Materials

Apigenin was obtained from Sigma–Aldrich Lab. (MO, USA). Carboxymethyl-PEG2000-1,2-Distearoyl-sn-glycero-3-phosphoethanolamine or (DSPE-PEG2000-COOH) was purchased from Laysan Bio Inc. (AL, USA). 1-Ethyl-3-(3-dimethylamino propyl) carbodiimide, chloride (EDC, HCl), N-hydroxy succinimide (NHS), cholesterol, dimethyl sulfoxide, butylated hydroxytoluene (BHT), ethanol, chloroform, sodium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate were procured from E Merck Specialties Ltd. (Mumbai, India). Fluorescein isothiocyanate (FITC) and soya-l-α-lecithin (SPC) were acquired from Hi-Media Lab. Pvt. Ltd. (Mumbai, India).

The 26-mer- AS1411 DNA aptamer, having sequence 5′-GT GGT GGT GGT TGT GGT GGT GGT GG-3′ (molecular weight: 8674.3 g/mole) was custom-synthesized as phosphorothioate backbone and 3′-amino-modified form by GCC Biotech (Kolkata, India).

Cell lines

All the in vitro cell-based studies were conducted on human liver cancer cell lines, Hep-G2/Huh-7 cell lines purchased from NCCS (National Centre for Cell Science, Pune, India). The cell lines were sub-cultured in DMEM media, supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic solution, and maintained at 5% CO2 containing humidified air at 37 °C.

Hep G2 is a well-differentiated immortal hepatocellular carcinoma cell line obtained from a Caucasian male aged 15 years [13]. The popularity of the use of Hep G2 cells for in vitro cellular study is related to the fact that it is primary liver cancer (well represented by hepatocellular carcinoma, HCC). Since Hep G2 cells have a high degree of morphological and functional differentiation in vitro, they are considered a suitable model for studying the intracellular trafficking and dynamics of various chemicals, including drugs, formulations, proteins, and ligand molecules in vitro [14]. Several studies reported Hep G2 as in vitro model for detecting cytoprotective, cytotoxic, genotoxic, and antigenotoxic agents in hepatocarcinogenesis and drug-targeting investigations [15, 16]. Thus, we selected Hep G2 cells for the in vitro investigation.

Huh 7 is also a well-differentiated hepatocellular carcinoma cell line, isolated from the tumor originated from liver hepatocytes, from a 57-year-old Japanese male.

Sprague Dawley (SD) rats for hepatocellular carcinoma (HCC) model

Sprague Dawley (SD) rats (120–150 g) were procured from NIN (National Institution of Nutrition), Hyderabad, India, and animal studies, conducted here were approved by the Institutional Animal Ethical Committee (AEC), Jadavpur University on submission of proper experimental protocol plan following guidelines of the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

Pre-formulation studies (FTIR-Fourier-transform infrared spectrometry)

Fourier -transform infrared spectrometric (FTIR) analysis was applied to examine if there was any molecular interaction between the drug and the excipients used during the formulation development [17]. FTIR spectroscopy was carried out with the drug (pure apigenin, Api), drug excipients (SLY, CHC, DSPE-PEG, BHT) individually along with their physical mixture, formulated plain nanoliposome (NLCs) with or without the drug, PEG-NLCs and finally, aptamer conjugated PEGylated nanoliposomes, Apt-NLCs, within the wave number array of 4000–400 cm−1 under inert atmospheric conditioning KBr palate by an FTIR spectrophotometer (Bruker-(Alpha), Ettlingen, Germany) and were analyzed.

Preparation of apigenin (Api)-loaded nanoliposomes (NLCs, PEG-NLCs) and aptamer functionalized PEGylated Api-loaded nanoliposomes (Apt-NLCs)

Apigenin-encapsulated plain nanoliposomes (NLCs) and PEGylated NLCs were prepared by the thin-film hydration method [18]. For subsequent preparation of the aptamer functionalized PEGylated nanoliposomes (Apt-NLCs), we have used the PEGylated NLCs (PEG-NLCs). The coupling of aptamer on the liposomal surface was accomplished by a covalent attachment of the amino group of the aptamer (NH2-modified AS1411) with the carboxyl group of PEG-DSPE-COOH in the PEG-NLCS [19]. Surface grafting of amino-modified AS1411 aptamer on the prepared pegylated NLCs was conducted through the activation carboxyl group of PEG component (PEGylated-1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE-COOH)). The pegylated NLCs were suspended in a mixture of 150 mM N-hydroxy succinimide (NHS) and 300 mM ethyl (dimethyl aminopropyl) carbodiimide (EDC) at room temperature for 1 h by maintaining all through a neutral of 1 M Tris HCI buffer [19, 20]. The resulting solution was added to 25 µl aliquot from 100 µM of 3′amine-modified AS1411 aptamer stock solution (the final concentration of AS1411 in experimental nanoliposomes Apt-NLCs was 5 µM) [21]. Here, the portion of the sample aptamer solution was previously arranged by applying a denaturation–renaturation process so that it would interact in a biochemical reaction readily. The solution mixture was kept in a shaker incubator overnight at normal room temperature to accomplish aptamer ligation on the surface of PEGylated NLCs. Finally, we removed the excess and unconjugated aptamer from aptamer-conjugated nanoliposomes (Apt-NLCs) by centrifuging and washing the mixture thrice with nuclease-free deionized water and the aptamer conjugated formulations were stored at 4 °C for further analysis [22].

The blank aptamer conjugated liposomes (blank-Apt-NLCs) were also prepared following a similar process but without encapsulating apigenin in the liposomes. Both NLCs and Apt-NLCs were labeled with FITC (fluorescein isothiocyanate) dye by incorporating 10 µl of FITC (0.4% w/v ethanolic solution) into the organic phase during thin layer formation of liposome preparation [22].

Determination of aptamer conjugation by agarose gel electrophoresis

To determine the successful ligation of aptamer AS1411 with the PEGylated liposomes (PEG-NLCs), aptamer conjugated nanoliposome (Apt-NLCs), free aptamer (AS1411), and a commercial 50 bp DNA ladder were sequentially placed in the wells of previously prepared 1% agarose gel plate. Through running electrophoresis for 20 min at 100 V, we observed the migration pattern for different samples as mentioned above [22, 23]. Here, 0.5 mg/ml ethidium bromide was used in the agarose gel to visualize bands in the gel electrophoresis, and gel loading dye was used for staining DNA during the electrophoresis process. Finally, free DNA aptamer, DNA ladder, and the DNA-conjugated nanoformulations in gel plate were visualized using a UV transilluminator as reported earlier [24].

To determine the conjugation of AS1411 aptamer on to PEG-NLCs quantitatively, first we repetitively washed out (more than three times) the Apt-PEG-NLCs solution with nuclease free water to remove all the unconjugated free aptamer available in Apt-PEG-NLCs. Finally, with proper dilution adding nuclease free water to it, we compared the amount of AS1411 aptamer in aptamer conjugated nanoliposomes, Apt-PEG-NLCs along with 1 µl of a free AS1411 (DNA aptamer) and a plain PEGylated nanoliposomes, PEG-NLCs (without aptamer) by applying nanodrop UV spectrophotometric (nano 300 micro spectrophotometer, IGene Labserve Pvt Ltd. India) method [25, 26].

Percentage of drug loading and drug entrapment efficiency

To determine drug-loading, the accurately weight of lyophilized liposome (2 mg), NLCs/PEG-NLCs or Apt-NLCs was dissolved in 2 ml of ethanol-acetonitrile-dimethyl sulfoxide solvent mixture at a ratio of 0.5:1:1(v/v) as the best suit solvent composition for testing drug loading of the experimental formulations. It was determined by the trial-and-error method. The resulting solution was vortex-mixed for 1–2 h and then centrifuged for 20 min at 10,000 rpm, clear supernatant (1 ml) was collected to measure the absorbance intensity by the UV/VIS-spectrometer at the corresponding λmax of apigenin 340 nm, and the drug content in the test liposomes were analyzed from the corresponding standard calibration curve prepared early [7, 18]. Thus, percentage drug-loading and drug encapsulation efficacy were obtained using the formulae mentioned below.

$$}\left( } \right) \times $$

(1)

$$}\left( } \right) \, \times $$

(2)

Particle size distribution & ζ-potential measurements

Particle size distribution and ζ-potential of NLCs, PEG-NLCs, and Apt-NLCs were analyzed by Malvern Zetasizer Nano-ZS 90; (Malvern Instruments, UK) applying dynamic light scattering (DLS) technique with sample suspension of the experimental liposomes prepared by diluting them properly, vortex-mixing and sonication process. The results represented were the average particle size considering the standard deviation of at least three different batches of the experimental liposomes.

Field emission scanning electron microscopy (FESEM)

Surface morphology of prepared test nanoliposomes (NLCs, PEG-NLCs, and Apt-NLCs) were observed under FESEM by forming a very thin layer of samples coated with platinum with a platinum coater at an accelerating voltage of 10 kV [27].

Atomic force microscopy (AFM)

The three-dimensional architecture of Apt-NLCs was observed under atomic force microscopy (AFM) by placing one drop of properly diluted and air-dried formulation suspension on a mica surface coverslip plate. Thus, shapes of the Apt-NLCs were observed through AFM by fixing a resonance frequency at 150–250 kHz under ACAFM mode [28, 29].

Cryo-transmission electron microscopy (Cryo-TEM)

The internal bilayer morphology, membrane stability and surface functionalization characteristics of the prepared experimental nanoliposome, Apt-NLCs, were determined by cryo-TEM. Here test liposomes were dispersed (100 µg/ml) in Milli Q water, and an aliquot (4 μl) of it was placed on a glow-discharged 300 mesh carbon-coated copper grid (TEM grid), and the air-dried samples were visualized under the cryo-TEM instrument (200 kV Talos-Arctica electron microscope, FEI/Thermo Scientific) [30].

In vitro drug release

In vitro drug release study was conducted by taking 2 ml of experimental liposomal suspensions (which were prepared by dispersing 2 mg of lyophilized NLCs, PEG-NLCs, and Apt-NLCs individually in 2 ml of buffer solution, PBS phosphate buffer with pH 7.4/acetate buffer, with pH-5) into a dialysis bag (Dialysis Membrane-110, Himedia, India). Then, the sample was dialyzed into 50 ml of respective drug release media, PBS/acetic buffer, containing 0.01% (w/v) β-cyclodextrin at room temperature (37 °C) by placing the dialysis system on a magnetic stirrer with very slow stirring at 100 rpm. During each study, 1 ml of drug release media was collected from 50 ml of solution at predetermined intervals up to 96 h, and was replaced by 1 ml of fresh media immediately [31]. Finally, all the samples were analyzed by UV–VIS spectroscopy at 340 nm wavelength against the respective medium without drug, and drug concentration was determined using a calibration curve prepared earlier. In vitro cumulative drug release data were represented through various release kinetic models such as zero order, first order, Higuchi model, Korsmeyer–Peppas and Hixson–Crowell models, and the highest R2 (correlation coefficient) value was evaluated after plotting the drug released data to determine the best suit drug-release kinetic model [32].

Stability study

Stability assay of the intended experimental formulation, aptamer-conjugated PEGylated nanoliposomes (Apt-NLCs), was performed as per ICH guidelines [22], at the storage conditions (40 ± 2 °C and 75 ± 5% RH) and in the refrigerated form at (~ 4 °C) for 6 months. The samples were then subjected to examination for any changes in particle size or morphology using FESEM and variation in drug content in the prepared nanoliposome (Apt-NLCs) by conducting a drug loading assay.

MTT assay to evaluate in vitro antiproliferative activity of test liposomes

Two different liver cancer cell lines, Hep G2 and Huh-7, were procured from the National Centre for Cell Science, Pune, India. The cells were cultured in DMEM media, maintaining all necessary conditions [33]. The cell (taking 1 × 103 cells/well) viability expressed as IC50 doses for the free drug (apigenin), NLCs/PEG-NLCS, Apt -NLCs and blank-Apt-NLCs (Apt -NLCs without the drug) in both the cell lines were evaluated through MTT [(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide] assay using a wide range of individual equivalent drug concentration (1 µM, 10 µM, 20 µM, 30 µM, 40 µM, 50 µM) following the standard protocol [34].

Human peripheral blood mononuclear cell (PBMC) processing

This process is performed by isolating peripheral blood mononuclear cells (PBMC) from anticoagulated blood. Anticoagulated blood was added with an equal volume of Ficoll-Hypaque (Histopaque-1077) and centrifuged at 400g for 30 min to separate the cells from the whole blood. The PBMCs were collected from the interface of two liquids, washed with PBS (0.01 M, pH 7.4) twice, and resuspended in the RPMI 1640 medium [22]. The cytotoxic activity of different nanoliposomes suspended in PBS in the presence of a very minute amount of dimethyl sulfoxide, DMSO (final DMSO concentration < 0.1%), was evaluated for PBMC using an MTT assay.

Apoptosis assay

To verify the apoptotic potential of aptamer functionalized nanoliposome, Apt-NLCs, in comparison with the nonconjugated ones (NLCs/Peg-NLCs) in Hep G2 cells, we executed a flow cytometric (BD LSRFortessa™, BD Biosciences) study using FITC-Annexin V staining assay kit (BD Biosciences). The apoptotic activity of the experimental nanoliposomes containing an equivalent amount of active drug (considering the IC50 values from MTT) was determined upon 48 h of prior treatment and following the protocols [29].

Determination of cell-targeting potential of the aptamer (AS1411) assessing in vitro cellular uptake of the experimental nanoliposomes

In vitro uptake of FITC-labeled PEG-NLCs and FITC-labeled Apt-NLCs by HepG2 cells treated with the formulations containing the equivalent amount of drug was evaluated quantitatively at the three successive time points (1 h, 2 h, 4 h) by flow cytometric method. To confirm the receptor-mediated cellular uptake of an aptamer (AS1411) functionalized PEGylated nano-liposome (Apt-NLCs) over the other non-functionalized pegylated nanoliposome (PEG-NLCs), a competitive assay was performed. In this competitive assay, Hep G2 cells were preincubated with an excess amount (0.2 µg/well) of free aptamer (AS1411) for 2 h to block available coupling receptors (majorly nucleolin) [34, 35]. After that, it was treated with Apt-NLCs maintaining all other conditions the same. A quantitative comparison of in vitro cellular uptake between FITC-labeled formulations PEG-NLCs and Apt-NLCs in Hep G2 cells and FITC-labeled Apt-NLCs against aptamer-pretreated Hep G2 cells were estimated to verify receptor-mediated cellular uptake of aptamer-conjugated experimental nanoliposomes, Apt-NLCs. FACS Diva software (BD Biosciences) was used to analyze the data [35]. We have also captured confocal microscopic (Olympus Fluo View FV10i, Olympus) images of Hep G2 cells treated with FITC-labeled PEGylated-NLCs and -Apt-NLCs at 1 h and 4 h intervals to visualize the cellular uptake qualitatively.

Study of cell cycle arrest and apoptosis-related proteins

FACS (BD Biosciences, USA) analyzer was also used to analyze different phases of cell cycle during cell propagation, referring to apoptotic behavior of test nanoliposomes (an equivalent IC50 dose of apigenin containing in the respective amount of NLCs, PEG-NLCs and Apt-NLCs was used to treat 1.5 × 105 Hep G2 cells per well in six-well plates for 24 h). After washing in 50 µl RNase-free water and treating with Anexin-V, propidium iodide (PI) solution, the cells were subjected to analysis [36].

Similarly, apoptosis-related signaling proteins (p53, cleaved caspases, Bcl-2) were also estimated using respective protein markers by flow cytometric assay. These proteins have a major influence on the genetic regulation process (DNA synthesis, damage, or repair) during apoptosis. The experiments were conducted in Hep G2 cells, treated with Apt-NLCs, PEG-NLCs, and NLCs, taking an equivalent dose of apigenin for 24 h following the published protocol and guidelines [36].

Development of DENA-induced hepatocellular carcinoma in Sprague Dawley (SD) rats

The Institutional Animal Ethical Committee Jadavpur University approved all the animal studies. Development of hepatocellular carcinoma induced by chemical carcinogens such as diethyl nitrosamine and 2-acetylaminofluorine (AAF) has been well-known in animal models for the last several decades [37, 38]. In the present study, we used Sprague Dawley rats. Carcinogen-induced initiation-promotion model was used. Hepatocellular carcinoma (HCC) was developed in Sprague Dawley male rats using chemical carcinogens, applying initially a single intraperitoneal dose of 200 mg/kg of diethyl-nitrosamine (DENA), followed by a bi-weekly oral administration of 0.5% of w/w 2-acetylaminofluorene (2-AAF) for 16 weeks [38, 39]. Experimental groupings (A–G) were done as normal control rats (Group A), carcinogen control rats (Group B), carcinogen-treated rats received free apigenin (Group C), carcinogen-treated received NLCs (Group D), carcinogen-treated rats received PEG-NLCs (Group E), carcinogen-treated rats received Apt-NLCs (Group F), and normal rats received Apt-NLCs (Group G). All the carcinogen-treated groups were injected (through the i.v. route) 20 mg/kg body weight of free apigenin or an equivalent dose of the experimental formulations once a week for 8 weeks after the 16th week of carcinogen-treated animals [7, 22].

Pharmacokinetic study

The plasma and liver pharmacokinetic profiles for free drug, NLCs, PEG-NLCs, and Apt-NLCs were evaluated in HCC-induced Sprague Dawley (SD) rats (body weight, 125–150 g). Plasma and liver samples were collected from the experimental animals after injecting a single i.v. bolus dosage of 2 mg/kg of body weight of apigenin or corresponding amount of NLCs, PEG-NLCs, and Apt-NLCs containing equivalent apigenin, at 2, 4, 8, 12, 24, 48, 72, 96, and 120 h. The final analytical samples were prepared from the biological samples through the liquid–liquid extraction process (using TBME, t-butyl methyl ether as a volatile solvent for drug extraction from plasma and liver homogenate) and analyzed by LC–MS/MS method applying naringenin as an internal standard [22]. The experiments were repeated in triplicate.

Assessing biodistribution of NLCs, PEG-NLCs, and Apt-NLCs by Gamma scintigraphy study

To investigate the accumulation efficiency of liposomes, NLCs, PEG-NLCs and Apt-NLCs, toward the target organ (liver), the formulations were radiolabeled by technetium-99 m (99mTc) following the stannous chloride reduction method with a radiolabeling accuracy almost above 90% [33]. Radiolabeled formulations (equivalent drug dose) were injected through the cannulation process to carcinogen-treated rats to ascertain the pattern of bio-distribution of different test nanoliposomes (NLCs/PEG-NLCs/Apt-NLCs) at 4 h and 8 h after their administrations. The results were articulated as % administered dose distributed per gram (%ID/g) of tissues or organs. Gama-scintigraph images of the animals were captured at 4 h and 8 h post-injection through GE Infinia γ Camera facilitated along with Xeleris Work Station, USA [29, 33].

In vivo hepatocyte uptake of fluorescent-labeled formulations by confocal microscopic study

To elucidate the accumulation pattern of different test nanoliposomes in the neoplastic hepatocytes, we have injected FITC-labeled NLCs, PEG-NLCs, and Apt-NLCs into the carcinogen-induced HCC animals through the tail veins. Taking a time interval of 4 h and 8 h post-injection, tumors and tumor-adjacent tissues were collected by sacrificing the animals and stored at 10% formaldehyde [22]. The tumors and tumor-adjacent tissue sections were fixed on slides and evaluated under confocal microscopy.

Assessing antitumor efficacy of the experimental nanoliposomes using a prepared animal model

To ascertain the antitumor potencial of the test nanoliposomes, we divided the animals into seven groups, Gr.A, normal (control), Gr. B, HCC positive control animals, HCC control, Gr.C, HCC induced animals treated with the free drug, Gr.D, HCC induced animals treated with NLCs, Gr.E, HCC induced animals treated with PEG-NLCs, Gr. F, HCC-induced animals treated with Apt-NLCs, Gr G, normal animals treated with Apt-NLCs. In the case of Gr C, D, E, F, and G, we followed a treatment schedule of once-weekly i.v. dose of 20 mg/kg body weight of free apigenin or equivalent amount formulations for twelve weeks. There were seven experimental animals in each group. At the end of the treatment, we collected the whole liver, and liver sections from the sacrificed experimental animals were assessed for the degree of antitumor potency of Apt-NLCs compared to NLCs/PEG-NLCs or free drug.

Gross and histopathological examination for liver morphology

We performed a gross macroscopic examination of the whole liver to identify the tumor incidences. Further, microscopic observation of tumor tissue sections by histopathological staining (with hematoxylin and eosin, periodic acid-Schiff) was conducted to evaluate the microscopic changes in the liver histopathology and to identify hepatic altered focal lesions [7].

Identifying apoptotic gene expression level through the qRT-PCR analysis

Total RNA was extracted from frozen (stored at − 70 °C) liver tissue sample (from Gr B, C, D, E & F animals) with Trizol reagent and was evaluated through nanodrop (QI Aexpert) after following the manufacturer’s protocol. Then, with the required amount of sample RNA, cDNA was prepared and using this template, RT-PCR studies were performed with the aid Bio-Rad SYBR green PCR master mix along with corresponding primers as per manufacturer’s instructions [40, 41]. The specific synthetic primers (for p53, F:5′-ATGTTTTGCCAACTGGCCAAG-3′, R:5′-TGAGCAGCGCTCATGGTG-3′); (for caspase 3, F: 5′-GTGGAACTGACGATGATATGGC-3′, R: 5′-CGCAAAGTGACTGGATGAACC-3′); (for Bcl-2, F:5′-TGTGGATGACTGACTACCTGAACC-3′, R:5′-CAGCCAGGAGAAATCAAACAGAGG-3′) and (for β-actin, F:5'-AAGATCCTGACCGAGCGTGG-3′, R:5′-CAGCACTGTGTTGGCATAGAGG-3′) were purchased from Saha Gene, Hyderabad, India and maintaining the thermo-cycling conditions as per the manufacturer’s protocol. The qRT-PCR studies were performed on Rotorgene (Qiagen) instrument. The fluorescence activity in qRT-PCR demonstrated the relative gene expression level for corresponding target genes and it was determined by applying 2-∆∆Ct calculative process, where β-actin was taken as the housekeeping gene [42].

Hepatic function test

Blood samples were collected from all the groups of experimental animals (Gr A-G), and after processing as per the reported method [33], the level of AST (aspartate aminotransferase), ALT (alanine transaminase), and ALP (alkaline phosphatase) were determined using commercially available bioassay kits (Coral Clinical Systems, Goa, India) following manufacturer protocols [33].

Statistical analysis

The data were statistically analyzed using ORIGIN 8.0 software, one-way ANOVA followed by post hoc Dunnett’s test using Graph Pad Prism Software, considered 5.0. p < 0.05 was as a minimum level of significance.