the ZnO, TfR Ab could be labeled on the ZnO nanorods via an amide link between the –COOH of the above carboxylic group functionalized ZnO nanorods and the –NH₂ of TfR Ab.³⁵ Thus the TfR Ab immobilized ZnO nanorods were synthesized as a candidate drug carrier to endow the special targeted function of HCC cells. Our previous investigations have demonstrated the capacity of the ZnO nanomaterials as drug carriers for loading Dox, due to the metal binding sites of the quinone and the phenolic oxygen molecules on both sides of the Dox aromatic moiety.² Thus, followed by Dox loading, the TfR Ab/Dox/ZnO nanocomposites were fabricated as a targeted DDS for HCC. When the TfR Ab/Dox/ZnO formed, the zeta potential decreased from −17.5 mV to −2.1 mV, which strongly supported the fact that the Dox was nicely combined with the ZnO nanorods. Fig. 1A shows the schematic procedure of the preparation of TfR Ab/Dox/ZnO. The encapsulation efficiency and loading efficiency of the TfR Ab/Dox/ZnO nanocomplexes were assessed and calculated to be 71.02 ± 5.36% and 20.05% ± 2.61%, respectively.

Drug release performance

As shown in Fig. 2, the release of drug molecules depended on the pH of the medium and the release time. Dox release at pH 7.4 was slow and sustained, with a release ratio at about 17% within 48 hours. However, at a lower pH, its release rate was much faster, with approximately 68% (pH 6.3) and 85% (pH 5.2). The pH-triggered drug release behavior is preferred in practical cancer treatment. Under normal physiological conditions, drugs could remain in the DDS and minimize the side effects of the drug to normal organs. Once the DDS reached the targeted tumor and entered the cancer cells, intracellular lysosomes and endosomes, which have acidic micro-environments, would trigger the drug release, leading to a significant improvement in cancer treatment efficacy.^2,14,38

Fig. 2 In vitro Dox release behavior at pH 7.4, 6.3, and 5.2.

Targeted intracellular uptake

Chemotherapy agents frequently encounter important problems such as low specificity and non-selective biodistribution. To enhance the targeted cellular uptake of nanomedicine DDSs in cancer cells is a good strategy for cancer therapy.^39–45 TfR is a specific target in tumor biotherapeutics including HCC,^27–29 and TfR expression levels in hepatocarcinoma SMMC-7721 cells were confirmed by immunofluorescence (Fig. 3A). To further verify TfR-mediated uptake and the intracellular accumulation of Dox, the fluorescent nature of the molecule was examined in SMMC-7721 cells. The flow cytometry histograms of the cell-associated Dox fluorescence for SMMC-7721 cells are shown in Fig. 3B and the relative intracellular fluorescence intensity is given in Fig. 3C to make a quantitative comparison. As learned from Fig. 3B and C, the minimal fluorescence was seen in SMMC-7721 cells treated with Dox alone. Cells treated with Dox/ZnO nanocomplexes (relative intracellular fluorescence intensity of 42.01 ± 2.21) at an equivalent Dox concentration exhibited a higher fluorescence intensity than those treated with Dox alone (relative intracellular fluorescence intensity of 23.83 ± 1.43), producing a 76.29 ± 3.58% enhancement due to the nonspecific uptake of the nanomedicine DDS. In contrast, TfR Ab/Dox/ZnO nanocomposites were readily taken up by TfR-overexpressing SMMC-7721 cells. As indicated by the intense fluorescence, the cellular uptake in cells treated with TfR Ab/Dox/ZnO (th relative intracellular fluorescence intensity of 73.61 ± 3.23) produced a 75.22 ± 5.74% enhancement compared with those treated with Dox/ZnO, and a 192.37 ± 6.82% enhancement than those treated with Dox alone. Taken together, our results demonstrated that ZnO nanorods bound with TfR Ab could target hepatocarcinoma cells by virtue of special recognition.

Fig. 3 TfR expression levels in hepatocarcinoma SMMC-7721 cells detected by immunofluorescence (A), the flow cytometry histograms of cell-associated Dox fluorescence for SMMC-7721 cells treated by DOX alone, Dox/ZnO, and TfR/Dox/ZnO (B), and a comparison of the respective average intracellular fluorescence intensity of SMMC-7721 cells (C). *P < 0.05.

Enhanced cytotoxic effect of ZnO nanorods combined with X-ray

HCC has good radiation sensitivity, and RT has gradually become an integral part of multi-modality treatment for HCC.^19,46 However, currently RT is not frequently performed for HCC due to the risk of radiation-induced liver disease.^47,48 Radiosensitizers are intended to enhance tumor cell killing while having much less effect on normal tissues.⁴⁹ To investigate whether ZnO has a radiosensitizing effect, the cell viability of the SMMC-7721 cells was examined after exposure to 3 Gy X-ray using 6 MV X-ray equipment (VARIAN600CD, USA) in the presence of ZnO nanorods. As shown in Fig. 4, no significant cytotoxicity was observed for the various concentrations of the ZnO nanorods (up to 100 to μg mL⁻¹). The lack of cytotoxicity of the ZnO nanorods ensures a wide potential range of applications in the field of biomedical science and cancer therapy. The X-ray irradiation itself (3 Gy) only showed a little cytotoxic effect on the SMMC-7721 cells, whose surviving fraction was about 85%. A sharp decrease in the cell viability was observed when the cancer cells were under X-rays irradiation in the presence of ZnO nanorods, which indicates that the radiosensitizer activity of ZnO nanorods can promote mortality of cells. Meanwhile, our results also validate that with increasing concentrations of ZnO nanorods, the lethality increases, suggesting a dose-dependent effect in vitro. The hypothesized mechanism is that the interaction of high-energy electron beams of X-ray irradiation with ZnO nanorods could further excite the Auger electron ejections.^24–26 These short-range secondary electrons could generate large quantities of free radicals that damage the cancer cells, which leads to the effects on radiation therapy.^24–26 As shown in the Fig. 4B, under X-ray irradiation, ZnO nanorods could induce ROS within SMMC-7721 cells which was easily visualized by fluorescence microscopy. However, there was no ROS production in the absence of X-ray irradiation and X-ray alone. The involved ROS could result in the cascade of cellular and molecular events relevant to tumor destruction, which is believed to be responsible for promoting the mortality of cancer cells.

Fig. 4 ZnO nanorods as radiosensitizers for cancer radiotherapy. (A) Cell viability of human hepatocarcinoma SMMC-7721 cells exposed to the ZnO nanorods in the absence and presence of X-ray irradiation. (B) The intracellular ROS production of ZnO nanorods under X-ray irradiation visualized under a fluorescence microscope.

ZnO nanorod-mediated concurrent chemoradiotherapy

Since single modalities have not always been sufficiently effective, combinations of cancer therapy modalities are attractive, which increases attention to improve the outcome of treatment. The National Comprehensive Cancer Network (NCCN) guidelines for HCC have recommended a combination of RT and chemotherapy for patients with unresectable tumors, who are not transplant candidates or if they have inoperable local diseases.¹⁹ In the current case, as well illustrated above, ZnO nanorods could not only be the drug carrier for chemotherapy but also the radiosensitizer for radiotherapy. To explore the possibility of the combination of chemotherapy and radiotherapy, the anticancer effect of TfR Ab/Dox/ZnO nanocomposites was investigated as a strategy for concurrent chemoradiotherapy for HCC. We cultured SMMC-7721 cells with ZnO, Dox alone, Dox/ZnO, or TfR/Dox/ZnO with an equivalent Dox concentration of 0.5 μg mL⁻¹, with or without X-ray irradiation, for 48 hours. Cells without any treatments were used as the control. The cytotoxicity results were estimated by a MTT assay and shown in Fig. 5. Compared with those receiving Dox alone, the viability of the SMMC-7721 cells treated with Dox/ZnO and TfR Ab/Dox/ZnO was decreased, especially with TfR Ab/Dox/ZnO. The obviously increased cytotoxicity of TfR Ab/Dox/ZnO is linked to the improved TfR-mediated cellular uptake of Dox, which was illustrated above. In contrast, X-ray irradiation could clearly decrease the viability of SMMC-7721 cells much more than that without X-ray irradiation, indicating that the radiosensitizer activity of the ZnO nanorods could promote mortality of cells in addition to the mortality effects induced by Dox. The synergistic effect of anticancer activity demonstrated that ZnO nanorods could mediate concurrent chemoradiotherapy for HCC.

Fig. 5 Concurrent chemoradiotherapy mediated by ZnO nanorods against SMMC-7721 cells. *P < 0.05.

Apoptosis induced by ZnO nanorod-mediated concurrent chemoradiotherapy

Next, we investigated the potential mechanisms of chemoradiotherapy by ZnO nanorod-mediated concurrent chemoradiotherapy. To verify whether apoptosis was involved in cell death triggered by ZnO nanorod-mediated concurrent chemoradiotherapy, further investigation with flow cytometry was carried out with SMMC-7721 cells. As shown in Fig. 6, no obvious apoptosis was induced by ZnO nanorods, and treatment of SMMC-7721 cells with Dox alone only weakly induced cell apoptosis (8.2 ± 0.3%). Apoptosis of cells treated by Dox/ZnO was increased by 11.5 ± 0.3%. Furthermore, TfR Ab/Dox/ZnO induced apoptotic cells significantly increased by 17.7% ± 0.4%. In contrast, X-ray irradiation could clearly increase the apoptosis of SMMC-7721 cells much more than without X-ray irradiation with 5.5 ± 0.3%, 12.6 ± 0.4%, 16.3 ± 0.3%, and 26.5 ± 0.4%, respectively, demonstrating that chemoradiotherapy can promote synergistic mortality of cancer cells.

Fig. 6 Apoptosis assay by flow cytometry (A) and a comparison of the induced apoptosis (B) with different treatments. *P < 0.05.

Chemoradiotherapy enhanced suppression of tumor growth in orthotopic HCC xenograft models

X-ray has the deepest tissue penetration compared with visible and UV light. It is also safe and causes minimal damage to the biological specimen involved.⁵⁰ Indeed, nanotechnology-based radiosensitizers are foreseen to improve the outcomes of cancer as a noninvasive treatment for deep tumor.⁵¹ Encouraged by the remarkable anticancer efficacy illustrated above in vitro, we next further performed an investigation of the noninvasive treatment of hepatocellular carcinoma in situ by ZnO nanorod-mediated concurrent chemoradiotherapy in vivo. In order to develop a clinically applicable intervention strategy for HCC, an animal model bearing orthotopic HCC was created in the current study. The tumor appearance, after sacrificing the animals at the end of the study, is shown in Fig. 7A. The changes in the relative tumor volume for different treatments are plotted in Fig. 7B. It was found that mice in the saline control group treated with ZnO nanorods showed severe tumor burden. Mice treated with Dox alone showed a small reduction in tumor load. The Dox/ZnO showed a better inhibition effect than Dox alone at the equivalent dose. Compared with other groups, mice treated with TfR Ab/Dox/ZnO nanocomposites showed a considerable slow-down of tumor growth. This desired treatment effect might be attributed to a specific targeting effect in tumor, allowing the DOX to accumulate selectively in the tumor tissues. In contrast, X-ray irradiation could clearly enhance suppression of tumor growth better than that without X-ray irradiation, demonstrating that concurrent chemoradiotherapy can promote tumor inhibition. Therefore, a short-term and noninvasive treatment strategy could be available for HCC.

Fig. 7 Appearance of tumor bodies at the end of the study after 14 days treatment (A), and relative tumor volume of mice treated by saline as control, ZnO, Dox alone, Dox/ZnO, or TfR/Dox/ZnO nanocomposites (with equivalent Dox dose of 5 mg kg⁻¹), with or without X-ray irradiation (B). *P < 0.05.

Besides, in the present study, we used AFP, a tumor marker for HCC, as a prognostic factor response to chemoradiotherapy.^52,53 As shown in Fig. 8 of the AFP levels at the endpoint, in accordance with the above tumor burden, high levels could be found in mice in the saline control group treated with ZnO nanorods. Compared with Dox alone and Dox/ZnO which showed a small decrease, mice treated with TfR Ab/Dox/ZnO nanocomposites showed a considerable decline. In contrast, X-ray irradiation could clearly decrease the levels of AFP more than treatment without X-ray irradiation. Similar to the study of the tumor appearance, the changes in AFP levels upon treatment correlated positively with the observed efficacy of the different treatments.

Fig. 8 AFP levels of mice treated by saline as the control, ZnO, Dox alone, Dox/ZnO, or TfR/Dox/ZnO (with equivalent Dox dose of 5 mg kg⁻¹), with or without X-ray irradiation.

Attenuation of side effects

The liver, a critical organ with various functions, is known to exhibit low radiation tolerance, which hinders the application of RT involving multimodality treatments for HCC. The radiation tolerance level of approximately 30 Gy for the whole liver does not allow the delivery of therapeutic doses to HCC.¹⁹ Therefore, efforts should be made to improve the radiotherapy with a low dose while preserving normal liver functions. As shown in Fig. 9A, there was no apparent histopathologic damage to the liver after different treatments. Meanwhile, serum biochemistry tests were also performed for quantitative evaluation about important indicators for hepatic function. The resulting data are shown in Fig. 9B and C and there were also no apparent effects on hepatic function in all treatments. This indicates that the ZnO nanorod-mediated concurrent chemoradiotherapy could improve therapeutic efficacy, while avoiding its toxic side effects on liver with short term and low dose X-ray. The great cancer therapy performance without side effects makes it a promising candidate for HCC.

Fig. 9 Histopathologic examination to determine the liver toxicity of different treatments at the end of the experiments (A); serum biochemistry data ALT (B) and AST (C) for hepatic function and kidney function.

Consequently, based on the above studies, Fig. 10 schematically illustrates the possible processes of ZnO nanorod-mediated concurrent chemoradiotherapy for the noninvasive treatment of hepatocellular carcinoma in situ. Firstly, TfR Ab/Dox/ZnO nanocomposites, in which TfR Ab functionalized ZnO nanorods were loaded with Dox, were prepared to act as a targeted DDS. ZnO nanorods have a dual function in the DDS i.e., not only as the role of the drug carrier to deliver Dox into the targeted cancer cells, but also as the radiosensitizer under X-ray irradiation for deep tumor radiotherapy. Thereby, the increased intracellular drug enhanced the anticancer efficiency of the chemotherapeutic agent. Moreover, the excellent radiosensitizer activity of the ZnO nanorods further attack the cancer cells, demonstrating excellent prospects for radiotherapy. Then apoptosis, a preferred mode of killing the cancer cells, is induced synergistically by the concurrent chemoradiotherapy, resulting in a distinct improvement in anticancer activity.

Fig. 10 Schematic illustration of the possible mechanism of the noninvasive treatment of hepatocellular carcinoma in situ by ZnO nanorod-mediated concurrent chemoradiotherapy.

Conclusions

In summary, in this study noninvasive treatment strategy of concurrent chemoradiotherapy was designed based on ZnO nanorods for hepatocellular carcinoma in situ. The results demonstrate that the TfR Ab modified ZnO nanorods can delivery Dox into the targeted HCC SMMC-7721 cells to enhance its chemotherapeutic efficiency. In addition, ZnO nanorods act an adjunct to radiotherapy under X-ray irradiation. Since X-rays have the deepest tissue penetration, low dose and short term X-ray irradiation could further attack the tumor in situ by the radiosensitizer property of ZnO nanorods, noninvasively. The synergistic effect could induce a distinguished improvement in anticancer activity by concurrent chemoradiotherapy. Therefore the TfR Ab/Dox/ZnO nanocomposites could present a promising strategy for comprehensive cancer treatment of HCC.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31200750, 81371678) and the Natural Science Foundation of Jiangsu Province (BK2012332).

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